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1
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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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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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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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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Williams, A. C. "The importance of cyclic nucleotides (cyclic GMP and cyclic AMP) in the development of malignant disease." Thesis, University of Bristol, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379348.
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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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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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Wu, Albert Ya-Po. "Molecular mechanism of cyclic nucleotide binding to the GAF domains of phosphodiesterases 2 and 5 /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/5012.
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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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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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Fujishige, Kotomi. "Metabolism of Cyclic Nucleotides : Association of Cyclic GMP with Chondrogenic Differentiation and Identification of a Novel Cyclic Nucleotide Phosphodiesterase." Kyoto University, 2000. http://hdl.handle.net/2433/151601.
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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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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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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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Kostick, Jessica. "Characterization of cyclic-di-GMP signaling with the Lyme spirochete, Borrelia burgdorferi." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/272.
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Lyme disease is a tick-borne infection caused by Borrelia burgdorferi, B. garinii, and B. afzelii. These spirochetes experience environmental fluctuations as they are passed between mammalian and Ixodes tick hosts throughout their enzootic cycle. Recent studies have suggested cyclic diguanylate (c-di-GMP), a ubiquitous secondary messenger, is a key modulator of B. burgdorferi adaptive responses and may play a significant role in cycle progression. In this study, we examined the impact of the sole diguanylate cyclase (Rrp1), c-di-GMP binding proteins (PlzA and PlzB), and HD-GYP-containing phosphodiesterase (PdeB) in disease establishment of both murine and Ixodes tick systems. Strains harboring targeted gene deletions or plasmid-based constitutive gene expression constructs were generated. Rrp1 was required for tick colonization, yet overexpression abolished murine disease, thus implicating the requirement of finely regulated c-di-GMP levels for enzootic cycle progression. Deletion of rrp1 disrupted translational motion and swarming patterns by causing extended cell runs, eliminating stops/flexes, and reducing swarming capabilities. This was attributed to a defect in N-acetyl-D-glucosamine (NAG) metabolism and chemotaxis. NAG is a major source of nutrition for B. burgdorferi within the tick environment; therefore this defect would impede spirochete migration towards feeding ticks, as well as pathogen uptake and survival within the Ixodes vector. In contrast, the downstream c-di-GMP effector, PlzA, was critical for murine disease but nonessential for survival within ticks nor functionally complemented by PlzB. Deletion of plzA altered strain motility and swarming similarly to the rrp1 deletion mutant, yet had a distinct phenotype with significantly slower translational motion and no affect on NAG chemotaxis and metabolism. This indicates B. burgdorferi could possess alternate c-di-GMP effectors or Rrp1 could be directly influencing these cellular processes. Uniquely, PdeB did not abolish murine infection via needle inoculation, but wasrequired for natural transmission from ticks. This defect was linked to the decreased tick colonization efficiency upon pdeB deletion. Together, these analyses indicate that c-di-GMP signaling is an important virulence mechanism of Borrelia burgdorferi and demonstrate the complexity of this signaling pathway in an arthropod-borne pathogen. The data presented here additionally provide significant new insight into the gene regulatory mechanisms of the Lyme disease spirochetes.
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Ryjenkov, Dmitri A. "Cyclic dimeric GMP, a novel bacterial second messenger enzymology of its turnover /." Laramie, Wyo. : University of Wyoming, 2006. http://proquest.umi.com/pqdweb?did=1188872151&sid=1&Fmt=2&clientId=18949&RQT=309&VName=PQD.
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Barnes, Christopher Simon. "The potential influence of cyclic GMP on arrhythmias associated with mycardial ischaemia." Thesis, University of Liverpool, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359292.
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Gallagher, Thomas. "REGULATION OF SATIETY QUIESCENCE: CYCLIC GMP, TGF BETA, AND THE ASI NEURON." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/3254.
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The worm Caenorhabditis elegans is a well-studied model organism in numerous aspects of its biology. This small free living nematode has less than 1,000 cells, but shows clear conservation in both signaling and behavior to mammals in aspects of appetite control. This is of importance to humans, where failure of appetite control is a major factor in the unprecedented obesity epidemic that we see today. In general, worm behavior reflects its internal nutritional state and the availability and quality of food. Specifically, worms show a behavioral state that mimics aspects of the mammalian behavioral satiety sequence, which has been termed satiety quiescence. We have used locomotion tracking and Hidden Markov Model analysis to identify worm behavioral state over time, finding quiescence along with the established worm locomotive behaviors roaming and dwelling. Using this analysis as well as more conventional cell biology and genetic approaches we have further investigated satiety signaling pathways. We have found that the neuron ASI is a major center of integration of signals regarding the internal nutritional state of the worms as well as the nutritional content of its environment. Our results show that cGMP causes levels of the TGFβ ligand to be increased in fasted worms, which is then released and binds to its receptor on the RIM and RIC neurons. This signaling connects nutritional state to behavioral response, promoting the sleep-like behavioral state satiety quiescence. Additionally, we have begun a candidate approach examining several other groups of signaling molecules for potential roles in satiety quiescence signaling including cannabinoids, multidrug resistance proteins, and neuropeptides. The result of this investigation is a better understanding of mechanisms of satiety quiescence signaling as well as a new tool that provides highly quantitative, unbiased, and automated data to aid in our ongoing work.
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Gibbs, Sarah Margaretha. "Regulation of Drosophila visual system development by nitric oxide and cyclic GMP /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/10651.
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Gallagher, Thomas L. "Regulation of satiety quiescence| Cyclic GMP, TGF beta, and the ASI neuron." Thesis, Virginia Commonwealth University, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=3610909.
