Dissertations / Theses: 'GMPE'

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Akyuz, Emre. "Development Of Site Specific Vertical Design Spectrum For Turkey." Master's thesis, METU, 2013. http://etd.lib.metu.edu.tr/upload/12615403/index.pdf.

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Vertical design spectra may be developed in a probabilistic seismic hazard assessment (PSHA) by computing the hazard using vertical ground motion prediction equations (GMPEs), or using a vertical-to-horizontal spectral acceleration (V/H) ratio GMPEs to scale the horizontal spectrum that was developed using the results of horizontal component PSHA. The objective of this study is to provide GMPEs that are compatible with regional ground motion characteristics to perform both alternatives. GMPEs for the V/H ratio were developed recently by Gü
lerce and Abrahamson (2011) using NGA-W1 database. A strong motion dataset consistent with the V/H ratio model parameters is developed by including strong motion data from earthquakes occurred in Turkey with at least three recordings per earthquake. The compatibility of GA2011 V/H ratio model with the magnitude, distance, and site amplification scaling of Turkish ground motion dataset is evaluated by using inter-event and intra-event residual plots and necessary coefficients of the model is adjusted to reflect the regional characteristics. Analysis of the model performance in the recent moderate-tolarge magnitude earthquakes occurred in Turkey shows that the Turkey-Adjusted GA2011 model is a suitable candidate V/H ratio model for PSHA studies conducted in Turkey. Using the same dataset, a preliminary vertical ground motion prediction equation for Turkey consistent with the preliminary vertical model based on NGA-W1 dataset is developed. Proposed preliminary model is applicable to magnitudes 5-8.5, distances 0-200 km, and spectral periods of 0-10 seconds and offers an up-to-date alternative to the regional vertical GMPEs proposed by Kalkan and Gü
lkan (2004).

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Moratto, Luca. "Ground motion estimation in the eastern-southern alps:from ground motion predictive equations to real-time shake maps." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2688.

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2006/2007
Lo scopo di questa tesi di dottorato è la stima del moto forte del suolo nell’area delle Alpi Sud-Orientali. A tal fine sono state proposte delle relazioni empiriche che stimano i parametri del moto in funzione della magnitudo, della distanza dall’epicentro e della classificazione geologica del suolo; successivamente tali relazioni sono state usate per calibrare il software ShakeMaps con il fine di generare in tempo reale le mappe di scuotimento del terreno per la regione Friuli-Venezia Giulia. Le GMPEs (Ground Motion Predictive Equations) per PGA, PGV e SA sono state calcolate nell’area delle Alpi Sud-Orientali utilizzando registrazioni del moto forte del terreno. Sono state selezionate 900 forme d’onde accelerometriche filtrate tra 0.1 Hz e 30 Hz; la distanza epicentrale varia tra 1 km a 100 km, mentre la magnitudo locale, opportunamente calibrata confrontando diversi cataloghi, varia in un intervallo relativamente ampio (3.0 <= ML <= 6.3). Sono stati testati diversi modelli di attenuazione e il miglior risultato è stato individuato utilizzando specifici criteri di valutazione derivanti da considerazioni di carattere statistico (valore di R2, uso dell’ANOVA test, analisi dei residui). I coefficienti del modello finale sono stati determinati oltre che da ML, dalla distanza epicentrale e dagli effetti dovuti al sito, anche dalla saturazione della magnitudo, dalla correlazione tra magnitudo e distanza e dagli effetti di “near-source”. I coefficienti delle GMPEs sono stati calcolati per le componenti verticali ed orizzontali (rappresentata sia con la componente maggiore sia con la somma vettoriale delle due componenti); la tecnica dell’analisi dei gruppi ha permesso di ridurre l’incertezza finale sulle relazioni empiriche. Il confronto con i risultati ottenuti precedentemente evidenzia come le relazioni ottenute in questa tesi abbiano una maggiore attenuazione a basse magnitudo e a grandi distanze; risultati analoghi sono stati ottenuti per le relazioni ricavate dai dati registrati in tutta l’Italia Settentrionale. L’evoluzione recente delle reti sismiche rende oggi disponibile una grossa mole di dati acquisiti in tempo reale, per cui risulta fattibile stimare velocemente lo scuotimento del terreno tramite mappe; il software “ShakeMap” è stato adattato alle Alpi Sud-Orientali implementato allo scopo di ottenere una stabile interfaccia con il sistema di acquisizione dati “Antelope” che garantisca l’estrazione dei parametri del moto dalle forme d’onda e la creazione delle mappe di scuotimento entro 5 minuti dall’evento sismico. Questa procedura richiede una fitta e uniforme distribuzione spaziale degli strumenti di registrazione sul territorio e una classificazione geologica del suolo fatta usando le velocita’ medie, Vs30, dei primi 30m del mezzo immediatamente sotto gli strumenti. La classificazione geologica del suolo prevede la suddivisione in tre categorie (suolo rigido, suolo addensato e suolo soffice) mentre i coefficienti di amplificazione sono stati calcolati usando le relazioni proposte da Borcherdt (1994). Le relative mappe vanno calcolate usando le GMPEs e le relazioni empiriche che legano il moto del terreno all’intensità macrosismica, basate ambedue su dati registrati nella regione alpina. Le GMPEs discusse in precedenza sono state inserite nel software “ShakeMap” per la produzione delle mappe di scuotimento in tempo reale e quasi-reale nell’Italia Nord-Orientale. Per valutare l’effetto della densità di stazioni sulle mappe di scuotimento sono stati calcolati dei sismogrammi sintetici relativi al terremoto di Bovec 2004 variando il passo di griglia e la geometria dei ricevitori. I risultati ottenuti indicano come una distribuzione fitta e uniforme di strumenti sul territorio e una scelta accurata delle dimensioni della griglia dei ricevitori siano cruciali per calibrare le mappe di scuotimento in una ben determinata area geografica. Le mappe di scuotimento del suolo sono state generate per otto terremoti avvenuti nell’area considerata negli ultimi 30 anni; inoltre per gli eventi del Friuli 1976 e Bovec 1998 è stato utilizzato il modello di faglia finita con i parametri di sorgente stimati in precedenti studi. La validazione del modello è stata fatta calcolando il misfit tra le intensità macrosismiche osservate (catalogo DBMI04) e quelle “strumentali” che sono state ottenute dai sismogrammi sintetici tramite relazioni empiriche tra moto del suolo ed intensità. L’analisi è stata fatta per i terremoti del Cansiglio (1936), del Friuli (1976) e di Bovec (1998). I sismogrammi sintetici sono stati calcolati ad una frequenza massima di 10 Hz applicando il modello della riflettività; i parametri del moto sono stati estratti dai segnali sintetici calcolati nelle attuali stazioni di registrazione e successivamente sono state generate le mappe di scuotimento. L’intensità macrosismica “strumentale” è stata ricavata applicando diverse relazioni; il minor misfit è stato ottenuto usando le relazioni proposte da Kästli and Fäh (2006) per tutti e tre i terremoti considerati, il che sembra validare il nostro modello di Shake Maps.
The aim of this PhD thesis is to estimate ground motions in the South-Eastern Alps area. For this purpose we purposed empirical relationships that estimate the ground motion parameters as function of the magnitude, the epicentral distance and the soil geological characterization. Later on these relationships are used to calibrate the ShakeMaps software to generate ground motion shake maps in real time for the Friuli-Venezia Giulia region. The GMPEs (Ground Motion Predictive Equations) for PGA, PGV and SA are computed in the South-Eastern Alps area using strong motion observations. 900 accelerometric waveforms are selected and filtered between 0.1 Hz and 30 Hz; the epicentral distance varies from 1 km to 100 km, while the local magnitude, calibrated by comparison with various catalogues, varies in a relatively wide range (3.0 <= ML <= 6.3). Various attenuation models are tested and the best result is selected by the adoption of specific evaluation criteria derived from statistical considerations (R2 value, ANOVA test, residuals analysis). The coefficients of the final model are determined from ML, the epicentral distance, the site effects, the magnitude saturation, the correlation between the distance and the magnitude and the near-source effects. The coefficients of the GMPEs are computed from vertical and horizontal components (the latter represented both as the largest horizontal component and the vectorial addiction); the cluster analysis reduces the final uncertainties on the empirical relations. The comparison with the previous results evidences that the obtained relationships are characterized by a strong attenuation at low magnitudes and large distances. Similar results are obtained for the relationships derived from data recorded all over Northern Italy. The recent evolution of the seismic networks provides a large number of data, available in real time, so it is possible to quickly estimate shake maps. The “ShakeMap” software has been adapted to the South-Eastern Alps region and implemented to obtain a stable interface with the “Antelope” acquisition system in order to extract the ground motion parameters from the waveforms and the generation of the shake maps within 5 minutes from the earthquake occurrence. This procedure requires a dense and uniform spatial distribution of the recording instruments in the field and a geological classification of the soil derived from the average velocities of the S waves in the first 30m below the recording instruments (Vs30). In the geological classification the soil is divided into three classes (bedrock, stiff soil and soft soil), and the amplification coefficients are computed using the relationships proposed by Borcherdt (1994). The related maps are generated using the GMPEs and the empirical relations that predict the macroseismic intensity from the ground motion, both derived from data observed in the Alpine region. The GMPEs that are obtained in this thesis are inserted in the ShakeMap software to generate shake maps in real time or quasi real time in North-Eastern Italy. To evaluate the effects of the station coverage on the shake maps, synthetic seismograms are computed for the Bovec 2004 earthquake by varying the grid size and the network geometry. The results indicate that a dense and uniform spatial distribution in the field and a careful choice of the grid size are crucial to calibrate the shake maps in a given geographical area. The shake maps are generated for eight earthquakes occurred in the studied area in the last 30 years. Furthermore, the finite-fault model is utilized for the seismic events of the Friuli 1976 and Bovec 1998 selecting the source parameters proposed in previous studies. The model validation is done computing the misfit value between the observed macroseismic data (DBMI04 catalogue) and the “instrumental” intensities that are obtained from the synthetic seismograms using empirical relationships between the ground motion and intensity. This analysis has been done for the earthquakes of Cansiglio (1936), Friuli (1976) and Bovec (1998). The synthetic seismograms are calculated for an upper cutoff frequency of 10 Hz applying the reflectivity model. The ground motion parameters are extracted from synthetic signals computed at the presently operating seismic stations and the shake maps are generated. The macroseismic intensity is derived from various relationships; the lowest misfit is obtained using the relation proposed by Kästli and Fäh (2006) for all considered seismic events and this seem to validate our Shake Maps model.
XX Ciclo
1978

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Moubarak, Majed. "Étude des effets du peptide natriurétique atrial sur les fibroblastes : implication physiopathologique dans le remodelage cardiaque." Thesis, Poitiers, 2014. http://www.theses.fr/2014POIT2312/document.

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L'ANP est une hormone cardiaque libérée lors de l'insuffisance cardiaque. Les fibroblastes cardiaques, responsables de la synthèse des composants de la matrice extracellulaire (MEC), acquièrent dans les conditions pathologiques la capacité de se différencier en myofibroblastes, conduisant ainsi à une fibrose cardiaque. Les mécanismes de régulation impliquant l'ANP et ses récepteurs (NPR) restent peu connus et font l'objet de ce travail. Les fibroblastes ventriculaires ont été isolés à partir de coeurs de rats Wistar et mis en culture afin d'induire leur différenciation. Les cultures ont ensuite été soumises à différents traitements impliqués dans la voie ANP/NPR. L'ANP diminue le taux de prolifération, la migration cellulaire, et la sécrétion de collagène des myofibroblastes. Cet effet est mimé par le 8-Br-GMPc. L'analyse protéomique et génomique a permis de confirmer la présence des récepteurs natriurétiques A et B dans nos cellules. Par ailleurs, l'expression de dix isoformes de phosphodiestérases dans les myofibroblastes a été révélée par un criblage génomique. L'inhibition non sélective de ces phosphodiestérases provoque une diminution de la prolifération et de la sécrétion de collagène. Enfin, les concentrations intracellulaires de GMPc et d'AMPc ont été trouvées augmentées en présence de l'ANP. En parallèle, la caractérisation des courants ioniques présents sur les myofibroblastes a montré une absence des courants sodique rapide et potassique ATP-dépendant. Cette étude montre le rôle de la voie ANP/NPR/GMPc dans la modulation des propriétés fibroblastiques et illustre la complexité des processus de différenciation cellulaire au cours de la fibrogenèse cardiaque
ANP is a cardiac hormone released during heart failure and acts as a regulator of the extracellular matrix (ECM). Cardiac fibroblasts are responsible for the synthesis of ECM components and acquire under pathological conditions the capacity to differentiate into myofibroblasts, leading to cardiac fibrosis. Regulatory mechanisms involving ANP and its receptors (NPR) are poorly known and make the subject of our work. Ventricular fibroblasts were isolated from Wistar rat hearts and cultured to induce differentiation. The cultures were then subjected to various treatments involved in the ANP/NPR pathway. ANP decreases the proliferation rate, cell migration and collagen secretion. This effect was mimicked by 8-Br-cGMP. In addition, genomic and proteomic analysis confirmed the presence of the natriuretic receptor A and B in our cells. Furthermore, the expression of ten phosphodiesterases isoforms in the myofibroblasts was revealed by genomic screening. The non-selective inhibition of these phosphodiesterases causes a decrease in the proliferation and secretion of collagen. Finally, the intracellular concentrations of cAMP and cGMP were increased in the presence of ANP. In parallel, the characterization of ionic currents present in myofibroblasts revealed the absence of rapid sodium and potassium ATP-dependent currents. This study shows the role of the ANP/NPR/cGMP pathway in modulating fibroblast properties and exposes the complexity of the cell differentiation process during cardiac fibrogenesis

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Mantler, Mathias. "Der GMP-Vertrag aus bauvergaberechtlicher Sicht /." Frankfurt ; New York : Lang, 2004. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=010635104&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.

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Dissertation Koln, Universität, 2003. Zugl.: Köln, Universiẗat, Diss., 2003 u. d. T.: Der GMP-Vertrag als neue Vertrags- und Wettbewerbsform aus bauvergaberechtlicher Sicht.
Includes bibliographical references (p. 187-204).