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The worm Caenorhabditis elegans is a well-studied model organism in numerous aspects of its biology. This small free living nematode has less than 1,000 cells, but shows clear conservation in both signaling and behavior to mammals in aspects of appetite control. This is of importance to humans, where failure of appetite control is a major factor in the unprecedented obesity epidemic that we see today.
In general, worm behavior reflects its internal nutritional state and the availability and quality of food. Specifically, worms show a behavioral state that mimics aspects of the mammalian behavioral satiety sequence, which has been termed satiety quiescence. We have used locomotion tracking and Hidden Markov Model analysis to identify worm behavioral state over time, finding quiescence along with the established worm locomotive behaviors roaming and dwelling. Using this analysis as well as more conventional cell biology and genetic approaches we have further investigated satiety signaling pathways. We have found that the neuron ASI is a major center of integration of signals regarding the internal nutritional state of the worms as well as the nutritional content of its environment. Our results show that cGMP causes levels of the TGFβ ligand to be increased in fasted worms, which is then released and binds to its receptor on the RIM and RIC neurons. This signaling connects nutritional state to behavioral response, promoting the sleep-like behavioral state satiety quiescence. Additionally, we have begun a candidate approach examining several other groups of signaling molecules for potential roles in satiety quiescence signaling including cannabinoids, multidrug resistance proteins, and neuropeptides. The result of this investigation is a better understanding of mechanisms of satiety quiescence signaling as well as a new tool that provides highly quantitative, unbiased, and automated data to aid in our ongoing work.
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Ng, David Dean Wing. "Studies on the role of cyclic GMP in the regulation of contractility in heart and blood vessels." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26507.
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This thesis is mainly concerned with the study of the role of cGMP in regulating contractility in the heart and blood vessels. A novel cGMP lowering agent, LY83583 (6-anilino-5,8-quinolinedione), was employed as a tool to determine the involvement of cGMP in mediating pharmacological and biological responses in the tissues being examined.
In the first study, the role of cGMP in atriopeptin II-induced vascular relaxation was investigated. Atriopeptin II is believed to produce its vasorelaxant effect by virtue of its ability to elevate cGMP. However, the ability of the guanylate cyclase inhibitor, methylene blue, to inhibit the atriopeptin II-induced vasorelaxation has not been conclusively demonstrated. In the present study, LY83583 was found to partially prevent the rise in cGMP level caused by atriopeptin II but was without effect on the extent of the relaxation. This lack of correlation between cGMP elevation and relaxation may indicate either functional compartmentalization of the cyclic nucleotide or the existence of a cGMP-independent pathway for relaxation. Alternatively, the attenuated cGMP level may still be sufficient to elicit full relaxation. The inability of atriopeptin II to relax KC1-contracted bovine coronary arteries agrees with other reports in the literature and may indicate that the drug is less effective in antagonizing vascular responses associated with a marked degree of cell membrane depolarization.
In the second study, the role of cGMP in mediating the endothelium-dependent inhibition of contractile responses of vascular tissue to alpha adrenoceptor stimulation was examined. There are reports in the literature that EDRF released from the endothelium elevates cGMP and depresses the response of the vessels to vasoconstrictors such as clonidine and norepinephrine. In the present study, LY83583 was used to examine the role of cGMP in mediating this effect. The treatment with LY83583 significantly lowered basal levels of cGMP and markedly enhanced the contractile response of endothelium-containing rat arteries to clonidine and norepinephrine. cGMP measurements indicate that
clonidine did not elevate cGMP levels; hence the drug is unlikely to stimulate EDRF release. On the other hand, the depressant action of LY83583 on basal cGMP levels supports the hypothesis that inhibition of contractile responses may be a result of spontaneous release of EDRF, which results in tonic elevation of cGMP. The ability of 8-bromo-cGMP to reverse LY83583-induced enhancement of contractile responses to clonidine and norepinephrine further supports the involvement of cGMP in EDRF-induced vascular relaxation. In the final study, the role of cGMP in regulating cardiac contractility of amphibian ventricles was examined. The importance of cGMP in controlling mammalian cardiac function is controversial. However, a remarkable correlation between cGMP and contractile force has been reported in hypodynamic frog ventricles, and cAMP and cGMP were reported to act in a reciprocal fashion to regulate contractility. The present investigation attempted to verify whether such a relationship actually exists in the frog ventricles. Carbachol elicited a dose-dependent reduction in contractility without altering cGMP levels. On the
contrary, sodium nitroprusside (100µM) did not reduce cardiac contractility despite a significant elevation of cGMP. At 1mM sodium nitroprusside, a huge elevation of cGMP and a small reduction in contractile tension were observed. Qualitatively similar results were obtained with a degraded sample of sodium nitroprusside. cAMP/cGMP ratios were not correlated with contractility. Hence, these findings were inconsistent with those found in earlier studies on hypodynamic frog hearts and do not support the proposed role of cGMP as a second messenger. The disparate findings may be caused by differences in experimental design. Alternatively, functional compartmentalization of cGMP (in the case of sodium nitroprusside) and the involvement of other cGMP-independent pathways (in the case of carbachol) cannot be ruled out. All these findings suggest that cGMP may play a more crucial role in regulating vascular than cardiac contractility.Pharmaceutical Sciences, Faculty of
Graduate
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Bailin, Adam. "Regulation of the Pseudomonas aeruginosa type III secretion system by cyclic-di-GMP." Thesis, University of Iowa, 2017. https://ir.uiowa.edu/etd/5411.
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Pseudomonas aeruginosa is a gram-negative pathogen that causes opportunistic infections in immunocompromised individuals. Whereas clinical isolates from acute infections are characterized by host cell cytotoxicity and motility, isolates from chronic infections are characterized by biofilm formation and persistence. The type III secretion system (T3SS) causes cytotoxicity by injecting effectors into host cells. T3SS gene expression is activated by ExsA, an AraC family transcriptional regulator. Transcription of exsA is controlled by two promoters, PexsC and PexsA, which are regulated by ExsA and the cAMP-Vfr system, respectively. Additional global regulatory systems also influence T3SS including the second messenger signaling molecule c-di-GMP and the RsmAYZ regulatory system. c-di-GMP signaling increases biofilm production and decreases acute virulence factor expression. A previous study found that c-di-GMP alters cAMP levels and affect cAMP-Vfr signaling. Other studies found that c-di-GMP signaling alters expression of the small non-coding regulatory RNAs, rsmY and rsmZ. The RsmAYZ post-transcriptional regulatory system regulates ExsA translation. We hypothesize that c-di-GMP regulates T3SS expression by altering exsA transcription through the cAMP-Vfr dependent PexsA promoter. Overexpression of YfiN, a c-di-GMP synthase, decreases T3SS reporter activity in PA103 and requires a functional GGDEF active site for full inhibition. Inhibition by YfiN does not require rsmYZ. YfiN expression decreases cAMP-Vfr signaling and coordinately inhibits PexsA-lacZ reporter activity. Consistent with the proposed model, YfiN expression in a vfr mutant does not further decrease T3SS reporter activity. These data indicate that the YfiN alters T3SS expression through transcriptional control of the cAMP-Vfr dependent PexsA promoter.
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au, K. Powers-Martin@murdoch edu, and Kellysan Powers-Martin. "Nitric oxide and central autonomic control of blood pressure: A neuroanatomical study of nitric oxide and cGMP expression in the brain and spinal cord." Murdoch University, 2008. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20090220.204446.
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Essential hypertension is defined as a chronic elevation of blood pressure of unknown cause. Though a definitive trigger for this change in blood pressure has not been established, there is a strong association with an upregulation of sympathetic output from the central nervous system. There are a number of central autonomic nuclei involved in the maintenance of blood pressure, including the brainstem regions of the nucleus tractus solitarii (NTS), caudal ventrolateral medulla (CVLM), rostral ventrolateral medulla (RVLM), the sympathetic preganglionic neurons (SPNs) within the intermediolateral cell column (IML) of the spinal cord, as well as forebrain regions such as the paraventricular nucleus (PVN) of the hypothalamus. Within these centers, a vast number of neurotransmitters have been identified that contribute to the control of blood pressure, including glutamate, angiotensin II, serotonin, neurotensin, neuropeptide Y, opioids and catecholamines. Recognition of the role of nitric oxide (NO) and its multiple influences over the neural control of blood pressure is gaining increasing significance.
Nitric oxide is a unique modulatory molecule that acts as a non-conventional neurotransmitter. As NO is a gas with a short half-life of 4 6 seconds, its synthesising enzyme, nitric oxide synthase (NOS) is often used as a marker of location of production. Once activated, the best-known receptor for NO is soluble guanylate cyclase (sGC), which drives the production of cyclic guanosine monophosphate (cGMP). Identifying the presence of cGMP can therefore be used to determine sites receptive to NO. Previous studies examining the role of NO in the central autonomic control of blood pressure have focused predominantly upon application of either excitatory or inhibitory drugs into the key central autonomic regions and assessing pressor or depressor effects. This thesis aims instead to study the neuroanatomical relationship and functional significance of NO and cGMP expression in the brain and spinal cord of a hypertensive and normotensive rat model.
In the first experimental chapter (Chapter 3), a comparative neuroanatomical analysis of neuronal NOS expression and its relationship with cGMP in the SPN of mature Spontaneously Hypertensive Rats (SHR) and their controls, Wistar Kyoto (WKY) was undertaken. Fluorescence immunohistochemistry confirmed the expression of nNOS in the majority of SPN located within the IML region of both strains. However, a strain specific anatomical arrangement of SPN cell clusters was evident and while there was no significant difference between the total number of SPN in each strain, there were significantly fewer nNOS positive SPN in the SHR animals. All nNOS positive SPN were found to express cGMP, and a novel subpopulation of nNOS negative, cGMP-positive SPN was identified. These cells were located in the medial edge of the IML SPN cell group. These results suggest that cGMP is a key signalling molecule in SPN, and that a reduced number of nNOS positive SPN in the SHR may be associated with the increase in sympathetic tone seen in essential hypertension.
The second experimental chapter (Chapter 4) aimed to determine if reduced numbers of nNOS containing SPN translated into reduced detectable cGMP. The functional significance of cGMP signalling in the two strains was then examined. Based on previous work by our group, it was predicted that reduced nNOS in the SHR would translate into reduced cGMP and that intrathecal administration of exogenous cGMP in the spinal cord would drive a differential pressor response in the two animal strains. Immunohistochemical techniques confirmed that within each SPN, the relative level of cGMP expression was significantly reduced in the SHR when compared to the WKY. Intrathecal application of 8-bromo-cGMP, a drug analogous to cGMP, increased blood pressure in both strains and had a differential and dose dependent effect, causing only a small increase in blood pressure in anaesthetised WKY animals, while driving a significant pressor response in the SHR. This finding raised the novel hypothesis that in the SHR, reduced nNOS expression is not a driver of hypertension, but is instead a protective mechanism limiting the potent pressor effects of cGMP within SPN.