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Forner, Francesco <1996&gt. "GME: A Pump and Dump Scheme?" Master's Degree Thesis, Università Ca' Foscari Venezia, 2021. http://hdl.handle.net/10579/19937.

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At the end of January 2021, an important page in financial history was written for the records as a moment in which private investors attacked hedge funds. The operation "headquarter" was Wallstreetbets, a forum of people looking for high return investments and it played a central role in the GME rally. From an economical perspective, the main reason of the exponential increase of the stock price is Short-squeeze; to make it happen, what is need is pressure from the market. This research focused on finding a relationship between activity on the Reddit platform and the change in GME stock price. Many different analysis were used, starting with a Sentiment analysis measuring the similarity beten different NLP dictionaries and verbiage within the Reddit platform. Lastly, a Fama and French model was run to find whether or not, there was an economical reason behind the return amounts for this event. Relevant data was found from all the tests conducted and the result led to believe that this event was a pump and dump scheme.

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Volant, Karine. "Les sécrétions intestinales GMPc-dépendantes chez le rat, in vivo." Lyon 1, 1997. http://www.theses.fr/1997LYO1T066.

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Kameni, Tcheudji Jacques Fulber. "Etude comparative des phosphodiestérases spécifiques du GMPc : relation structure-fonction." Université Louis Pasteur (Strasbourg) (1971-2008), 2001. http://www.theses.fr/2001STR13754.

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Tang, Katherine Mary. "Targets of cyclic GMP in blood platelets, photolabelling, mutagenesis and pharmacological analysis of the cyclic GMP-inhibited phosphodiesterase." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0016/NQ30173.pdf.

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Engelhardt, Thomas. "The regulation of cyclic GMP during anaesthesia." Thesis, University of Aberdeen, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409272.

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The glutamate-NO-cyclic GMP pathway has previously been identified as a potential major target for general anaesthetic agents. An animal model of type I NOS gene disrupted mice was employed to investigate in vivo effects of the general anaesthetic agents isoflurane, ketamine, pentobarbital, and propofol on cyclic GMP in the presence or absence of the isoform specific NOS inhibitor, 7-nitroindazole. G protein function was studied ex vivo in whole brains. Primary neuronal cell cultures were employed to investigate the effects of anaesthetic agents on glutamate stimulated cyclic GMP production. The influence of anaesthetic agents on the metabolism of cyclic GMP via phosphodiesterases was studied in vitro. The effects of anaesthetic agents on the regulation of cyclic GMP in humans are unknown. Cyclic GMP was measured in human oral mucosal transudate in volunteers and patients undergoing short general anaesthesia. A prospective double-blind placebo controlled crossover trial was conducted assessing the effects of selective PDE5 inhibition on propofol sedation requirements in healthy volunteers. Both studies indicate a potential role of cyclic GMP mediating consciousness in humans. The work presented in this thesis indicates substantial effects of anaesthetic agents on the regulation of cyclic GMP in in vivo, ex vivo, in vitro and human studies and provides new insights into the mechanisms involved in modulating general anaesthesia. However, a great deal of further work remains before the complex processes underlying general anaesthesia are fully elucidated.

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Hennan, James Kenneth. "Role of cyclic GMP, cyclic GMP-dependent protein kinase and protein phosphorylation in the control of smooth muscle tension." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0017/NQ56558.pdf.

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Таран, О. І., and Олена Михайлівна Проскурня. "Реалізація нових вимог GMP на прикладі процесу "підготовка та отримання чистих середовищ" на фармацевтичному підприємстві"." Thesis, Національний фармацевтичний університет, 2015. http://repository.kpi.kharkov.ua/handle/KhPI-Press/25029.

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Clerc, Armel. "Mécanisme d'activation de la phosphodiestérase spécifique du GMPc du bâtonnet rétinien." Grenoble 1, 1991. http://www.theses.fr/1991GRE10119.

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Le mecanisme d'activation de la pde specifique du gmpc (pde) par la proteine g (g) a ete etudie en utilisant trois approches differentes: 1) par des mesures d'activite en systemes reconstitues dans lesquels la concentration de g et de pde varient, nous avons montre que l'activation totale de la pde necessite l'intervention de deux molecules d'activateurs (sous-unite alpha de la g avec un gtp lie). Le premier activateur se fixe sur la pde avec une forte affinite (kd voisin de 100 mm), entrainant une faible activation de la pde, tandis que le second se fixe avec une plus faible affinite (kd voisin de 600 nm), mais active de facon totale la pde (la vitesse d'hydrolyse du gmpc est alors 10 a 20 fois superieure a celle obtenue lors de la fixation du premier activateur). Les deux etats actives de la pde ont le meme km pour le gmpc, mais different par leur sensibilite vis-a-vis du gmp; 2) des experiences de fixation aux membranes de disques d'activateurs, en presence de pde et/ou de sous-unites inhibitrices de la pde (pde gamma) ont ete realisees, afin de preciser la nature des etats activites de la pde: complexe regroupant la pde et l'activateur ou sous-unites catalytiques de la pde (alpha et beta) liberees des sous-unites inhibitrices (gamma). Les resultats obtenus lors de la fixation d'activateurs aux membranes en presence de pde gamma (absence de fixation) et de pde native (fixation de 2 activateurs par pde), nous incitent a penser que l'activation de la pde native (fixation de 2 activateurs par pde), nous incitent a penser que l'activation de la pde se fait au sein d'un complexe stable membranaire, compose de pde et de g alpha; 3) des esperiences de reticulation de g alpha et de pde ont permis de mettre en evidence des complexes composes de pde alpha et beta avec une ou deux molecules de g alpha. Ces complexes correspondent aux deux etats actifs de la pde decrit en (1). L'ensemble de ces resultats prouve que la pde active est sous la forme d'un complexe membranaire stable compose de pde (alpha-beta-gamma) et de un ou deux activateurs (g alpha avec un gtp lie). L'activation de la pde se ferait par modification des interactions entre les sous-unites gamma et alpha beta de la pde au sein du complexe

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Schüler, Susanne. "Qualitätskontrolle von Blutkomponenten nach GMP ("Good Manufacturing Practice")." [S.l.] : [s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=95979025X.

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Zolle, Lapuente Olga C. "Cyclic GMP and calcium homeostasis in endothelial cells." Thesis, University of Liverpool, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367654.

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Park, Ji S. "CYCLIC GMP: A SATIETY SIGNAL IN C. ELEGANS." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3851.

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Appetite control and satiety mechanisms help animals maintain energy homeostasis; however, these mechanisms can be misregulated, leading to overweight and obesity. Caenorhabditis elegans is an excellent model system to study appetite and satiety because of its conserved behavioral aspects of satiety and conserved molecular mechanisms. ASI senses nutrition and its activity is required for the behavioral state of satiety quiescence. The purpose of this thesis project was to elucidate the function of cGMP signaling in ASI by looking at behavioral effects from the pharmacological use of sildenafil (Viagra), a PDE inhibitor, and the effects on ASI activation from mutating guanylyl cyclase DAF-11. Sildenafil treatment increases satiety quiescence and decreases fat storage in a PDE-dependent manner. The daf-11 mutation decreased overall fluorescence intensity of ASI activation and the frequency at which ASI activated by about 50% compared to wild-type worms, suggesting that DAF-11 plays an important role in ASI to promote satiety.

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Freedman, John. "Cyclic-di-GMP Signaling in the Borrelia Spirochetes." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/269.

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Lyme disease is the most common tick-borne disease in North America, with approximately 35,000 cases reported to the Centers for Disease Control in 2008. The genome of its causative agent, Borrelia burgdorferi, encodes for a set of genes involved in the metabolism and regulatory activities of the second messenger nucleotide, cyclic-di-GMP (c-di-GMP). Rrp1 is a response regulatory-diguanylate cyclase, and its regulatory capability is likely mediated via production of c-di-GMP, as it lacks a DNA-binding domain. One known class of c-di-GMP effector/binding proteins are those that harbor a PIlZ domain. The genome of B. burgdorferi strain 5A4 encodes for one chromosomally-carried PilZ domain, which we have designated PlzA. Additionally, certain B. burgdorferi strains encode for a second PilZ domain-containing protein (PlzB) which is plasmid-carried. Both PlzA and PlzB were found to bind specifically to c-di-GMP, and c-di-GMP binding by PlzA was found to be dependant upon arginine residues in the c-di-GMP binding region. Additionally, expression of PlzA was found to be upregulated by tick feeding and was constitutive in the mammalian host. We next constructed two deletion/allelic exchange mutants – one with the targeted deletion of PlzA, and on ethat replaced PlzA with PlzB in a strain lacking the plzB gene. Our studies demonstrated that ΔplzA was deficient in motility and was also non-infectious in the mouse model of B. burgdorferi infection. Additionally, this strain remained viable in larval Ixodes ticks. Also, B31-plzB KI was deficient in motility, as well as infectivity, demonstrating that PlzB is unable to complement for functions fo PlzA in vitro and in vivo and that it may play other roles in the biology of B. burgdorferi strains carrying the plzB gene. These studies represent the first identification of a c-di-GMP binding protein in any spirochete, but also represent the first demonstration of the importance of PilZ domain proteins in a spirochetal system. We additionally examined the effects of c-di-GMP synthesis and breakdown in the related bacterium, B. hermsii, a causative agent of tick-borne relapsing fever (TBRF). Deletion mutants in Rrp1 (B. hermsii’s sole diguanylate cyclase) and PdeA (B. hermsii’s only EAL domain-containing phosphodiesterase) were created. These strains were analyzed in order to determine: 1) the effect(s) of the losse of Rrp1/PdeA on intracellular spirochete c-di-GMP levels, and 2) the effects of Rrp1/PdeA on the establishment of murine infection and on gross motility/chemotaxis. It was demonstrated that c-di-GMP accumulates intracellularly in the cells lacking PdeA. Additionally, spirochetes were shown to chemotax towards N-acetyl-glucosamine (NAG) and they did not form soft agar swarms. In contrast, cells lacking Rrp1 did not accumulate detectable levels of c-di-GMP, demonstrated a reduced ability to chemotax towards NAG, and swarmed on soft agar in a fashion indistinguishable from wild type. Despite these differences in phenotype, both mutant strains display an attenuated murine infectivity. These results indicate that c-di-GMP is indeed important in the TBRF spirochete, B. hermsii and this vital second messenger plays key roles in virulence, motility, and chemotaxis. These studies also pave the way for future investigation of B. hermsii through use of targeted genetic manipulation.

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Schüler, Susanne. "Qualitätskontrolle von Blutkomponenten nach GMP ("Good Manufacturing Practice")." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2000. http://dx.doi.org/10.18452/14490.

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Um die Qualität von Blutprodukten nach GMP zu überprüfen, wurden 72 Blutspenden bzw. ihre Folgeprodukte Erythrozytenkonzentrat in PAGGS-M, gefiltertes Erythrozytenkonzentrat und Fresh Frozen Plasma untersucht. Zusätzlich wurden an jeweils 12 Blutderivaten, die z.T. nicht zum Routineprogramm des Blutspendedienstes der Abteilung für Klinische Hämostaseologie und Transfusionsmedizin in Homburg/Saar gehören, Messungen durchgeführt, um ihre Qualität am Herstellungstag und am Ende der Haltbarkeit zu bestimmen. Als letztes wurden schließlich auch rheologische Parameter an 21 Erythrozytenkonzentraten in PAGGS-M und Fresh Frozen Plasma über einen Zeitraum von 28 Tagen gemessen und dokumentiert. Die Messung der Parameter, sowohl hämatologische als auch hämostaseologische und hämorheologische, erfolgte nach Standardmethoden. Die Ergebnisse waren insgesamt nicht zufriedenstellend. Ausschlaggebende Grundlage der Untersuchung sind die GMP-Richtlinien, die sich an den europäischen Normen orientieren. Zwar zeigten die Produkte, die routinemäßig hergestellt werden, wie auch die, die nur für diese Untersuchung produziert wurden, in vielen Bereichen ein Übereinstimmen mit den Vorschriften. Speziell auch die Qualität der gefilterten Erythrozytenkonzentrate, die besonders für Immunsupprimierte wegen ihres niedrigen Gehalts an Leukozyten verwendet werden, war in vielen Fällen gut. Die Ergebnisse der rheologischen Messungen glichen denen anderer Studien: so wurde ein Abfall der Fließfähigkeit über den Meßzeitraum hinweg beobachtet, was auf ein leichtes Schwellen der Zellen und eine Zunahme der Rigidität zurückzuführen ist. Fast durchgängig wich der Meßwert für den Hämatokrit der EK von der Richtlinie ab, am stärksten in PAGGS-M. Als Grund hierfür wurde eine Meßungenauigkeit des elektronischen Zellzählers bzw. die Methode der Bestimmung angesehen, so daß eine echte Abweichung als unwahrscheinlich anzusehen ist. Jedoch wurde die vorgeschriebene maximale Hämolyserate mit Ablauf der Haltbarkeit häufig überschritten, ebenso wie die gemessenen Leukozytenzahlen. Eine Vielzahl der Plasmen war durch Erythrozyten und Leukozyten kontaminiert. Durch häufige Waschvorgänge beim Auftauen von tiefgefrorenen EK wurde ein erhöhter Hb-Überstand erzeugt, das geforderte Mindestvolumen wurde dabei unterschritten. Daher ist festzuhalten, daß für jede untersuchte Produktgruppe Abweichungen festgestellt wurden, die in diesem Maße nicht tolerabel sind. Damit ist die Qualität der Produkte insgesamt als unzureichend zu bezeichnen. Der hier geführte Nachweis dieser Abweichungen ermöglichte die Einführung geänderter Herstellungsverfahren, so daß die heute erzeugten Produkte den Qualitätsvorschriften entsprechen.
72 blood donations and their follow-up products (red blood cell concentrates resuspended in PAGGS-M, leukocyte-reduced red cell concentrates and fresh frozen plasma) were investigated to check their quality. Additionally, tests were performed on blood components of which most were not part of the routine program in the Department of Hemostaseology and Transfusion Medicine in Homburg/Saar. Finally, rheological parameters of red blood cell concentrates in PAGGS-M and of fresh frozen plasma were measured and documented over a period of 28 days. The results were not satisfying. Both the routine products and the blood components which were only produced for this study met the GMP-guidelines in many cases. Especially the quality of leukocyte-reduced red blood cell concentrates which are used especially for the transfusion for immunocompromised patients was good. The results of the rheological measurements were similar to those of other studies: decreasing flow over a period of time which was put down to slight swelling of the cells and to a rise in erythrocyte rigidity. The average hematokrit of red cell concentrates was generally too low especially in PAGGS-M. The reason for this was thought to be the electronic cell counter or rather the method of measuring. Hemolysis after storage and mean leucocyte count often exceeded the official dates. Many samples of the FFP were contaminated with red and white blood cells. Repeated washing after thawing of frozen red blood cell concentrates caused an excess of extracellular haemoglobin and low concentrate volumes. Any deviation from the guidelines is not tolerable. After this study adjustments in the production could be made so blood products now meet all of the requirements.