The third experimental chapter (Chapter 5) examines the expression of neuronal and inducible isoforms of NOS (nNOS, iNOS) within the RVLM of SHR and WKY rats. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyse the level of mRNA expression and immunohistochemistry was then used to further analyse protein levels of nNOS. Total RNA was extracted and reverse transcribed from the RVLM of mature male WKY and SHR. Quantitative real-time PCR indicated that relative to WKY, mRNA levels for nNOS was significantly higher in RVLM of the SHR. This was confirmed immunohistochemically. When compared to iNOS, nNOS was expressed at significantly higher levels overall, however there was no difference in iNOS mRNA expression between the two strains. This demonstration of differential expression levels of nNOS and iNOS in the RVLM raises the possibilities that (i) NO production is up-regulated in the RVLM in SHR in response to increased sympathetic activity in order to re-establish homeostatic balance or alternatively that (ii) an alteration in the balance between nNOS and iNOS activity may underlie the genesis of augmented sympathetic vasomotor tone during hypertension.
The fourth experimental chapter (Chapter 6) extends the observations in Chapter 5 through examination of the expression of cGMP and sGC within the RVLM. There is strong functional evidence to suggest that NO signalling in the RVLM relies on cGMP as an intracellular signalling molecule and that this pathway is impaired in hypertension. Immunohistochemistry was used to assess cGMP expression as a marker of active NO signalling in the C1 region of the RVLM, again comparing SHR and WKY animals. Fluorescence immunohistochemistry on sections of the RVLM, double labelled for cGMP and either nNOS or phenylethylamine methyl-transferase (PNMT) failed to reveal cGMP positive neurons in the RVLM from aged animals of either strain, despite consistent detection of cGMP immunoreactivity neurons in the nucleus ambiguus from the same or adjacent sections. This was demonstrated both in the presence and absence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) and in young vs. aged animals. In-vitro incubation of RVLM slices in the NO donor DETA-NO or NMDA did not reveal any additional cGMP neuronal staining within the RVLM. In all studies, cGMP was prominent within the vasculature. Soluble guanylate cyclase immunoreactivity was found throughout the RVLM, although it did not co-localise with the PNMT or nNOS neuronal populations. Overall, results suggest that within the RVLM, cGMP is not detectable in the resting state and cannot be elicited by phosphodiesterase inhibition, NMDA receptor stimulation or NO donor application. A short time course of cGMP signalling or degradation not inhibited by the phosphodiesterase inhibitor utilised (IBMX) in the RVLM cannot be excluded.
The final experimental chapter (Chapter 7) examines cGMP expression in magnocellular and preautonomic parvocellular neurons of the PVN. Retrograde tracing techniques and immunohistochemistry were used to visualise cGMP immunoreactivity within functionally, neurochemically and topographically defined PVN neuronal populations in Wistar rats. Basal cGMP immunoreactivity was readily observed in the PVN, both in neuronal and vascular profiles. Cyclic GMP immunoreactivity was significantly higher in magnocellular compared to preautonomic neuronal populations. In preautonomic neurons, the level of cGMP expression was independent on their subnuclei location, innervated target or neurochemical phenotype. The data presented in this chapter indicates a highly heterogeneous distribution of basal cGMP levels within the PVN, and supports work by others indicating that constitutive NO inhibitory actions on preautonomic PVN neurons are likely mediated indirectly through activation of interneurons.
Summary
Together, these studies comprise a detailed analysis of the neuroanatomical expression of NO and its signalling molecule cGMP in key central autonomic regions involved in the regulation of blood pressure. Under resting or basal conditions, the studies demonstrate notable differences in the expression of NO synthesising enzymes between normotensive and hypertensive animals, and correlating changes in the downstream signalling molecule cGMP. In the spinal cord, novel functional differences in cGMP activity were also demonstrated. In the RVLM, although differences in nNOS were demonstrated, cGMP expression could not be readily detected in either the WKY or SHR, while in contrast within the PVN, cGMP was detected in both magnocellular and parvocellular neuronal populations.
Conclusion
This thesis gives insight into the physiological role of NO and cGMP as mediators of central blood pressure control. The results presented indicate that the NO-cGMP dependent signalling pathway may not be the dominant driver responsible for maintaining high blood pressure in the SHR model of essential hypertension, and that there is no globally consistent pattern of expression, and indeed the role of NO as a mediator of pressor and depressor function may vary between the autonomic regions examined. Further, it is possible that this pathway is only recruited during activation of reflex homeostatic pathways or during times of marked physiological stress, and that the differences we see in basal expression between the normotensive and SHR animals are instead a result of compensatory mechanisms.
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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 AntunesDissertação (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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Skotnicka, Dorota [Verfasser], and Lotte [Akademischer Betreuer] Søgaard-Andersen. "Regulation by cyclic di-GMP in Myxococcus xanthus / Dorota Skotnicka. Betreuer: Lotte Søgaard-Andersen." Marburg : Philipps-Universität Marburg, 2016. http://d-nb.info/1097531015/34.
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Trampari, Eleftheria. "Allosteric control of type III secretion systems by the second message cyclic-di-GMP." Thesis, University of East Anglia, 2016. https://ueaeprints.uea.ac.uk/61544/.