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Gallardo, Galdames Christian E. "Rol del sistema No-GMPc en la antinocicepción orofacial experimental del ketorolaco." Tesis, Universidad de Chile, 2011. http://repositorio.uchile.cl/handle/2250/132096.

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Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista
Autor no autoriza el acceso a texto completo de su documento
Introducción: Los fármacos más ampliamente usados en el manejo del dolor son los corticoides, AINEs y analgésicos opioides. Otros fármacos moduladores de neurotransmisores, algunos de uso experimental, se han desarrollado como alternativa en analgesia. En este estudio se evaluará la actividad antinociceptiva de ketorolaco (AINE pirrolacético) en presencia y ausencia de LNAME (modulador nitridérgico) en el ensayo de la formalina orofacial en ratones. Material y método: Se utilizaron 120 ratones machos Mus musculus cepa CF/1 a los cuales se les administró i.p. soluciones salinas de ketorolaco en dosis de 10-200 mg/kg, L-NAME 1-5 mg/kg, y la combinación de ambos fármacos (6-8 muestras por cada dosis de los fármacos) 30 minutos previo al test algesiométrico de la formalina, que consiste en la inyección subcutánea en labio superior de formalina al 1%. Se realizó la observación y registro con cronómetro digital del tiempo total de frotamiento perinasal posterior a la inyección de la noxa en fase I algésica y fase II inflamatoria. Posteriormente se realizó análisis de regresión lineal mediante curvas dosisrespuesta para cada fármaco independiente para evaluar el efecto antinociceptivo; para la combinación de fármacos se utilizó el método de potenciación de Zelcer para evaluar la interacción sinérgica o antagónica de la coadministración farmacológica. Los resultados para el análisis estadístico fueron expresados como promedio ± EEM con límite de confianza al 95% (LC =95%) y los cálculos fueron realizados mediante programa computacional del laboratorio (Pharm Tool Pro, versión 1.27, McCary Group Inc.). La significancia estadística considerada fue de un 5% (p<0,05) a través de análisis de varianza y pruebas t de Student. Resultados: El análisis algesiométrico y de las dosis efectivas al 50% (DE ) determinó efecto antinociceptivo dosis-dependiente de ketorolaco y L-NAME al administrarse aislados, respecto al grupo control. La coadministración de ketorolaco con L-NAME 1 mg/kg dio como resultante DE 50 50 de 18,63 ± 1,20 mg/kg en fase I y de 25,47 ± 2,88 mg/kg en fase II; la coadministración de ketorolaco con L-NAME 5 mg/kg dio como resultante DE de 4,90 ± 2,07 mg/kg en fase I y 15,42 ± 2,89 mg/kg en fase II, comparadas con las DE 50 50 de ketorolaco aislado de 22,08 ± 3,08 mg/kg en fase I y de 20,56 ± 4,96 mg/kg en fase II. El análisis estadístico determinó un efecto antagónico de L-NAME sobre la antinocicepción generada por ketorolaco. Conclusiones: La administración i.p. de ketorolaco y de L-NAME aislados produce un efecto antinociceptivo dosis-dependiente en el test de la formalina orofacial. La coadministración de LNAME produce un efecto antagónico en la antinocicepción generada por ketorolaco, respecto a la administración de ketorolaco de manera aislada.

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Matsuyama, Bruno Yasui. "Caracterização estrutural e funcional de FleQ, fator de transcrição envolvido na expressão de genes flagelares e de formação de biofilme." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-02052016-111714/.

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Bactérias podem formar comunidades bacterianas sésseis, conhecidas como biofilmes, os quais possuem um grande impacto tanto na indústria, por serem responsáveis pelo entupimento e corrosão de tubulações, quanto na saúde, por serem resistentes ao tratamento com antibióticos. Nos últimos anos, o mensageiro secundário c-di-GMP foi descoberto como um importante regulador nas vias de sinalização envolvidas na capacidade da bactéria alternar entre esses dois fenótipos: livre nadante ou em biofilme. Um dos alvos moleculares de c-di-GMP é FleQ, uma Enhancer binding protein bacteriana (bEBP), que regula a expressão dos genes flagelares de forma dependente do fator σ54, em um processo dependente de ATP. FleQ também atua no controle da transcrição dos genes envolvidos na síntese do exopolissacarídeo PEL, principal constituinte da matriz protetora que reveste o biofilme bacteriano em Pseudomonas aeruginosa, possivelmente em um processo dependente do fator σ70. A atividade de FleQ é regulada pelos níveis de c-di-GMP e por uma segunda proteína, FleN, que se liga diretamente à FleQ. Apesar do papel do complexo FleQ:FleN na regulação do operon pel ter sido estudado, seu efeito na transcrição dos genes flagelares ainda é desconhecido. Para um melhor entendimento do mecanismo regulatório de FleQ, foram resolvidas as estruturas cristalográficas do domínio REC e do domínio AAA+ em seu estado apo e em complexo com ADP, ATPγS e c-di-GMP. Também foi identificado e confirmado o sítio ativo da proteína, as diferentes formas oligoméricas de FleQ, além da proposição de como a atividade da proteína é controlada por FleN. Esses resultados sugerem um mecanismo único na transcrição mediada por uma bEBP não convencional que atua tanto com o fator σ54 e σ70, no qual a oligomerização do complexo FleQ:FleN possui um papel essencial na regulação dos genes flagelares e de formação do biofilme. Estes estudos também levam à hipótese que outras bEBPs, associadas a diferentes fenótipos, podem ser reguladas por c-di-GMP.
Bacteria can form sessile communities known as biofilm, which has a major industrial impact, causing tubing obstruction and corrosion, and in the health, due to its resistance against antibiotic treatment. In recent years, a novel secondary messenger molecule named c-di-GMP has been discovered as a key regulator in signaling pathways related to the bacteria capacity to alternate between two distinct phenotypes: free-swimming cells and biofilm community. One of the molecular targets of c-di-GMP so far identified is FleQ, a bacterial enhancer binding protein (bEBP), which regulates flagellar gene expression in a σ54 dependent mechanism. FleQ acts also controlling transcription of genes involved in PEL exopolysaccharide synthesis, main component of the protective matrix, possibly in a σ70 dependent process. FleQ activity is regulated by a second protein, FleN, which directly interacts with FleQ. Although the role of FleQ:FleN complex in regulating pel operon has been studied, its effect in flagellar gene transcription has not. For a better understanding of FleQ regulatory mechanism, we obtained the crystallographic structure of the REC domain and the AAA+ domain in its apo state and bound to ADP, ATPγS and c-di-GMP. It has also been identified and confirmed c-di-GMP binding site, the distinct oligomers of FleQ, and also proposed a mechanism by which FleN regulates FleQ. These results suggest an unique mechanism in the transcription mediated by an unusual bEBP that acts in conjunction with both σ54 and σ70 factors, in which oligomerization of FleQ:FleN complex has a crucial role in the regulation of flagellar and biofilm formation genes. These studies also lead to the hypothesis that others bEBP, related to distinct phenotypes, may be regulated by c-di-GMP.

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Torres, Naiara Utimura. "Estudo da ativação de biossíntese do polissacarídeo PEL na formação de biofilme de Pseudomonas aeruginosa PA14." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-18032016-155144/.

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Nos últimos anos, um estilo de vida celular vem ganhando destaque no mundo científico: o biofilme. O biofilme é uma comunidade celular em que as células permanecem aderidas a um substrato e envoltas em uma matriz de biopolímeros. Bactérias no estado de biofilme possuem algumas características alteradas, como o aumento da resistência a antibióticos e a facilidade de troca de genes de resistência, sendo um grande inconveniente na área médica e industrial. O patógeno humano Pseudomonas aeruginosa é uma bactéria gram-negativa causadora de infecções associadas a pacientes que apresentam o sistema imune debilitado, sendo muito comum em casos de fibrose cística. Além disso, P. aeruginosa é um organismo modelo no estudo de formação de biofilme, podendo produzir três tipos distintos de exopolissacarídeos: alginato, PSL e PEL. Devido ao pouco conhecimento do polissacarídeo PEL, a cepa P. aeruginosa PA14 vem sendo amplamente estudada, uma vez que essa é a única cepa em que PEL é o principal polímero responsável pela estabilidade da matriz de polissacarídeos. No processo de produção e exportação de PEL, sete proteínas são necessárias: Pel(A-G). Segundo predições computacionais e comparações com outros tipos de complexos biossintéticos de exopolissacarídeos, um modelo de arranjo molecular das proteínas Pel foi proposto, embora algumas proteínas não possuam uma função bem determinada no processo de síntese de PEL. Nesse contexto, o trabalho buscou estudar as proteínas envolvidas na formação de PEL para a melhor elucidação do mecanismo de ativação, produção e exportação desse polissacarídeo, com destaque para a enzima glicosiltransferase PelF e a investigação de uma possível interação de PelF com a proteína reguladora de produção do polissacarídeo, PelD, e a transportadora de membrana interna, PelG. Foram realizados diversos testes de interação entre PelF, PelG e construções solúveis de PelD por cromatografia de exclusão molecular, crosslinking e pull-down que não apresentaram interações entre tais construções, indicando que frações de membrana de PelD ou outros parceiros de interação podem ser necessários para a formação do complexo de síntese e exportação de PEL da membrana interna. Adicionalmente, mutantes de PelD sítio-dirigidos foram construídos em P. aeruginosa PA14 e tiveram sua capacidade de formação de biofilme avaliadas para investigação do mecanismo de ativação de PelD. A diminuição da formação de biofilme de mutantes de algumas regiões de PelD como a hélice S (resíduos 158-176), o core hidrofóbico ocupado pela hélice S e resíduos que estabilizam a ligação de c-di-GMP nos levou a propor um mecanismo de ativação desse importante regulador proteico de formação de biofilme em P. aeruginosa nunca descrito anteriormente.
In the last years, a cellular lifestyle has been in the spotlight in the scientific world: the biofilm. Biofilm is a cellular community in which cells are attached to a substrate and surrounded by a biopolymeric matrix. Bacteria in a biofilm lifestyle has some altered characteristics, as a higher antibiotics tolerance and facility in resistance genes exchange, turning them into a big problem in medical and industrial fields. The human pathogen Pseudomonas aeruginosa is a gram-negative bacterium which causes infections associated to patients with an impaired immune system, as frequently found in patients with cystic fibrosis. Moreover, P. aeruginosa is a model organism in biofilm formation studies, producing three distinct types of exopolysaccharides: alginate, PSL and PEL. Since there is few information about PEL polysaccharide, the strain PA14 has been broadly studied because this is the unique strain in which PEL is the main polymer that gives stability of the polysaccharide matrix. In the process of PEL production and exportation, seven proteins are required: Pel(A-G). Computational predictions and comparison with other similar exopolysaccharides biosynthetic complexes led to a model of molecular complex of Pel proteins, though some proteins do not have a clear role in the PEL synthesis process. In this context, the work aimed to study the proteins related to PEL synthesis for a better understanding of the mechanism of production and exportation of this polysaccharide, focusing on the glycosyltransferase PelF and the investigation of its possible interaction with PelD, the regulatory protein of the polysaccharide production, and PelG, the putative inner membrane transporter. Several interaction assays were performed with PelF, PelG and soluble constructs of PelD using size exclusion chromatography, crosslinking and pull-down. No interaction was detected, showing that membrane fractions of PelD or other interaction partners can be required to the inner membrane complex of synthesis and export of PEL. Additionally, site-directed mutants of PelD in P. aeruginosa PA14 were constructed to evaluate their biofilm formation ability and investigate PelD activation mechanism. Mutants in regions as S-helix (residues 158-176), hydrophobic core occupied by the S-helix and residues of c-di-GMP stabilization presented a decrease of biofilm formation compared to the wild type strain. Those results allowed us to propose an activation mechanism of this important regulator of biofilm formation in P. aeruginosa never described before.

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Dahinden, André Tony. "Umfassendes Qualitätsmanagement im GMP-Bereich unter Anwendung von Qualitätskosten." [S.l.] : [s.n.], 2003. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=15045.

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Dahinden, André Tony Dahinden André Tony. "Umfassendes Qualitätsmanagement im GMP-Bereich unter Anwendung von Qualitätskosten /." [S.l.] : [s.n.], 2003. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=15045.

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23

Simm, Roger. "Characterization of c-di-GMP signalling in Salmonella typhimurium /." Stockholm : Karolinska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-417-4/.

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Broderick, Kate Elizabeth. "Cyclic GMP - dependent signalling in D. melanogaster Malpighian tubules." Thesis, University of Glasgow, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252518.

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Taibi, Fatima. "Études des riborégulateurs c-di-GMP chez Clostridium difficile." Mémoire, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/8785.

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Chez une bactérie, la régulation de l’expression génétique est essentielle afin de maintenir l’équilibre, s’assurer du bon fonctionnement des processus cellulaires et mieux s’adapter aux changements environnementaux. Elle peut s’effectuer à plusieurs niveaux (la transcription, la traduction et la synthèse ou la dégradation des protéines), et par le bais de différents mécanismes, dont les protéines, font le plus grand part de cette régulation. Cependant, au début des années 2000, une découverte fascinante a mis en évidence un nouveau mécanisme de régulation dont l’ARN est l’acteur principal. Ce sont les riborégulateurs (riboswitches). Ces derniers sont localisés dans la partie non traduite de certains ARNmessagers (ARNm) et capable de lier un ligand spécifique sans l’intervention des protéines, afin de réguler l’expression génique du gène d’intérêt. Aujourd’hui, plusieurs familles de riborégulateurs sont caractérisées, entre autres les riborégulateurs c-di-GMP. Ces derniers sont présents chez plusieurs espèces bactériennes notamment les bactéries pathogènes telles que Clostridium difficile, une bactérie nosocomiale opportuniste qui a causé des problèmes majeurs durant les dernières années, vu sa multirésistance aux antibiotiques. Le séquençage de son génome a révélé la présence de 66 riborégulateurs dont 16 sont des riborégulateurs c-di-GMP. Il a été proposé que parmi ces derniers, certains régulent l’expression des gènes impliqués dans deux phénotypes essentiels chez C. difficile : la motilité et la formation du biofilm. La présente étude porte sur la caractérisation structurale et fonctionnelle de deux riborégulateurs c-di-GMP chez le C. difficile, le Cdi1-1 et Cdi1-12, qui se trouvent en amont du gène CD1990 et le gène CD2830 (ZmpI) respectivement. Au début, nous avons prédit les deux structures liées (en présence du ligand) et non liées (en absence du ligand). Nous avons ainsi démontré qu’un des deux riborégulateurs (Cdi1-12) est fonctionnel et capable de lier le c-di-GMP in vitro. Ensuite, nous avons caractérisé les changements structuraux potentiels lors de l’interaction riborégulateur Cdi1-12/ligand. Nous avons également caractérisé le mécanisme de régulation en cis du riborégulateur Cdi1-12 in vitro et nous avons constaté que c’est un riborégulateur transcriptionnel Rho-indépendant. À la fin de notre étude, nous avons confirmé le mode de régulation de ce riborégulateur in vivo dans la bactérie modèle Bacillus subtilis.

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Tamaki, Gabriela Mól Avelar. "Estudo da sinalização por GMP cíclico em Blastocladiella emersonii." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-25032015-133924/.

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O segundo mensageiro cGMP está envolvido em diversas funções celulares incluindo a visão em mamíferos. Embora trabalhos anteriores mostrassem variações nos níveis de cGMP durante o ciclo de vida de Blastocladiela emersonii e evidências da existência de enzimas específicas envolvidas na sua síntese (guanilato ciclase) e degradação (cGMP fosfodiesterase), nenhum genoma de fungo publicado até o momento mostrou a existência de genes codificando estas enzimas. Este fato é atribuído por evolucionistas à completa perda de motilidade dos fungos em geral, já que cGMP está primordialmente associado a células com cílios. Blastocladiomicetos, como Blastocladiella, apresentam células móveis em pelo menos um estágio do seu ciclo de vida, o que poderia explicar a existência dessa via nesses fungos. Uma investigação no banco de ESTs de B. emersonii revelou a existência de cDNAs codificando parte de prováveis guanilato ciclases (BeGC1, BeGC2 e BeGC3) e uma possível cGMP fosfodiesterase (BePDE). Assim, este trabalho buscou confirmar a existência destas enzimas e caracterizar a sinalização por cGMP em B. emersonii. A proteína recombinante selvagem correspondente ao domínio catalítico de BePDE mostrou atividade de degradação sobre cGMP e a mutação E389A foi capaz de alterar a especificidade por cGMP. Com o sequênciamento do genoma de B. emersonii obteve-se as sequências completas das guanilato ciclases. Em BeGC2 não foi possível identificar o ligante responsável por sua ativação. Em BeGC3, a presença de um domínio Heme-Pas sugeriu sua ativação por óxido nítrico. A presença de um domínio rodopsina em BeGC1 sugeriu sua ativação por luz. Experimentos de microscopia por imunofluorescência localizaram BeGC1 no \"eyespot\", BeGC2 no capacete nuclear e BeGC3 no citoplasma de zoósporos de B. emersonii. Verificamos também que zoósporos realizam fototaxia em direção à luz verde e que a adição de hidroxilamina, inibidor de rodopsina, ou do inibidor de guanilato ciclase LY83583 tem efeito negativo na fototaxia, bem como impede o aumento dos níveis de cGMP observado em zoósporos expostos à luz verde. O bloqueio da síntese de retinal por Norflurazon também inibiu a fototaxia sendo esta restaurada quando adicionamos retinalA1. Estes dados, juntamente com o fato de o domínio rodopsina de BeGC1 ser a única rodopsina presente no genoma, indicam que BeGC1 é responsável pela fototaxia nos zoósporos de B. emersonii. O genoma do fungo apresenta ainda um possível canal de potássio ativado por cGMP (BeCNG1) localizado na membrana plasmática de zoósporos, similar ao canal regulado por cGMP envolvido na visão em humanos. Ensaios de microfluorimetria também evidenciaram a presença de um canal ativado por cGMP relacionado com o influxo de potássio e a motilidade dos zoósporos. Um modelo para a via de sinalização da fototaxia em B.emersonii foi proposto e comparado com a sinalização presente na visão de mamíferos, destacando a existência de cGMP e rodopsina em ambos os processos e sugerindo uma possível origem comum. Portanto, os resultados obtidos suportam a existência da sinalização por cGMP em B. emersonii, além de indicar o papel dessa sinalização na fototaxia dos zoósporos, sendo esta a primeira via de sinalização por cGMP caracterizada em fungos.
The second messenger cyclic GMP is involved in a wide array of cellular processes including vision in mammals. Although previous studies demonstrated changes in cGMP levels during the life cycle of Blastocladiela emersonii and evidences of specific enzymes involved in its synthesis (guanylyl cyclase) and hydrolysis (cGMP-phosphodiesterase), no fungal genome published so far shows the presence of genes encoding these enzymes. Evolutionists attribute the absence of cGMP signaling pathways in higher fungi to the sedentary life style of these organisms, since cGMP is primarily associated with ciliated cells. However, blastocladiomycetes like Blastocladiella, have motile cells in at least one stage of their life cycle, which could explain the existence of this pathway in these primitive fungi. Inspection of B. emersonii EST data bank, revealed cDNAs encoding part of three putative guanylyl cyclases (BeGC1, BeGC2 e BeGC3) and one possible cGMP phosphodiesterase (BePDE). Thus, the purpose of this study was to confirm the existence of these enzymes and characterize the cGMP signaling pathway in this model. The recombinant protein containing the wild type catalytic domain of BePDE presented activity towards hydrolysis of cGMP and the E389A mutation of this domain changed the cGMP specificity of this enzyme. The complete nucleotide sequence of the guanylyl cyclases were obtained by sequencing of B. emersonii genome. In BeGC2 we were unable identify the ligand responsible for its activation, but in BeGC3, the presence of a Heme-Pas domain suggested its activation by nitric oxide. The presence of a rhodopsin domain in BeGC1 suggested its activation by light. Immunofluorescence microscopy localized BeGC1 in the \"eyespot\" structure, BeGC2 in the nuclear cap and BeGC3 in the cytoplasm of zoospores of B. emersonii. We found that Blastocladiella zoospores performed phototaxis toward green light and photobleaching of rhodopsin function using hydroxylamine prevented both phototaxis and the increased cGMP levels observed when zoospores were exposed to green light. The same effect was observed using the guanylyl cyclase inhibitor LY83583. Inhibition of retinal synthesis using Norflurazon prevented the phototaxis response, which could be restored by zoospore complementation with retinalA1. The BeGC1 gene is the only rhodopsin found in the draft assembly of B. emersonii genome, which indicates that BeGC1 is responsible for phototaxis observed in zoospores. We also found in the genome a possible cGMP-activated potassium channel (BeCNG1), localized in the plasma membrane of the zoospores, which is similar to the cGMP-activated channel involved in human vision. In addition, microfluorimetry assays revealed the presence of a cGMP-activated potassium channel involved in potassium influx and zoospore motility. The signaling model of B. emersonii phototaxis was proposed and compared with the mammalian vision system, with cGMP and rhodopsin acting in both signaling pathways, suggesting a common origin. Altogether our data indicate that Blastocladiella emersonii has a cGMP signaling system involved in phototaxis, being the first cGMP signaling pathway characterized in fungi.

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Deal, Justin. "Second Messenger Cyclic-di-GMP Regulation in Acinetobacter baumannii." Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/honors/534.

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Over time, “superbugs,” or bacteria that have become resistant to antibiotics, have become a great concern in modern medicine. Viable alternates are currently being looked into as effective and safe ways to prevent or treat infections caused by these superbugs. One such method is through the utilization of the second messenger molecule cyclic-di-GMP (c-di-GMP) that has been shown to regulate phenotypes within other bacteria that may control surface colonization in Acinetobacter baumannii. Through a series of experiments, the active enzymes that create c-di-GMP - diguanylate cyclases - and break down c-di- GMP - phosphodiesterases - have been inactivated in mutants to test phenotypes including biofilm formation, motility, antibiotic resistance, and desiccation survival. The research’s objective is to show that manipulation of c-di-GMP within the multi-drug resistant strain of Acinetobacter baumannii may serve as a means to control this bacteria.

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28

Cook, Neil James. "Le gmp cyclique et la phototransduction chez les vertebres." Strasbourg 1, 1986. http://www.theses.fr/1986STR13147.

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29

Cook, Neil James. "Le GMP cyclique et la phototransduction chez les vertébrés." Grenoble 2 : ANRT, 1986. http://catalogue.bnf.fr/ark:/12148/cb375968043.

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30

Huang, Daming. "Molecular determinants of cGMP-binding to chicken cone photoreceptor phosphodiesterase /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/5095.

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31

Liu, Kuan-Yu. "Generalized Many-Body Expansion: A Fragment-Based Method for modeling Large Systems." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1560442158764827.

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32

Bau, Fernando Ricardo. "Avaliação do efeito relaxante do BAY 41-2272 em detrusor isolado de coelhos." [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/308919.

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Orientador: Edson Antunes
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A síndrome da bexiga hiperativa atinge grande parte da população mundial, e gera sintomas que prejudicam a qualidade de vida dos portadores. Está associada com a hiperatividade do detrusor que se dá por um aumento das contrações espontâneas. Alguns estudos têm mostrado que a deficiência de NO é um dos fatores responsáveis por gerar estas contrações espontâneas. É sabido que o mecanismo de sinalização do NO envolve a ativação da guanilil ciclase solúvel e produção de GMPc. Atualmente, algumas drogas têm sido sintetizadas para mimetizar o efeito exercido pelo NO, tal como o BAY 41-2272, um potente estimulador da guanilil ciclase solúvel independente de NO. Vários trabalhos mostraram que o BAY 41-2272 causa relaxamento de vários tipos de musculatura lisa, podendo ser um composto com grande potencial terapêutico em doenças onde a via do NO/GMPc está prejudicada. O objetivo deste trabalho é investigar a capacidade do BAY 41-2272 de relaxar detrusor isolado de camundongo, coelho e rato in vitro e os mecanismos farmacológicos envolvidos na resposta relaxante. Camundongos C57b6 machos (30-40 g), coelhos New Zealand machos (2-3 kg) e ratos Wistar machos (250-300 g) foram anestesiados e mortos. As bexigas foram removidas e fragmentos de detrusor foram montados em banho para órgãos isolados contendo 10 ml de solução de Krebs. Curvas concentração-resposta ao BAY 41-2272 (10-9 - 10-4 M) foram construídas em tecidos précontraídos com carbacol (10 µM) ou KCl (80 mM), na ausência ou na presença de LNAME (inibidor da óxido nítrico sintase; 100 µM), ODQ (inibidor da guanilato ciclase solúvel; 100 µM), Sildenafil (inibidor da fosfodiesterase tipo-5; 10 µM), ou inibidores de canais de potássio (0,1 µM charibdotoxina + 1 µM apamina; 1µM tetraetilamônio; ou 10 µM glibenclamida). Curvas concentração-resposta ao nitroprussiato de sódio (SNP; 10-8 - 10-4 M), gliceril trinitrato (GTN; 10-8 - 10-4 M) e 8Br-GMPc (10-8 - 10-4 M) foram também construídas. Contrações induzidas por CaCl2 extracelular foram avaliadas na presença do BAY 41-2272, bem como o efeito no influxo de cálcio em plaquetas isoladas de coelho. Níveis de GMPc e AMPc foram avaliados após a estimulação do detrusor com BAY 41- 2272 (10 e 100 µM) e SNP (100 µM) na ausência ou na presença de ODQ (100 µM), através de imunoensaio enzimático (ELISA). O BAY 41-2272 produziu relaxamento de detrusor isolado de camundongos, ratos e coelhos de maneira concentração-dependente, com valores de resposta máxima de 61,3 ± 6,6%, 91,7 ± 5,9% e 95,1 ± 9,9%, respectivamente. Detrusor de coelhos foram selecionados para os experimentos subseqüentes. Os doadores de NO, SNP e GTN, bem com o 8Br-GMPc produziram um discreto relaxamento comparado ao BAY 41-2272. O tratamento dos tecidos com L-NAME (100 µM) ou sildenafil (10 µM) não afetou de maneira significativa o relaxamento induzido pelo BAY 41-2272. Entretanto, o ODQ (100 µM), reduziu significativamente a resposta ao BAY 41-2272. Os bloqueadores de canais de K+ (apamin + charibdotoxina, glibenclamida ou tetraetilamônio) também não afetaram a resposta relaxante do BAY 41-2272. O BAY 41-2272 (10 e 100 µM) elevou os níveis de GMPc em cerca de 14 e 20 vezes respectivamente, sem afetar os níveis de AMPc. Na menor concentração do BAY 41-2272 (10 µM), o ODQ aboliu a elevação dos níveis de GMPc, ao passo que na maior concentração do BAY 41-2272 (100 µM), o ODQ inibiu parcialmente a elevação dos níveis de GMPc. A adição de CaCl2 (0,01-30 mM) extracelular em detrusor isolado de coelhos causou contração de maneira concentração-dependente que foi significativamente reduzida pelo tratamento prévio com BAY 41-2272 (1 e 10 µM), sendo que este efeito não foi prevenido pelo ODQ. O BAY 41-2272 reduziu significativamente o aumento dos níveis intracelulares de cálcio em plaquetas de coelho induzido por trombina. Em resumo, o BAY 41-2272 produz relaxamento em detrusor isolado de camundongos, coelhos e ratos através da produção de GMPc e da inibição do influxo de cálcio que independe de GMPc
Abstract: Overactive bladder (OAB) is a highly prevalent condition that affects millions of people worldwide with a profound effect on quality of life. The bladder overactivity is related to spontaneous contractions of the detrusor smooth muscle causing an increase in the intravesical pressure and consequently stimulation of the micturirion reflex. Evidences suggest that impairment of nitric oxide (NO) signaling pathway may account for OAB. It is well established that NO signaling pathways involves soluble guanylate cyclase (sGC) stimulation and cyclic GMP production. Recently, pharmacological agents capable of directly stimulating soluble guanylate cyclase independenly of NO, such as BAY 41-2272 has been reported to produce relaxation of different types of smooth muscle, showing great therapeutic potential in disturbs which NO pathway is impaired. The present study aimed to evaluate the capacity of BAY 41-2272 to relax isolated mouse, rat and rabbit DSM and the mechanism underlying these response. C57b6 male mice, Wistar male rats and New Zealand male rabbits were anesthetized, and urinary bladder removed. DSM was transferred to 10-mL organ baths containing oxygenated and warmed Krebs-Henseleit solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. Concentration-response curves to BAY 41-2272 (10-9 - 10-4M) were constructed, in previously contracted tissues with carbachol (10 µM) or KCl (80 mM), in the absence and in the presence of L-NAME (Nitric Oxide Synthase inhibitor; 100 µM), ODQ (sGC inhibitor; 100 µM), Sildenafil (phosphodiesterase type-5 inhibitor; 10 µM), or potassium channel blockers (0.1 µM charybdotoxin + 1 µM apamin; 1 µM tetraethylammonium; or 10 µM glybenclamide). Concentration-response curves to sodium nitroprusside (SNP; 10-8 - 10-4 M), glyceryl trinitrate (GTN; 10-8 - 10-4 M) and 8Br-cGMP (10-8 - 10-4 M) were also constructed. CaCl2-induced contractions in DSM and calcium influx in rabbit isolated platelets were evaluated in the presence of BAY 41-2272. Levels of cAMP and cGMP in DSM strips were determined after treatment with BAY 41-2272 (10 and 100 µM), SNP (100 µM) in the absence or in the presence of ODQ (100 µM) using specific EIA kit. BAY 41-2272 (0.001-100 µM) produced concentration-dependent DSM relaxations in mouse, rat and rabbit with maximal responses of 61.3 ± 6.6%, 95.1 ± 9.9% and 91.7 ± 5.9%, respectively. The NO-donors sodium nitroprusside and glyceryl trinitrate, as well as 8-bromo-cGMP also produced concentration-dependent rabbit DSM relaxations, but to a lesser extent than BAY 41-2272. Pretreatment with L-NAME (NO synthesis inhibitor) or sildenafil (phosphodiesterase-5 inhibitor) had no effect in BAY 41-2272- induced responses. However, the soluble guanylyl cyclase inhibitor ODQ significantly reduced BAY 41-2272-induced relaxantions. BAY 41-2272 (10 and 100 µM) increased the bladder cGMP levels by about of 14- and 20-fold, respectively, without affecting the cAMP levels. The cGMP increases in response to BAY 41-2272 and SNP were markedly reduced by ODQ. CaCl2 caused a concentration-dependent contraction in DSM strips and BAY 41- 2272 significantly reduced the contractile responses to extracellular Ca2+ in an ODQinsensitive manner. BAY 41-2272 also significantly reduced the increase of intracellular calcium levels induced by thrombin. This inhibitory effect was completely reverted after the treatment with ODQ. BAY 41-2272 relaxes DSM of the three animal species studied. BAY 41-2272-induced DSM relaxation involves mainly cGMP production, but an additional mechanism involving Ca2+ influx blockade independently of cGMP production appears to be involved
Mestrado
Farmacologia
Mestre em Farmacologia