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Cyclic di-GMP (cdG) is a ubiquitous second messenger in bacteria, regulating transcriptional and post-transcriptional processes and allosterically controlling protein function. While the mechanisms of cdG metabolism are well understood, the downstream targets of this molecule are poorly characterised. To understand the role of cdG signalling in plant-associated species, cdG-capture compound pull-down experiments were performed to identify potential binding proteins in Pseudomonas fluorescens. One of the top targets identified was the flagella export AAA+ ATPase FliI, which was shown to bind specifically and tightly to cdG. FliI-cdG interaction was demonstrated for diverse bacterial FliI homologs. Excitingly, high-affinity binding was observed for the type-III secretion system (T3SS) homolog, HrcN and the type-VI ATPase, ClpB2. A combination of techniques was used to predict the FliI cdG binding site at the interface between two FliI subunits. Although the addition of cdG inhibits the ATPase activity of both FliI and HrcN in vitro, this occurs at a non-physiological cdG concentration suggesting that this does not represent the in vivo role of binding. However, when cdG concentrations are artificially increased, the export of flagellin subunits is significantly reduced, suggesting a link between cdG binding and protein export. Changes in the in vitro multimerization state of the protein were also observed upon the addition of cdG. As part of this study, novel and highly specific tools for nucleotide-protein interactions were developed and existing biochemistry techniques were optimised. These assays were employed to characterise cdG binding to four more proteins, which were identified as cdG binders. The results generated in this study broaden the existing knowledge about cdG binding protein diversity. The identification of FliI as a cdG binder suggests a novel cdG-dependent control mechanism for the function of bacterial export pathways including the flagellum and the T3SS, through allosteric interaction with export ATPase proteins.
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Zayas, Ventura Ricardo Manuel. "Nitric oxide/cyclic GMP signaling in the central nervous system of Manduca sexta larvae /." Thesis, Connect to Dissertations & Theses @ Tufts University, 2003.
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Thesis (Ph.D.)--Tufts University, 2003.Adviser: Barry A. Trimmer. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 147-164). Access restricted to members of the Tufts University community. Also available via the World Wide Web;
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Morgan, Robert Owen. "The role of cyclic GMP in regulating vascular smooth muscle and blood platelet function." Thesis, Cardiff University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389404.
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Bedri, Babiker A. "The role of the enteric nervous system in intestinal cyclic GMP-dependent secretory processes." Thesis, University of Aberdeen, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265847.
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This study investigated enteric nervous system (ENS) involvement in intestinal secretion induced by cyclic GMP-dependent secretagogues. The investigation was based upon the study of transepithelial ion transport in rat small and large intestine and in guinea pig caecum using voltage-clamped in vitro preparations mounted in Ussing chambers. ENS participation was established from the use of neural blocking agents (tetrodotoxin (TTX), bicuculline and capsaicin). The relative contribution of the myenteric plexus was assessed by selectively stripping tissues of the longitudinal muscle layer. All tissues, both unstripped and stripped, responded to Escherichia coli STa enterotoxin, guanylin and the nitric oxide (NO) donor sodium nitroprusside (SNP) with a dose-dependent increase in inward short circuit current (ISC). Regarding STa/guanylin, TTX inhibited this ISC in unstripped rat distal colon, ileum and guinea pig caecum, demonstrating that the ENS plays an important role in these tissues. In rat distal colon, TTX induced an abolition of the STa/guanylin response in both preparations, indicating submucous plexus involvement. In rat proximal colon there was no discernible TTX-sensitive component observed. The ileum displayed partial control from both the myenteric and submucous plexuses, whereas the caecum exhibited partial control from the myenteric plexus alone. Bicuculline inhibited STa action to a significant degree in the caecum while capsaicin inhibited secretion in the proximal colon. In rat small intestine, the SNP-induced ISC was inhibited by TTX in both unstripped and stripped tissues. In contrast, inhibitory pathways were shown to exist in distal colon exposed to SNP, TTX revealing an enhancement of SNP-induced secretion in the stripped preparation. Thus, although there is clear involvement of the ENS in the actions of STa/guanylin and SNP, it is not possible to make a general statement regarding its contribution throughout the length of the alimentary canal due to the extent of inter segmental and inter species variations.
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Skotnicka, Dorota Jagoda [Verfasser], and Lotte [Akademischer Betreuer] Søgaard-Andersen. "Regulation by cyclic di-GMP in Myxococcus xanthus / Dorota Skotnicka. Betreuer: Lotte Søgaard-Andersen." Marburg : Philipps-Universität Marburg, 2016. http://d-nb.info/1097531015/34.
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Reierson, Gillian W. "Role of the Phosphodiesterase (PDE) System in Mediating the Effects of Chronic Antidepressant Treatment in Rat Brain." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_dissertations/365.
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Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) act as second messengers in intracellular signaling cascades to influence neuronal responses. Hippocampal cAMP signaling is thought to underlie the pathophysiology of major depressive disorder (MDD) and antidepressant action; however, little is known about the possible role of cGMP signaling. Furthermore, circadian rhythm disturbances can occur as part of the clinical symptoms of MDD and resolve with antidepressant therapy. The pineal gland is relevant to circadian rhythms as it secretes the hormone melatonin following activation of cAMP signaling and the rate-limiting enzyme for its synthesis, arylalkylamine N-acetyltransferase (AA-NAT). Little is known about the contribution of the phosphodiesterase (PDE) system to antidepressant-induced alterations in pineal cAMP signaling and melatonin synthesis. There is a need to clarify the trajectory of cAMP and cGMP concentrations, their synthesis by cyclases, and degradation by PDEs to understand the role of cyclic mononucleotide signaling in the effect of chronic antidepressant therapy. Using quantitative real-time PCR and enzyme immunoassay, we systematically studied elements of intracellular signaling in the hippocampus of rats chronically treated with imipramine, fluoxetine, and amitriptyline and in the pineal gland of rats treated chronically with fluoxetine. In the hippocampus, we found chronic imipramine downregulated cAMP signaling with decreased cAMP, increased PDEs and decreased adenylate cyclase mRNA. In contrast, repeated fluoxetine and amitriptyline increased hippocampal cGMP signaling, with increased cGMP and decreased PDE mRNA. We conclude that in contrast to the assumption of antidepressant-mediated increases in cAMP levels, increased hippocampal cGMP signaling might underlie the efficacy of chronic antidepressant treatment. A follow up study using cultured embryonic rat hippocampal cells in vitro treated with the PDE type 5 inhibitor, sildenafil, demonstrated increased cAMP content following acute and chronic treatment, indicating either crosstalk between cAMP and cGMP pathways or a non-specific inhibitory effect of sildenafil on other PDEs. In the pineal gland, we found elevated melatonin synthesis with increased pineal AA-NAT mRNA and daytime plasma melatonin and downregulated cAMP signaling with increased PDE and unchanged AC pineal mRNA, and decreased pineal cAMP. We conclude that chronic fluoxetine increases daytime plasma melatonin and pineal AA-NAT mRNA despite downregulated pineal cAMP signaling.
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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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Kotikoski, Hanna. "Effects of nitric oxide donors and cyclic GMP on intraocular pressure and aqueous humor dynamics." Helsinki : University of Helsinki, 2003. http://ethesis.helsinki.fi/julkaisut/laa/biola/vk/kotikoski/.
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Lahiri, Tanaya. "Untangling the interactions| Structural basis of nitric oxide regulated cyclic-di-GMP metabolism in bacteria." Thesis, State University of New York at Stony Brook, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=3608293.
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Bacteria use numerous small molecules for cellular signaling. These primary and secondary messengers act within the cell to relay signal transduction and regulate distinct pathways. Among these the diatomic gas molecule nitric oxide (NO) and the nucleotide cyclic-di-GMP play central role in virulence, quorum sensing, and biofilm formation. In this dissertation, we have focused on H-NOX, a heme-nitric oxide/oxygen binding protein in the biofilm-dwelling bacterium Shewanella woodyi (Sw), which mediates NO-induced biofilm dispersal by modulating the activity of a dual-functioning diguanylate cyclase/phosphodiesterase enzyme that we have named HaCE, (H-NOX-associated cyclic-di-GMP enzyme). These enzymes tightly regulate the intracellular spatio-temporal concentrations of cyclic-di-GMP which is synthesized from 2 molecules of GTP by enzymes called Diguanylate Cyclases (DGC), and gets hydrolyzed to pGpG by enzymes called Phosphodiesterases (PDE), in turn controlling biofilm formation. Thus, H-NOX/HaCE represents a potential drug target for regulating biofilm formation. This is the first biophysical and structural study of an NO-bound SwH-NOX/SwHaCE complex. We have shown that SwH-NOX/SwHaCE associate in a α 2β2 (heterotetramer) stoichiometry. The SwH-NOX surface residues critical for binding to SwHaCE have been identified using NMR studies. Fluorescent quenching binding studies, co-immunoprecipitation and enzyme assays confirm this protein-protein interface and its importance for H-NOX/HaCE function.
Also described is the role of charged residues that are required for substrate binding and divalent metal ion coordination in the hydrolysis of cyclic-di-GMP by PDE domains found in bi-functional enzymes with an N-terminal DGC domain. Crystal structures of active PDE enzymes indicate a TIM-barrel type of fold. The catalytic pocket, situated towards the C-terminus, contains an unstructured loop, called "loop 6", which is conserved in this family of enzymes. The role of these conserved residues has been elucidated using mutational studies combined with biophysical studies and enzymatic analysis to show how structure affects enzyme function.
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Wang, Yuan. "Uroguanylin and cGMP signaling : a pathway for regulating epithelial cell renewal in the intestine /." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3036866.
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Clements, Peter James Mackenzie. "Molecular genetic investigations of rod cyclic GMP phosphodiesterase beta subunit in canine Generalised Progressive Retinal Atrophy." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307615.
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Cai, Yuming. "Molecular mechanism of nitric oxide-mediated regulation of intracellular cyclic-di-GMP in Pseudomonas aeruginosa biofilms." Thesis, University of Southampton, 2018. https://eprints.soton.ac.uk/419014/.