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33

Alves, Moscoso Joana. "Regulatory cascades involving cyclic di-GMP signalling in Pseudomonas aeruginosa." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/24816.

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Cyclic di-GMP (c-di-GMP) has emerged as a bacterial second messenger that regulates a variety of cellular processes, in particular those associated with the switch between a motile and a sessile lifestyle. At low levels of c-di-GMP the motile lifestyle is favoured whereas at high levels of c-di-GMP the formation of biofilms is promoted. In Pseudomonas aeruginosa, over 50 genes encoding proteins involved in the synthesis, hydrolysis or sensing of c-di-GMP are found and many remain uncharacterized. Herein, an analysis of a collection of mutants was performed and supported the idea that the sophisticated c-di-GMP network operates at high specificity. In addition to c-di-GMP, P. aeruginosa has a regulatory pathway, the Gac pathway, that is known to control the bacterium lifestyle switch. By investigating a particular mutant affected in this pathway, a link between the Gac pathway and c-di-GMP was established. Furthermore, the Gac pathway not only influences biofilm formation, but it is also crucial in determining the bacterium mode of infection. In other words, biofilm formation correlates to a chronic mode of infection where the bacterium has an active type VI secretion system (T6SS), and a motile phenotype correlates to an acute infection where the type III secretion system (T3SS) is active. Interestingly, by artificially modulating the levels of c-di-GMP it was demonstrated that c-di-GMP regulation goes beyond the control of the motile/sessile phenotypes and is able to inversely regulate the T3SS and T6SS. Finally, the link between c-d-GMP and the Gac pathway was consolidated by showing that the Gac system impacts the expression of a few c-di-GMP related proteins and one protein, SadC, was identified as a central component of the network. Overall this work largely contributed to reconcile two independent concepts involved in the regulation of the P. aeruginosa lifestyle.

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Simonsson, Filip, Azucar Eduardo Cuadra, and Johan Roos. "Motivational Factors and their Prioritization for GMP in Nordic SMEs." Thesis, Internationella Handelshögskolan, Högskolan i Jönköping, IHH, Företagsekonomi, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:hj:diva-39849.

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Purpose - The subject of green management and its connection to motivational factors is relatively unexplored in existing literature, especially regarding SMEs in the Nordic Countries. The paper aims to investigate frequencies of motivational factors as SMEs present them, suggest the theoretical prioritization of motivational factors for green management practices, and factors impact on different levels of green management. The reason for this is to help SMEs to better understand what motivational factors to prioritize to further their green management development. Design/Method - The chosen research method of this paper is quantitative, and the empirical data is collected through questionnaires distributed using business networks in the Nordic Countries. Furthermore, the research takes an abductive approach, with a positivist paradigm, and a mixture of ontological, epistemological, and method research assumption. Findings - This paper has found that at different levels of green management practices specific factors of motivation should theoretically be prioritized based on a regression model. Furthermore, the suggested prioritization is different from presented by the sample of this paper. Research Implications - Theoretically, the thesis suggests a conceptual map of existing frameworks and the motivational factors presented. Practically, SMEs can draw from this study to see if their prioritised motivation for implementation of green management is effective or if they can further improve it.

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Günay-Esiyok, Özlem. "Cyclic GMP signaling during the lytic cycle of Toxoplasma gondii." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/20740.

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Der cGMP-Signalweg ist als einer der Hauptregulatoren von diversen Funktionen in Eukaryoten bekannt; allerdings ist seine Funktionsweise in Protozoen wenig verstanden. Im Rahmen dieser Arbeit wurde eine Guanylatcyclase, gekoppelt mit N-terminalen P4-ATPase, in intrazellulären Parasiten Toxoplasma gondii gemeldet. Eine in silico-Analyse wies auf eine Aktivierung der Guanylatcyclase durch Heterodimerisierung ihrer Cyclasedomänen hin und ermöglichte wertvolle Einsichten in mögliche Funktionen ihrer ATPase-Domäne. Dieses Protein (477-kDa) bezeichnet als TgATPaseP-GC in dieser Studie, lokalisiert in der Plasmamembran am apikalen Pol des Parasiten. TgATPaseP-GC ist unempfänglich gegenüber genetischer Deletion und seine CRISPR/Cas9 unterstützte Spaltung beendet den lytischen Zyklus von T. gondii vorzeitig. Darüber hinaus reduzierte ein Cre/loxP-vermittelter Knockdown von TgATPaseP-GC die Synthese von cGMP im Tachyzoiten und inhibierte das Parasitenwachstum aufgrund von Beeinträchtigungen Motilitäts-abhängiger Prozesse des Austretens und Eindringens. Trotz seiner zeitlich beschränkten Funktion ist TgATPaseP-GC konstitutiv während des ganzen lytischen Zyklus exprimiert, welches eine post-translationale Regulierung des cGMP-Signalweges bedingt. Nicht zuletzt impliziert das Vorhandensein von TgATPaseP-GC-Orthologen in anderen Alveolata eine divergente Umfunktionierung der cGMP-Signalwege in Protozoen. Darüber hinaus wurde ein optogenetischer Ansatz verwendet, um den cGMP-Weg durch eine photo-aktivierte Rhodopsin-Guanylat-Cyclase (RhoGC) in T. gondii zu exprimiert. Dieses System erlaubte eine kontrollierte Erhöhung von cGMP durch Licht in einer schnellen und reversiblen Weise. Die Anregung von RhoGC stimulierte signifikant die Parasitenmotilität, deren Auswirkung auch mit erhöhten Eindringen und Austreten überwacht wurde; im Gegensatz zum genetischen Knockdown von TgATPaseP-GC. Das System ermöglicht die Vermittler des cGMP-Signalwegs durch Phosphoproteomics zu identifizieren.
cGMP signaling is known as one of the master regulators of diverse functions in eukaryotes; however, its architecture and functioning in protozoans remain poorly understood. In the scope of this thesis, an exclusive guanylate cyclase coupled with N-terminal P4-ATPase was reported in an obligate intracellular parasite Toxoplasma gondii. In silico analysis indicated an activation of the guanylate cyclase by heterodimerization of its two cyclase domains and offered valuable insights into possible functions of its ATPase domain. This bulky protein (477-kDa), termed in this study as TgATPaseP-GC to reflect its envisaged multifunctionality, localizes in the plasma membrane at the apical pole of the parasite. TgATPaseP-GC is refractory to genetic deletion, and its CRISPR/Cas9-assisted disruption aborts the lytic cycle of T. gondii. Besides, Cre/loxP-mediated knockdown of TgATPaseP-GC reduced the synthesis of cGMP in tachyzoites and inhibited the parasite growth due to impairments of motility-dependent egress and invasion events. Notably, despite its temporally restricted function, TgATPaseP-GC is expressed constitutively throughout the lytic cycle, entailing a post-translational regulation of cGMP signaling. Not least, the occurrence of TgATPaseP-GC orthologs in several other alveolates implies a divergent functional repurposing of cGMP signaling in protozoans. Furthermore, an optogenetic approach was utilized to induce cGMP pathway by a photo-activated rhodopsin-guanylate cyclase (RhoGC) in T. gondii. The system enabled a light-control of cGMP elevation on crucial steps of lytic cycle in a fast, spatial and reversible manner. Excitation of RhoGC significantly stimulated the parasite motility of which impact was also monitored with an increased host-cell invasion and egress; as opposed to the genetic knockdown of TgATPaseP-GC. Having an established optogenetic system in the parasite allows to identify downstream targets of cGMP signaling via phosphoproteomic analysis.

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Goes, Júnior Francisco de Assis Mendes. "Relaxamento do corpo cavernoso de coelho induzido pela fração alcaloidal F3-5 de Aspidosperma ulei Markgr." reponame:Repositório Institucional da UFC, 2007. http://www.repositorio.ufc.br/handle/riufc/7320.