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Biofilms are defined as multicellular communities encased by a self-produced extracellular matrix. It is now well understood that most bacteria found in natural, clinical and industrial settings preferentially attach to surfaces or adhere to each other to grow as biofilms, causing serious problems due to their tolerance to conventional antibiotics. Previous studies have shown that low dose nitric oxide (NO) can trigger Pseudomonas aeruginosa biofilm dispersal by modulating the level of the intracellular secondary messenger cyclic dimeric guanosine monophosphate (c-di-GMP). Diguanylate cyclase (GGDEF motif) and phosphodiesterase (EAL/HD-GYP motif) activities are responsible for the synthesis and hydrolysis of c-di-GMP, respectively. Various sensor domains have been found to link environmental cues to modulation of GGDEF and EAL/HD-GYP activities, of which PAS and MHYT domains were of our interest due to their potentials to bind NO. In P. aeruginosa PAO1, a total of 14 proteins containing either PAS-DGC+/PDE or MHYT-DGC+/PDE were thought to be responsible for the NO-induced biofilm dispersal and were selected as our targets for investigation of their relationships between NO responses and biofilm phenotypes. To investigate the response of P. aeruginosa biofilms to NO, a range of NO donors were first tested for their efficacies, of which 250μM Spermine NONOate (S150) showed outstanding results in dispersing ~60% batch cultured PAO1 biofilms within only 2 hrs. S150 was further applied to some cystic fibrosis P. aeruginosa clinical isolates (CF PA) biofilms in vitro. The results showed it successfully triggered the dispersal of surface-attached biofilms formed by 14 out of 17 CF PA strains tested, implying its potential for wide applications in clinical settings. However, S150 failed to disperse the non-attached cell aggregates formed by 4 CF PA in aqueous medium, suggesting a different mechanism might exist for clinical isolates to defend the drugs that requires much more attention. In order to facilitate future studies, a quantifying index, Concentration Coefficient, was proposed to evaluate the degree of cell aggregation by simply using one stacked CLSM image for a certain planktonic culture. This index may become widely applied for researchers to compare the cell aggregates more easily and accurately. For mechanism studies, gene deletion was applied to 14 candidates for phenotypic analysis of mutants. An efficient gene knockout technique that minimizes cloning steps was developed and allowed for rapid generation of mutants. Phenotypic assays for 14 mutants suggested that PA0861 (RbdA) and PA5017 (DipA) play central roles in P. aeruginosa PAO1 for reducing intracellular c-di-GMP levels, enhancing swarming motility and triggering biofilm dispersal in response to NO. PA0847 and PA4601 (MorA) are involved in the regulation of biofilm dispersal and swarming/swimming motility, which were suspected to come from localized c-di-GMP pools due to their altered motility phenotypes without changes in intracellular levels. PA0285 and PA4959 (FimX) are responsible for both twitching and swimming motility, which contribute greatly to the 3D structures of the biofilms formed. Deleting either of these two proteins led to much enhanced biofilm dispersal upon NO treatment, providing insight that the deficiency in both Type IV pili and flagella functions may facilitate the elimination of P. aeruginosa biofilms. In summary, evaluation of a broad range of commercially available NO donors showed that S150 was the most effective in dispersing laboratory and clinical P. aeruginosa strains in our experimental system. By using S150 and gene-deleted mutants, our research suggested several models whereby different proteins may be responsible for either coarse-tuning on intracellular c-di-GMP pools or fine-tuning on localized ones in response to NO, which collaboratively regulate the motility and biofilm dispersal in PAO1. This work has enhanced our understanding of the NO-c-di-GMP-swarming-dispersal pathway and its regulators in PAO1, shedding light on NO signaling mechanisms and providing potential new targets for therapeutic drug design.
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Rascón, Ana. "Cyclic GMP-inhibited cAMP phosphodiesterase further characterization and identification of the phophorylation site for cAMP-dependent protein kinase /." Lund : Dept. of Medical and Physiological Chemistry, University of Lund, Sweden, 1992. http://books.google.com/books?id=KfFqAAAAMAAJ.
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Günay-Esiyok, Özlem [Verfasser], Friedrich W. [Gutachter] Herberg, Richard [Gutachter] Lucius, and Nishith [Gutachter] Gupta. "Cyclic GMP signaling during the lytic cycle of Toxoplasma gondii / Özlem Günay-Esiyok ; Gutachter: Friedrich W. Herberg, Richard Lucius, Nishith Gupta." Berlin : Humboldt-Universität zu Berlin, 2019. http://d-nb.info/1200406303/34.
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Juilfs, Dawn Marie. "Cyclic GMP-stimulated phosphodiesterase isoforms : distinct subcellular distribution, localization in mouse brain, and identification of a novel olfactory signaling pathway /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/6254.
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Yim, Seung-Ae. "Multimodal study of the interactions between the hepatitis B virus and the cyclic GMP-AMP synthase cGAS." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAJ041/document.
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Le virus de l’hépatite B (HBV) est l’agent étiologique de l’hépatite B. Ce virus est responsable d’hépatite chronique B, de cirrhose et de cancer du foie au niveau mondial. L’absence d’activation de la voie Interféron (IFN) suite à l’infection par HBV est encore mal comprise. Récemment, le senseur cellulaire cytosolic GMP-AMP synthase (cGAS) a été décrit comme un senseur efficace de DNA double brin possédant également une activité antivirale envers des virus à ADN et à ARN. Le but de mes travaux de thèse a été de contribuer à la compréhension des relations existants entre le HBV et cGAS, à des stades précoces et tardifs de l’infection HBV en utilisant des expériences de perte- et gain- de function ainsi que du profilage génomique des génes apparentés à cGAS dans un modéle cellulaire permissif au HBV. Mes travaux ont démontré (1) que cGAS exerce une forte activité antivirale envers le HBV incluant une réduction de la forme nucléaire du génome, le cccDNA; (2) alors que le rcDNA génomique nu est reconnu par la voie cGAS/STING et induit une réponse IFN efficace, la nucléocapside virale protège le DNA génomique viral et l’empêche d’être détecté par la réponse immunitaire innée; et (3) que l’infection par HBV diminue l’expression des acteurs de la voie cGAS-STING et des gènes impliqués dans la réponse immunitaire innée in vitro et in vivo. Ce dernier point met en lumière le rôle de cGAS dans un nouveau mécanisme d’échappement du HBV au système immunitaire inné dans les cellules hépatocytaires et dans ce mécanismeChronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic GMP-AMP synthase (cGAS) was identified as a DNA sensor. In this PhD work, we aimed to investigate the functional role of cGAS in sensing of HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss- and gain-of-function experiments combined with cGAS effector gene expression profiling in an HBV infection-susceptible cell culture model. Collectively, our data show that (1) the cGAS-STING pathway exhibits robust antiviral activity against HBV infection including reduction of viral cccDNA levels; (2) naked HBV genomic rcDNA is sensed in a cGAS-dependent manner whereas packaging of the viral genome during infection abolishes host cell recognition of viral nucleic acids; (3) HBV infection down-regulates the cGAS/STING pathway actors as well as innate immune effector gene expression in vitro and vivo. Overall, this work led to describing new aspects of the complex interaction between HBV and the DNA sensor cGAS in hepatocytes
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Grange, Robert Matthew Henry. "Targeting multidrug resistance proteins and C-type natriuretic peptide to optimise cyclic GMP signalling in cardiovascular disease." Thesis, Queen Mary, University of London, 2016. http://qmro.qmul.ac.uk/xmlui/handle/123456789/23786.