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GOES JÚNIOR, Francisco de Assis Mendes. Relaxamento do corpo cavernoso de coelho induzido pela fração alcaloidal F3-5 de Aspidosperma ulei Markgr. 2007. 65 f. Dissertação (Mestrado em Cirurgia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2007.
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Erectile dysfunction is a worldwide public health issue and, in spite of advances brougth by the utilization of type-5 phosphodiesterase inhibitors, there is still much interest in new treatment alternatives, especially when derived from natural products. Some investigators observed the pro-erectile effects of an alkaloidal-rich fraction from Aspidosperma ulei Markgr., named F3-5. In this study, it was evaluated the degree of relaxation induced by F3-5 on rabbit corpus cavernosum, as well as possible Pharmacological mechanisms involved, in vitro. Several experimental assays were performed, utilizing the methods of cascade tissue superfusion and isolated tissue bath. Wíth strips ot corpus cavernosum pre-contracted with phenilephrine (5µM), dose-response curves of relaxation for F3-5 papaverine and DMSO were produced. were There were also assays fo, evaluation of the effects of L-NAME, 7-NI, 000, propranolol, atropine, sildenafil and F3-5 on the relaxations mediated by sodium litroprusside, acetylcholine, isoproterenol, sildenafil and F3-S. With depolarized preparations of corpus cavernosum, in a calcium-free, potassium-rich (60 mM) medium, the effects of F3-S and DMSO on the contractions induced by the progressive increase in Ca2+ concentration (1 - 300 mM) were observed. F3-5 was capable of inducing complete relaxation of rabbit corpus cavernosum with magnitude similar to that of papaverine for all doses tested, except 3 mg, when ít presented a significantly higher relaxation. Superfusions of L-NAME, 7-NI and ODQ did not significantly inhibit the relaxatíons provoked by F3-5, suggesting that it acts independently of the nitrergiê pathway. Propranolol and atropine also did not significantly interfere with relaxations mediated by F3-5 indicating that B-adrenergic or rnuscarinic receptors also might not be involved. F3-5 did not significantly amplify the relaxations promoted by sodium nitroprusside or acetylcholine, as opposed to sildenafil, suggesting that it does not act through inhibition of type-5 ohosphodiesterase. Finally, in cavernous tissue pre-incubated with F3-5, the minimum dose of Ca2+ necessary for muscular contraction was thirty times superior to that utilizedon the tissue without previous treatment, or treated with OMSO. This inhibition of the contractions of corpus cavernosum mediated by Ca2+ suggests, therefore, that F3-S can act through blockade of voltage-dependent calcium channels.
A disfunção erétil é um problema mundial de saúde pública e, apesar dos avanços trazidos pela utilização dos inibidores da fosfodiesterase tipo-5, ainda há muito interesse em novas alternativas de tratamento, especialmente quando derivadas de produtos naturais. Alguns pesquisadores observaram os efeitos pró-eréteis de uma fração rica em alcalóides de Aspidosperma ulei Markgr., denominada F3-5. Neste estudo, foi avaliado o grau de relaxamento induzido por F3-5 no corpo cavernoso de coelho, bem como possíveis mecanismos farmacológicos envolvidos, in vitro. Foram realizados diversos ensaios experimentais, utilizando-se os métodos de superfusão de tecido em cascata e de banho isolado de tecido. Com tiras de corpo cavernoso pré-contraídas com fenilefrina (5 μM), foram produzidas curvas de dose-resposta de relaxamento para F3-5, papaverina e DMSO. Também foram realizados ensaios para avaliação dos efeitos de L-NAME, 7-NI, ODQ, propranolol, atropina, sildenafil e F3-5 sobre os relaxamentos mediados por nitroprussiato de sódio, acetilcolina, isoproterenol, sildenafil e F3-5. Com preparações de corpo cavernoso despolarizadas, em um meio livre de cálcio e rico em potássio (60 mM), os efeitos de F3-5 e DMSO sobre as contrações induzidas pelo aumento progressivo na concentração de Ca2+ (1 – 300 mM) foram observados. F3-5 foi capaz de induzir o relaxamento completo do corpo cavernoso de coelho, com magnitude similar à da papaverina para todas as doses testadas, exceto 3 mg, quando apresentou relaxamento significantemente maior. As superfusões de L-NAME, 7-NI e ODQ não inibiram significantemente os relaxamentos provocados por F3-5, sugerindo que este age independentemente da via nitrérgica. Propranolol e atropina também não interferiram significantemente com os relaxamentos mediados por F3-5, indicando que receptores β-adrenérgicos ou muscarínicos também não devem estar envolvidos. F3-5 não amplificou significantemente os relaxamentos promovidos por nitroprussiato de sódio ou acetilcolina, ao contrário de sildenafil, sugerindo que não age por inibição da fosfodiesterase tipo-5. Finalmente, em tecido cavernoso pré-incubado com F3-5, a dose mínima de Ca2+ necessária para contração muscular foi trinta vezes superior àquela utilizada em tecido sem tratamento prévio ou tratado com DMSO. Esta inibição das contrações de corpo cavernoso mediadas pelo Ca2+ sugere, portanto, que F3-5 pode atuar através do bloqueio de canais de cálcio voltagem-dependente.

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Furini, Cristiane Regina Guerino. "Participação da via de sinalização NO/GMPc/PKG na memória de reconhecimento de objetos." Pontifícia Universidade Católica do Rio Grande do Sul, 2009. http://hdl.handle.net/10923/4609.

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Evidence suggests that the NO/cGMP/PKG pathway plays a key role in memory processing but the actual participation of this signaling cascade in the consolidation of declarative memory, such as object recognition (OR), remains unknown. Here we show that consolidation of long-term object recognition memory enhances the nitric oxide synthase neuronal activity (nNOS) in the CA1 region of the dorsal hippocampus of rats. Indeed, when infused immediately after but not 180 and 360 min after training in the OR task, the nNOS inhibitor, L-NN, hindered long-term memory retention without affecting locomotion, exploratory behavior, anxiety state or hippocampal functionality. The amnesic effect of L-NN was not due to statedependency and was equally produced by the hippocampal infusion of soluble guanylyl cyclase (sGC) inhibitor, LY83583, and by the protein kinase G (PKG) inhibitor, KT5823.Furthermore, post-training infusion of the non-hydrolysable analog of cGMP, 8Br-cGMP, reversed the L-NN and LY83583 effect but not that induced by KT5823. The noradrenaline (NA) regulate NO/sGC/PKG pathway activation in distinct preparations. In accordance with this fact, the amnesia induced by intrahippocampal administration of antagonist -adrenergic receptor, timolol, immediately after training in the OR task is not reversed by co-infusion of 8-Br-cGMP or of NO donator, SNAP. When administrated in the CA1 region of the dorsal hippocampus together with LY-83583, KT-5823 or L-NN, NA reversed the amnesic effect of these compounds. Our results indicate that the activation of NO/cGMP/PKG pathway in the CA1 region of the dorsal hippocampus is essential for consolidation of OR memory and suggests that the ß-adrenergic receptors, has a fundamental participation on regulation of these events.
Evidências sugerem que a via de sinalização NO/GMPc/PKG tem papel chave no processamento da memória, entretanto a atuação desta cascata de sinalização no hipocampo durante a consolidação de memórias declarativas, tais como a de reconhecimento de objetos (RO), permanece desconhecida. Aqui será mostrado que a aquisição da memória associada ao treino na tarefa de reconhecimento de objetos aumenta a atividade da óxido nítrico sintase neuronal (NOSn) na região CA1 do hipocampo dorsal de ratos. Ainda mais, quando infundido nesta região do cérebro imediatamente, mas não 180 ou 360 min após o treino na tarefa de RO, o inibidor da NOSn, L-NN, prejudica a retenção da memória em questão, sem afetar a locomoção, o comportamento exploratório, nem o estado de ansiedade dos animais ou causar lesão ao hipocampo. O efeito amnésico do L-NN não se deve a indução de dependência de estado e é mimetizado pela infusão intra-hipocampal do inibidor da guanilil-ciclase solúvel (GCs), LY83583 ou do inibidor da proteína cinase G (PKG), KT5823.Ainda, a infusão pós-treino do análogo não-hidrolizável do GMPc, 8- Br-GMPc, reverte o efeito de L-NN e LY5823, mas não o de KT5823. A noradrenalina (NA) regula a ativação da via NO/GMPc/PKG em distintas preparações. Em concordância com este fato a amnésia induzida pela administração intra-hipocampal do antagonista -adrenérgico, timolol, imediatamente após o treino na tarefa de RO não é revertida pela co-infusão de 8-Br-GMPc e do doador de NO, SNAP. Quando administrada na região CA1 do hipocampo dorsal juntamente com LY-83583, KT-5823 ou L-NN, a NA reverte o efeito amnésico destes agentes. Nossos dados indicam que a ativação da cascata de sinalização NO/GMPc/PKG na região CA1 do hipocampo dorsal é fundamental para a normal consolidação da memória de reconhecimento de objetos e sugerem que os receptores ß-adrenérgicos desempenham um papel fundamental na regulação destes eventos.

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Diaz, Lucena Daniela Del Valle. "Mechanisms involved in the remyelinating effect of sildenafil." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/400468.

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Multiple Sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system characterized by a coordinated inflammatory attack on the myelin sheaths with ensuing damage to the underlying axons. Using myelin oligodendrocyte glycoprotein (MOG)-induced chronic experimental autoimmune encephalomyelitis (EAE) as a MS model, it has been previously demonstrated that daily administration of the PDE-5 inhibitor sildenafil starting at peak disease rapidly ameliorates clinical symptoms whereas administration at the onset of symptoms prevents disease progression. These beneficial effects involved down-regulation of adaptive and innate immune responses and protection of axons and oligodendrocytes and promotion of remyelination. The aim of this work was confirm the remyelinating potential of sildenafil treatment and investigate mechanisms involved in this effect in CNS cells. Results show that sildenafil induces remyelination in EAE mice even when the administration of the drug starts during the chronic stage of the disease. Sildenafil also stimulates remyelination in cerebellar organotypic cultures demyelinated with lysophosphatidylcholine and this effect is prevented by inhibitors of nitric oxide-dependent guanylyl cyclase (NO-GC), NO synthase type 2 (NOS-2) and cGMP-dependent protein kinase (PKG), indicating the involvement of the endogenous NO-cGMP-PKG pathway. Maturation of oligodendrocytes as a potential mechanism implicated in the remyelinating effect of sildenafil was investigated by immunostaining for transcription factors involved in different stages of oligodendrocyte development. Results in the EAE model show that sildenafil treatment increases oligodendrocyte precursor cells (OPCs; Nkx2.2+ cells) and promotes the final stage of oligodendrocyte maturation (olig2-/MBP+ cells). These later result was confirmed in LPC-demyelinated cerebellar slices treated with cGMP increasing compounds. This work also shows that expression of the neurotrophic factor CNTF, that has been implicated in oligodendrocyte maturation, is increased in astroytes of sildenafil-treated EAE mice spinal cord, as well as in cerebellar slice cultures. Results also show that cGMP-increasing treatments alter expression of inflammatory phenotype markers (COX-2 and Arg-1) in microglia in demyelinated slice cultures. The potential of cGMP-increasing treatments for regulating the inflammatory phenotype of monocytes was confirmed in bone marrow derived macrophages (BMDM). In these cells sildenafil treatment induces arginase activity and potentiates the effect of IL-4 suggesting the promotion of an M2 phenotype. Analysis by flow cytometry of BMDM confirmed that cGMP augments the number of cells expressing an M2 phenotype marker (CD206). This work further demonstrates that sildenafil significantly increases the myelin phagocytic capacity of microglia/macrophages in EAE mice and in BMDM. Taken together these data suggest that promotion of oligodendrocyte maturation, growth factor expression, modulation of the inflammatory process and clearance of myelin debris may be relevant mechanisms involved in sildenafil enhancement of remyelination in demyelinated tissue.

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39

Soares, José Roberto. "Desenvolvimento de sistema para seguimento de produto e aquisição de dados do processo de irradiação em irradiadores de grande porte." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/85/85131/tde-11082011-154344/.

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A esterilização utilizando a radiação ionizante é uma técnica consolidada para o processamento de produtos médicos descartáveis. No Brasil há irradiadores gama em operação com capacidade entre 0.37 PBq (10kCi) a 185 PBq (5 MCi ) utilizando radioisótopos 60Co como fonte de radiação. O trabalho desenvolvido proporciona um controle e registro apurado da aplicação das Boas Práticas de Fabricação (BPF), durante todas as fases de um processo de irradiação, requeridos pelas normas da ANVISA , ISO e recomendações técnicas da AIEA durante o tratamento de alimentos e produtos médicos. Todas as etapas envolvidas no tratamento por irradiação estão mapeados em fluxos de processos (workflow) onde cada agente (participante) tem suas tarefas sistematizadas. A aquisição de dados do processo, o acompanhamento e controle, estão baseados em um conjunto de ferramentas (software livre de licenças) integradas por uma rede de comunicação eficiente, inclusive, utilizando-se recursos da WEB. O desenvolvimento foi realizado para uma unidade com capacidade de processamento a nível industrial , utilizando-se o Irradiador Gama Multipropósito do IPEN/CNEN/USP. O sistema permite a rastreabilidade do processamento, em tempo real, a qualquer participante e também o armazenamento dos registros correspondentes para serem auditados.
The sterilization of medical care products using ionizing radiation is a consolidated technique. In Brazil there are in operation gamma irradiators with capacity between 0.37 PBq (10kCi) 185 PBq (5 MCi) using radioisotopes 60Co as radiation source. The developed work provides an accurate control anda data acquisition for the application of Good Manufacturing Practices during all fases of an irradiadiation process, required by the standards of ANVISA , ISO and IAEA technical recommendations for the treatment of foods and medical products.. All the steps involved in the irradiation treatment are mapped into process flow (workflow) , where each agent (participant) has its systematized tasks. The data acquisition process, monitoring and control, are based on a set of tools (free software licenses) integrated by a network of efficient communication, including the use of Web resources. Using the Gamma Irradiator Multipurpose IPEN/CNEN/USP all the development was performed to be applied in irradiators facilities operating in industrial scale. The system enables a complete traceability of the process, in real time, for any participant and also the storage of the corresponding records to be audited.

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40

McLatchie, Linda. "Block of the cyclic GMP-activated conductance of salamander rod photoreceptors." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320405.

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41

Вабіщевич, Федір Федорович, Юрій Анатолійович Собко, and О. О. Салій. "Впровадження вимог GMP на ТОВ «БіоТестЛаб» для забезпечення якості ветеринарних препаратів." Thesis, ЦП «Компринт», 2018. https://er.knutd.edu.ua/handle/123456789/11225.

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З метою реалізації стратегічного курсу України щодо Євроінтеграції Міністерство аграрної політики та продовольства України наказом № 606 від 10.11.2017 р. встановило єдиний для всієї території України режим правового регулювання для виробників ветеринарних препаратів за правилами та вимогами GMP. Біотехнологічні кампанії мають гарантувати та вдосконалювати якість ветеринарних препаратів шляхом регулярного перегляду методів виробництва та контролю якості, враховуючи і використовуючи наукові, технічні та програмні інновації галузі. Впровадження вимог GMP обов’язкове для виробництва ветеринарних препаратів, що виготовляються в Україні для продажу на внутрішньому ринку та з метою експорту. Впроваджені заходи на ТОВ «Біотестлаб» дозволяють вдосконалювати методи виробництва та контроль якості вироблених ветеринарних препаратів. Побудована система з забезпечення якості поширює можливість реалізації новітніх біотехнологій та підвищує конкурентоспроможність продукції підприємства.

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42

Whiting, Nicola. "Characterisation of 'degenerate' cyclic di-GMP signalling proteins of Escherichia coli." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/7787/.

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43

Nold, Philipp [Verfasser]. "GMP-konforme Expansion und funktionelle Charakterisierung humaner mesenchymaler Stromazellen / Philipp Nold." Gießen : Universitätsbibliothek, 2017. http://d-nb.info/1149507276/34.

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44

Rescaldani, M. "RIDOTTA ATTIVITA' DEL SISTEMA OSSIDO NITRICO/GMP CICLICO PIASTRINICO NELL'IPERALDOSTERONISMO PRIMARIO." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/217624.

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Rationale: Nitric oxide (NO) exerts a vasodilating effect and inhibits platelet aggregation via the second messenger cyclic GMP (cGMP). Oxidative stress associated with hypertension and major cardiovascular risk factors inhibits NO/cGMP activity. Aldosterone (Aldo) exerts a potent pressor effect via its mineralocorticoid activity; moreover, Aldo increases oxidative stress and impairs endothelial NO availability in experimental and clinical conditions. Aim:To evaluate whether also platelet NO/cGMP activity is impaired by chronic Aldo excess, we compared platelet cGMP levels in patients with primary aldosteronism (PA) and in a control group of essential hypertensives (EH). Methods: In patients with PA (n=12 males) and in EH (n=32) matched for sex, age and clinic systolic and diastolic blood pressure values (SBP, DBP), venous blood was sampled for plasma renin activity (PRA), Aldo, aldo-renin ratio (ARR), potassium (K+) and platelet cGMP (RIA on acid extracts of washed platelets) after a 60 minute supine rest. Results: PRA was lower and Aldo was higher in PA than in EH (0.1± 0.1 vs 0.4±0.1 and 25.5±8.8 vs 8.1±0.7, respectively, p<0.05), with similar SBP and DBP values. Potassium was lower in IPA than in EH (3.5±0.2 vs 4.1±.05, p<0.05) Platelet cGMP, marker of NO activity, was lower in hypokalemic patients with PA than in EH (5.1±0.4 vs 7.1±0.5 pM/10^9 platelets, p<0.05). Conclusions: These data are compatible with an impairment of the platelet NO/cGMP system in primary aldosteronism as compared to essential hypertension. To the extent that this defect is associated with an increased platelet aggregability, these data may provide a pathogenetic mechanism for the prevalence of thrombotic events in patients with primary aldosteronism.

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45

Reyes, Irisarri Elisabet. "Fosfodiesterasas del AMPc y del GMPc en el cerebro: Expresión en procesos neuroinflamatorios y neurodegenerativos." Doctoral thesis, Universitat de Barcelona, 2007. http://hdl.handle.net/10803/891.

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Los nucleótidos cíclicos, AMPc y GMPc, están considerados segundos mensajeros que participan en la transducción de las vías de señalización intracelular, contribuyendo de esta manera en una gran variedad de procesos celulares. Los niveles del AMPc están regulados por la adenilato ciclasa y los del GMPc por la guanilato ciclasa a nivel de su síntesis, y para ambos por las fosfodiesterasas, a nivel de su degradación.
Entre la gran variedad de procesos fisiológicos en los que están involucrados, se ha demostrado su participación en reacciones inflamatorias. Niveles elevados de ambos nucleótidos cíclicos desencadenan respuestas antiiflamatorias y/o neuroprotectoras, por lo tanto, es de esperar que la regulación de los enzimas involucrados en su degradación, las fosfodiesterasas, influya indirectamente en estos procesos.
En este trabajo determinamos la expresión de los ARNm de fosfodiesterasas específicas de AMPc, como las variantes de splicing de la PDE4B y el isoenzima de la PDE7, la PDE7B, en cerebros controles de ratas para posteriormente determinar su regulación en cerebros de modelos animales con componente inflamatorio (LPS y EAE). Por otro lado estudiamos la expresíón de los ARNm de la PDE2 y de la PDE9, fosfodiesterasas que son capaces de hidolizar el GMPc, en cerebros postmortem de humanos control, y comparamos su expresión con cerebros de pacientes con enfermedad de Alzheimer.
La distribución de los ARNm de las cuatro variantes de splicing de la PDE4B en el cerebro de la rata presenta estructuras en común, como el núcleo anterior olfatorio, o la corteza piriforme. Sin embargo existen regiones en las que no encontramos todas isoformas como en el área postrema, donde no se expresa la PDE4B4, en diversos tractos de materia blanca, que hibrida intensamente con el oligonucleótido para la PDE4B3 y no presenta señal para la PDE4B1, o en el giro dentado del hipocampo, donde sólo se expresa la PDE4B2. Su estudio en los modelos animales utilizados reveló un aumento exclusivo de la PDE4B2 a las 2 y 3 h después de la administración de la toxina en los plexos coroideos en el modelo animal de inflamación por LPS, y en áreas perivasculares de cerebro en el modelo animal de EAE. La expresión de esta variante de splicing en este último modelo se localizó en algunos linfocitos T y macrófagos/microglía.
El ARNm de la PDE7B se localiza a lo largo de todo el cerebro de la rata, siendo más abundante en regiones cerebrales anteriores, especialmente en el tubérculo olfativo, los núcleo caudado-putamen, el giro dentado del hipocampo, algunos núcleos talámicos y las células de Purkinje del cerebelo. En estas áreas se expresa preferentemente en neuronas glutamatérgicas y GABAérgicas, pero no en colinérgicas. La expresión de los dos isoenzimas de la PDE7, la PDE7A y PDE7B, no presenta cambios en la expresión en áreas perivasculares del cerebro de los animales EAE.
Los ARNm de la PDE2 y de la PDE9 en el cerebro humano se localiza en áreas como la corteza cerebral, la formación hipocampal, el claustrum y los núcleos caudado y putamen. Sin embargo en el cerebelo encontramos expresión de la PDE9, pero no de la PDE2, en las células de Purkinje, en la capa granular del cerebelo y en el núcleo dentado. La comparación de su expresión entre cerebros postmortem control y cerebros diagnosticados con la enfermedad de Alzheimer en las regiones estudiadas (corteza frontal, cerebelo y diferentes regiones del área hipocampal) no mostró diferencias estadísticamente significativas.
"cAMP and cGMP fosfodiesterases in brain: Expression in neuroinflammatory and neurodegenerative processes"

cAMP and cGMP are second messengers involved in intracellular signalling pathways, contributing in a variety of cellular processes. Their synthesis is regulated by adenilyl-cyclase and guanilyl-cyclase, and their degradation by phosphodiesterases.
High levels of both nucleotides develop antiinfammatory and/or neuroprotective actions, so that, we would expect that the regulation of the enzymes involved in their degradation, phosphodiesterases, will act indirectly in those processes.
In this work we determined cAMP specific phosphodiesterase mRNA expression, PDE4B splicing variants and PDE7B isozyme, first, in control rat brain, and then in neuroinflammatory animal model brains (LPS and EAE) in order to determine their regulation. We also studied PDE2 and PDE9 mRNA expression, phosphodiesterases hydrolyzing cGMP, in post-mortem control human brains comparing with Alzheimer disease patient brains.
PDE4B splicing variant mRNA distribution in rat brain presents common structures: anterior olfactory nucleus or piriform cortex. However, some regions do not express all PDE4B isoforms. PDE4B4 is not expressed in area postrema; some white fibre tracts do not hybridize for PDE4B1 mRNA, or hippocampal dentate gyrus expresses exclusively PDE4B2 mRNA. Their expression in our animal models showed an exclusive increase for PDE4B2 mRNA levels in choroid plexus after toxin administration in the LPS inflammatory model, and in brain perivascular areas in the EAE animal model, where we found this splicing variant expression in some T cells and some macrophages/microglia.
PDE7B mRNA is localized along de whole rat brain, abundantly in anterior brain regions: olfactory tubercle, caudate-putamen nucleus, dentate gyrus of the hipocampus, some thalamic nucleus and Purkinje cells. This isoenzyme is preferently expressed in glutamatergic and GABAergic neurons, but not in cholinergic cells. PDE7 isoenzymes expression does not change in perivascular regions of the EAE animal brains.
PDE2 and PDE9 mRNA are localized in human brain regions: cerebral cortex, hipocampal formation, claustrum and caudate and putamen nuclei. However, we find PDE9 mRNA expression, but no PDE2 mRNA expression, in Purkinje cells, in granule cerebellar layer and in dentate nucleus of the cerebellum. When comparing their expression between post-mortem control and Alzheimer disease brains in studied regions we do not find statistically significant differences.

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46

Komas, Narcisse Patrice Joseph. "Role des phosphodiesterases specifiques de l'ampc et du gmpc dans le cur et les vaisseaux." Université Louis Pasteur (Strasbourg) (1971-2008), 1991. http://www.theses.fr/1991STR13016.

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L'objectif du present travail a ete d'etudier les nucleotides cycliques phosphodiesterases (pde) pouvant etre impliquees dans les effets inotrope et chronotrope positifs et vasodilatateur afin d'etablir une base moleculaire de dissociation des effets inotrope positif et vasodilatateur de l'effet chronotrope positif. Les pde peuvent etre caracterisees par leurs proprietes physiques, leur specificite de substrats (ampc ou gmpc), leur regulation par des modulateurs (calmoduline, gmpc) et par leur sequence primaire ce qui a permis leur classification en 5 familles: pde i hydrolyse l'ampc et le gmpc et est activable par la calmoduline; la pde ii hydrolyse aussi l'ampc et le gmpc et est stimulable par le gmpc; la pde iii, cible des drogues cardiotoniques, hydrolyse l'ampc et le gmpc et est inhibee par le gmpc; la pde iv hydrolyse specifiquement l'ampc et la pde v hydrolyse specifiquement le gmpc. Cette etude a ete faite sur les pde du ventricule gauche (fonction inotrope), les pde du nud sinusal (fonction chronotrope) et les pde d'aorte et d'artere mesenterique (vasodilatation). Quatre formes de pde cytosolubles sont obtenues dans le ventricule gauche et dans le nud sinusal de cur de chein (pde i, ii, iii et iv) ainsi que dans l'aorte et l'artere mesenterique de rat (pde i, ii, iv et v) alors que seules trois formes de pde existent dans les membranes du ventricule et du nud sinusal dont les pde iii et iv. L'etude pharmacologique avec les differentes classes d'inhibiteurs de pde montre que les pde iii (sensibles aux cardiotoniques) du ventricule et des vaisseaux sont differentes de la pde iii sinusale alors que la pde iv est similaire dans les quatre tissus etudies. Au niveau du ventricule gauche, la pde iii membranaire est plus sensible aux agents inotropes positifs que la pde iii cytosolique. Les resultats obtenus suggerent qu'il est possible, sur des bases moleculaires, de dissocier les effets chronotropes positifs des effe

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47

Rosseto, Flávio Rodolfo. "Estudos estruturais e funcionais de STM3615 de Salmonella enterica: uma proteína contendo ambos os domínios GGDEF-EAL envolvidos na biossíntese de c-di-GMP." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-23032017-093752/.

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A formação de biofilmes bacterianos é um fenômeno bem conhecido, caracterizado pela formação de uma comunidade bacteriana estática, embebida em uma matriz exopolimérica, regulada pela molécula sinalizadora c-di-GMP. Os domínios proteicos que catalisam a síntese (GGDEF) e degradação (EAL e HD-GYP) de c-di-GMP estão presentes em grande quantidade em quase todos os genomas bacterianos sequenciados até hoje. Dentre as diversas proteínas envolvidas nas vias de sinalização mediadas por esse nucleotídeo, uma grande parcela são proteínas transmembranares que possuem ambos domínios GGDEF e EAL. Funcionalmente, esses domínios se apresentam em todas combinações: ambos degenerados ou conservados e combinações GGDEF-degenerado/EAL-conservado ou vice-versa. Enquanto que domínios conservados potencialmente apresentam atividade catalítica, os degenerados geralmente convertem-se em domínios estruturais ou receptores de c-di-GMP. Embora recentes estudos estruturais revelaram detalhes de proteínas com ambos domínios degenerados (LapD) ou ativos (MorA), pouco se sabe sobre uma das combinações mais representativas: GGDEF-degenerado/EAL-conservado. Nesse trabalho, realizamos um estudo estrutural e funcional da proteína STM3615 de Salmonella enterica, que apresenta um domínio periplasmático de função desconhecida, seguido pelos domínios citoplasmáticos HAMP, GGDEF-degenerado e EAL-conservado. Através de diferentes construções citoplasmáticas solúveis de STM3615, confirmamos que essa proteína apresenta atividade fosfodiesterase, mesmo quando o domínio EAL encontra-se isolado. Corroborando com sua atividade catalítica, estudos em solução, tais como SAXS e cromatografia de exclusão molecular, mostraram que o EAL isolado de STM3615 é dimérico, um pré-requisito para ser ativo. Utilizando uma construção com os domínios GGDEF-EAL determinamos sua estrutura cristalográfica a uma resolução de 2,5 Å. Comparada com proteínas de arquitetura próxima, como o receptor de c-di-GMP LapD de Pseudomonas fluorescens, ou a enzima bifuncional MorA de Pseudomonas aeruginosa, sua estrutura se assemelha muito mais a essa última. Em particular, a hélice que conecta os domínios GGDEF e EAL possui a mesma extensão que a de MorA, maiores que a encontrada em LapD. Como a hélice pequena de LapD está relacionada com sua plasticidade conformacional interdomínios, a estrutura apresentada nesse trabalho sugere as proteínas dual domain cataliticamente ativas (EAL-mono ou bifuncionais) sejam estruturalmente rígidas. Combinando esses resultados com uma análise computacional feita em outras 150 sequências representativas de proteínas dual domain, propomos mecanismos catalíticos distintos para as enzimas bifuncionais e as EAL-monofuncionais. Enquanto que essas últimas formam dímeros estáveis através do domínio EAL, numa conformação apta para interagir e degradar c-di-GMP, as enzimas bifuncionais apresentam transições oligoméricas mediadas por interação de c-di-GMP com EAL, impondo atividades ciclase (GGDEF) e fosfodiesterase (EAL) excludentes. Por fim, baseados nesses mecanismos e na arquitetura de STM3615, ainda especulamos mecanismos funcionais in vivo compatíveis com o tema emergente de interações proteicas e localização do sinal nas vias de sinalização mediadas por c-di-GMP.
The formation of bacterial biofilms is a well-established phenomenon regulated by the signaling molecule c-di-GMP, characterized by the establishment of a static bacterial community embedded in a exopolymeric matrix. The domains responsible for the synthesis (GGDEF) or degradation (EAL and HD-GYP) of c-di-GMP are present in multiple proteins in nearly all bacterial genomes sequenced to date. Among the multiple and structurally diverse proteins involved in c-di-GMP signaling and biosynthesis, a large class are transmembrane proteins bearing both EAL and GGDEF domains. Functionally, these domains are presented in all combinations: both degenerate or conserved and combinations GGDEF-degenerated/EAL-conserved or vice versa. While the predicted conserved domains exhibit catalytic activity, the degenerate usually converted into structural domains or c-di-GMP receptors. While structural studies have revealed details of proteins with both domains degenerated (LapD) or conserved (MorA), little is known about one of the most representative combinations: GGDEF-degenerated/EAL-conserved. In this work, we conducted a structural and functional study of Salmonella enterica STM3615 protein, which has a periplasmic domain of unknown function, followed by cytoplasmic domains HAMP, GGDEF-degenerated and EAL-conserved. Through different soluble cytoplasmic constructs of STM3615, we confirmed that this protein has phosphodiesterase activity, even with the isolated EAL domain. In agreement with its catalytic activity, solution studies, such as SAXS and size exclusion chromatography, showed that STM3615 isolated EAL is dimeric, a prerequisite for phosphodiesterase activity. Using a construct with the isolated EAL-GGDEF domains, we determine its crystal structure to a resolution of 2.5 Å. Compared to the architectural closed c-di-GMP receptor LapD from Pseudomonas fluorescens and the bifunctional enzyme MorA from Pseudomonas aeruginosa, STM3615 structure is more similar to the latter. In particular, the α-helix connecting the domains GGDEF and EAL has similar extension, longer than the helix found in LapD. Given that this helix in LapD is essential for its inter-domain conformational plasticity, the structure presented in this study suggests the dual domain catalytically active proteins are structurally rigid. Combining these results with a computational analysis with 150 representative sequences containing the tandem GGDEF-EAL domains, we propose distinct catalytic mechanisms for bifunctional and monofunctional EAL enzymes. While the latter form stable dimers through the EAL domain, a conformation prompted to interact and degrade c-di-GMP, the bifunctional enzymes present oligomeric transitions mediated by interaction of c-di-GMP with EAL domain, imposing excluding cyclase (GGDEF) or phosphodiesterase (EAL) activities. Finally, based on these mechanisms and STM3615 architecture, we also speculated about functional mechanisms in vivo consistent with the emerging theme of protein interactions and localized signal involved in signaling pathways mediated by c-di-GMP.

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QUIRIN, LAURENCE. "Gmp cyclique et hypertension arterielle gravidique : variations des taux plasmatiques et urinaires au cours du peri-partum." Université Louis Pasteur (Strasbourg) (1971-2008), 1993. http://www.theses.fr/1993STR1M054.

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Cerqueira, Joao Batista Gadelha de. "Identificação dos mecanismos envolvidos no relaxamento da musculatura lisa cavernosa e da aorta de coelho, induzido por doadores de óxido nítrico do complexo nitrosil-rutênio." reponame:Repositório Institucional da UFC, 2008. http://www.repositorio.ufc.br/handle/riufc/7710.

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CERQUEIRA, João Batista Gadelha de. Identificação dos mecanismos envolvidos no relaxamento da musculatura lisa cavernosa e da aorta de coelho, induzido por doadores de óxido nítrico do complexo nitrosil-rutênio. 2008. 125 f. Tese (Doutorado em Cirurgia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2008.
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Endothelial dysfunction makes 56% of patients with erectile dysfunction decline treatment with PDE-5 inhibitors. New forms of treatment are necessary for this group of patients. The present study evaluates the relaxation in vitro induced in rabbit corpus cavernosum smooth muscle and aortic rings by sodium nitroprusside (SNP) and by two new NO-donor substances of the nitrosyl-ruthenium complex: Rut-Byp and Rut-Caf. Tissues immersed in isolated baths of Krebs-Henseleit solution (37oC; pH 7.4) were precontracted with 1uM phenylephrine (PE). Relaxation concentration/response curves were plotted for all concentrations (10-12 to 10-4 M). To explore the mechanisms involved in induced relaxation, the following substances were added: 3µM, 10µM, 30µM or 100µM ODQ (soluble guanylate cyclase-specific inhibitor), 3µM or 10 µM oxyhemoglobin (extracellular NO scavenger), 1 mM L-cysteine (nitrosyl anion-specific scavenger), 100µM hydroxycobalamin (NO free radical scavenger), glibenclamide (ATP-dependent potassium channel blocker), iberiotoxin (medium and high-conductance potassium channel blocker) and apamin (low-conductance potassium channel blocker). The tissue samples were frozen in liquid nitrogen in order to quantify GMPc and AMPc produced during relaxation. All the substances tested produced a significant level of relaxation in the aortic vascular endothelium. Similar results were found for corpus cavernosum smooth muscle, with the exception of Rut-Byp (Emax 30%). In this tissue, Rut-Caf e SNP induced dose-dependent relaxation with a potency (pEC50) of 4.2 and 5.2, and a maximum effect (Emax) of 100% and 80%, respectively. All substances acted through the activation of soluble guanylate cyclase (sGC); therefore, the addition of 100µM ODQ inhibited the relaxation effect completely in all cases. Oxyhemoglobin reduced relaxation induced by all substances. At 3µM, the maximum effect (Emax) was reduced by 26% on the average, and at 10µM the effect was reduced by another 50% (p<0.05), though not completely neutralized. L-cysteine failed to affect relaxation, but hydroxycobalamin abolished Rut-Caf-induced relaxation in aortic rings (Emax: 112% vs 10%; p<0.005). A significant reduction was observed in corpus cavernosum smooth muscle relaxation, though not as intense as in aortic rings. The addition of glibenclamide to the baths increased the potency of Rut-Caf significantly (4.09 vs. 7.9; p<0.005) with no significant change in maximum effect. Potency and maximum effect remained unchanged with the other ion channel blockers. The agents released cGMP in both tissues studied. NO-donor substances of the nitrosyl-ruthenium complex were shown to be potent vasodilators. One substance (Rut-Caf) induced significant relaxation in animal corpus cavernosum. The substances tested in the study act through the activation of soluble guanylate cyclase producing intracellular GMPc. During relaxation they release NO and its free radical intracellularly, but not nitrosyl. They do not act directly upon potassium ion channels. Rut-Caf acts independently of the endothelial integrity.
Disfunção endotelial provoca 56% de resistência ao tratamento da disfunção erétil pelos inibidores da PDE-5. Novas formas de tratamento são necessárias para este grupo de pacientes. O estudo avaliou o relaxamento, in vitro, induzido por novas substâncias doadoras de óxido nítrico (NO) do complexo nitrosil-rutênio (Rut-Byp, Rut-Caf) na musculatura lisa de corpos cavernosos humanos e de coelho e em anéis de aorta de coelho e do nitroprussiato de sódio (SNP). Os tecidos, imersos em sistemas de banhos isolados em solução de KHS (pH 7,4; 37oC), foram pré-contraídos com fenilefrina (PE;1uM) e curvas de concentração-resposta (10-12M a10-4M) foram obtidas. Para esclarecer o mecanismo de ação envolvido no relaxamento induzido pelos agentes, foram adicionadas aos banhos as substâncias: ODQ (3µM, 10µM, 30µM e 100µM), inibidor da guanilatociclase solúvel; oxi-hemoglobina (3µM e 10µM), removedor extracelular do NO; L-cisteína (100µM), removedor intracelular do ânion nitroxil; hidroxicobalamina (100µM), removedor de radical livre do NO; glibenclamida, bloqueador de canais de íons potássio ATP-dependente (KATP); iberiotoxina, bloqueador de canais de potássio de alta e média condutividade (KCA); apamina, bloqueador de canais de potássio de baixa condutividade (KCA). Amostras dos tecidos foram congeladas em nitrogênio líquido para mensurar a quantidade de GMPc e AMPc produzido no relaxamento. Todas as substâncias provocaram relaxamento estatisticamente significante da musculatura lisa dos anéis de aorta. Na musculatura lisa cavernosa, só Rut-Byp não conseguiu induzir relaxamento significativo (efeito máximo= 30%). Neste tecido a Rut-Caf e SNP provocaram relaxamento dose-dependente, com efeito máximo de 80% e 100% e pEC50 4,2 e 5,2, respectivamente. Todas substâncias mostraram atividade mediante a ativação da enzima guanilatociclase solúvel (GCs), pois a adição do ODQ 100µM aos banhos, inibiu totalmente o efeito relaxante. A oxi-hemoglobina em anéis de aorta de coelho na dose de 3µM, diminuiu o efeito máximo das substâncias em média 26% e, na dose de 10µM, houve uma redução adicional de 50% (p<0,05). A L-cisteína falhou em alterar o relaxamento induzido pelos agentes estudados. A hidroxicobalamina em anéis de aorta e em corpo cavernoso de coelho aboliu o efeito relaxante da Rut-Caf (Efeito máximo: 112% x 10%; p<0,005). A adição de glibenclamida em corpos cavernosos de coelho aumentou a potência da substância Rut-Caf (4,09 x 7,9; p<0,005), sem alterar o efeito máximo. Não houve alteração de potência ou efeito máximo das substâncias com a adição de outros bloqueadores de canais de íons. A remoção do endotélio não alterou a potência e o efeito máximo da substância Rut-Caf em anéis de aorta de coelho. As substâncias liberaram GMPc em maior intensidade no corpo cavernoso do que em anéis de aorta. As substâncias doadoras de NO do complexo nitrosil-rutênio são potentes vasodilatadores e uma delas (Rut-Caf), demonstrou relaxamento significativo no tecido cavernoso animal e humano. As substâncias atuam ativando a enzima guanilatociclase solúvel produzindo GMPc, liberam NO e radical livre do NO durante o relaxamento, mas não o ânion nitrosil. Os agentes não atuam diretamente nos canais de íons potássio. A substância Rut-Caf atua independentemente da integridade endotelial.

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50

NUNES, Ana Karolina de Santana. "Avaliação dos efeitos do inibidor de fosfodiesterase-5 sobre as células gliais e a re-mielinização, em modelo de desmielinização induzido em camudongos C57BL/6 WILD TYPE e KONCKOUT para iNOS." Universidade Federal de Pernambuco, 2012. https://repositorio.ufpe.br/handle/123456789/12599.

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CNPQ; FACEPE
O Sildenafil (Viagra®) promove acúmulo de monofosfato de guanosina cíclico (GMPc) através da inibição seletiva da fosfodiesterase-5 (PDE5). Embora esse medicamento mantenha um excelente nível de segurança e perfil de tolerabilidade, apenas disfunção erétil e, mais recentemente, hipertensão pulmonar são doenças tratadas atualmente com Sildenafil. Astrócitos e micróglia são células do SNC que exercem um papel importante em numerosas patologias do cérebro, incluindo a Esclerose Múltipla (EM). A EM é uma doença inflamatória crônica, caracterizada por desmielinização das células nervosas, que leva a severa deficiência psicomotora. Tem sido reportado que o acúmulo intracelular de GMPc modula a reação microglial e astrocitária e protege oligodendrócitos diferenciados (formadores da mielina), reduzindo danos causados à mielinização. Portanto, o presente estudo investigou os efeitos do Sildenafil em modelo de EM induzido pela ingesta de Cuprizona. Cinco camundongos C57BL/6 selvagens e cinco iNOS-/-, machos, com 7-10 semanas de idade, foram usados por grupo. Os grupos receberam, durante quatro semanas: 1) Cuprizona 0,2% misturada na ração, 2) Cuprizona na ração e Sildenafil 3, 25 ou 50 mg/Kg na água de beber (os iNOS-/- receberam apenas a dose de 25 mg/Kg), ou 3) Os controles receberam água e ração puras. Após os tratamentos, os cerebelos foram processados de acordo com a rotina para microscopia eletrônica de transmissão, western blotting, imunohistoquímica (parafina), imunofluorescência (congelação) ou coloração Luxol Fast Blue. Os resultados demonstraram que, nos animais selvagens, Cuprizona induziu temores, limitações motoras e alterações posturais nos animais, aumentou os níveis das proteínas GFAP e Iba-1, indicando gliose reativa e ativação microglial e aumentou COX-2, indicando um ambiente pró-inflamatório no cerebelo; além disso, provocou redução da espessura da mielina e da intensidade de mielinização e promoveu alterações ultraestruturais na bainha de mielina e nos axônios do cerebelo. Sildenafil protegeu significativamente o microambiente neural dos animais tratados com Cuprizona, apresentando efeito dose-dependente, cuja dose mais efetiva foi a de 25 mg/Kg. O tratamento com Sildenafil diminuiu os tremores e limitações motoras induzidos pela Cuprizona, manteve a expressão basal de GFAP e Iba-1, diminuiu fortemente a expressão de COX-2 e das citocinas pró-inflamatórias (IFN-γ, TNF-α, , IL-1β e IL-2), impediu a desmielinização e protegeu ultraestruturalmente a organização mielínica e axonal. Nos animais iNOS-/-, os efeitos clínicos induzidos pela Cuprizona foram semelhantes aos vistos nos animais selvagens e Sildenafil diminuiu esses efeitos. Cuprizona aumentou a expressão de GFAP, Iba-1 e IFN-γ. Nesses animais, entretanto, Sildenafil aumentou a marcação para GFAP, indicando que não teve efeito protetor nos astrócitos, na ausência do NO produzido pela iNOS. Por outro lado, diminuiu a expressão de Iba-1 e IFN-γ, indicando efeito anti-inflamatório e inativação da micróglia estimulada pela Cuprizona. Os animais iNOS-/- controle (sem nenhum tratamento) apresentaram alterações degenerativas na mielina, que foram agravadas pela Cuprizona. Sildenafil diminuiu a intensidade dessas alterações, embora não tenha resolvido-as completamente. Portanto, o aumento dos níveis de GMPc, pela inibição da PDE5, provavelmente agiu como antiinflamatório e agente protetor de astrócitos, micróglia e oligodendrócitos, diminuindo os danos ao tecido neural. Interessantemente, a via de ação do Sildenafil foi tipo celular-dependente, ou seja, foi independente do NO produzido pela iNOS em micróglia e oligodendrócitos e dependente em astrócitos. Portanto, após ensaios clínicos, o Sildenafil pode ser um medicamento compatível com a administração oral para a população com EM e outras doenças neurodegenerativas, promovendo efeitos benéficos adicionais aos tratamentos atuais. Esclarecer o mecanismo de ação do Sildenafil nas células neurais será alvo de estudos futuros do nosso grupo.

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