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Cyclic-3',5'-guanosine monophosphate (cGMP) is a fundamental intracellular signalling molecule that regulates vascular homeostasis through the tight control of vascular smooth muscle cell (VSMC) reactivity (i.e. vasoconstriction/relaxation) and proliferation. Aberrant VSMC growth and sustained vasoconstriction are hallmarks of cardiovascular disease, exemplified by pulmonary hypertension (PH). Multidrug resistance proteins (MRPs) are membrane bound transporters that facilitate cGMP cellular export thereby representing a potential mechanism that regulates intracellular cGMP-driven signalling. C-type natriuretic peptide (CNP) is an important vasoactive peptide released from the endothelium that maintains vascular homeostasis. CNP binds to natriuretic peptide receptor-B (NPR-B), generating cGMP, and NPR-C, which acts as a clearance receptor removing CNP from the circulation and a signalling pathway regulating vascular function via a cGMP-independent mechanism. Herein, I investigated two separate hypotheses: that MRPs play an important role in maintaining vascular homeostasis, and that endothelium-derived CNP and its cognate receptor, NPR-C, protects against the development of PH. The role of MRPs in regulating vascular homeostasis was investigated using organ bath pharmacology, human VSMC (hVSMC) proliferation and measuring mean arterial blood pressure (MABP) in conscious and anaesthetised mice. To investigate the role of endothelium-derived CNP and NPR-C in PH, male and female CNP and NPR-C knockout (KO) mice were used in two experimental models of PH: hypoxia plus Sugen5416 (SU5416) and bleomycin-induced. The severity of PH was measured using right ventricular systolic pressure (RVSP), MABP, right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. MRP inhibition resulted in concentration-dependent vasorelaxation of mouse aorta per se and increased the potency of cGMP-dependent vessel relaxation in response to activation of both particulate and soluble guanylate cyclases (pGC and sGC). MRP inhibition alone also caused concentration-dependent attenuation of hVSMC proliferation, and enhanced cGMP-mediated attenuation of hVSMC growth via pGC and sGC activation. MRP inhibition per se did not decrease MABP in either anaesthetised or telemeterised mice. However, MRP inhibition did dose-dependently enhance reductions in MABP due to pGC activation in anaesthetised mice. Deletion of endothelial cell-derived CNP (ecCNP KO) in male and female mice did not result in any significant differences in RVSP, RVH or pulmonary vascular remodelling between WT and KO in the hypoxia plus SU5416 model of PH. However, global deletion of NPR-C in both male and female mice caused a significant increase in RVH but not RVSP or vascular remodelling when compared to WT. Both male and female NPR-C KO mice developed significantly increased RVSP compared to WT in the bleomycin-induced model of PH. However, only females exhibited a significant increase in RVH and lung weight in addition to RVSP. In conclusion, MRP inhibition demonstrates potential therapeutic utility to treat cardiovascular diseases by potentiating the vasodilatory and VSMC antiproliferative actions of natriuretic peptides and nitric oxide. Endothelial cellderived CNP is not essential to host protection against PH, whereas its cognate receptor NPR-C demonstrates a cardioprotective capacity. NPR-C attenuates bleomycin-induced PH in both males and females, with a greater effect observed in females. Overall, NPR-C agonism could potentially be used to ameliorate the cardiac and vascular pathology associated with PH.
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Lusche, Daniel Felix. "Cyclic GMP in the development of the social amoeba Dictyostelium discoideum regulation of calcium homeostasis by cGMP. /." [S.l. : s.n.], 2004. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB11482073.
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Murphy, Caitlin Nolan. "The role of cyclic di-GMP in regulating type 3 fimbriae : a colonization factor of Klebsiella pneumonia." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/4703.
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Klebsiella pneumoniae is a Gram negative, enteric bacterium that frequently causes disease in immunocompromised individuals. These types of infections are often associated with the presence of indwelling medical devices, which provide a site for the organism to attach and subsequently form a biofilm. A key component in K. pneumoniae biofilm formation in vitro is type 3 fimbriae. The two main components of this project have been to determine if type 3 fimbriae are an in vivo virulence factor using a mouse model of catheter associated urinary tract infection (CAUTI) and to examine the mechanism by which the production of type 3 fimbriae are regulated.
Using a mouse model in which a silicone tube is implanted into the bladder of mice, mimicking the effects of catheterization, we have been able to show that type 3 fimbriae are required for colonization and persistence. Using different time points and conditions, we demonstrated that there are conditions when type 3 fimbriae alone are sufficient for colonization and other conditions where both type 1 and type 3 fimbriae have unique roles in colonization and persistence. Additionally, competition experiments showed that neither fimbrial mutant alone, or a double mutant in type 1 and type 3 fimbriae could compete with wildtype K. pneumoniae. In most animals, only wild-type bacteria were recovered by 24 hours post-inoculation. This work reinforced the role of type 1 fimbriae in pathogenesis and showed, for the first time, a role for type 3 fimbriae using an in vivo model.
Our early work has indicated that type 3 fimbriae are regulated at least in part by the intracellular levels of the secondary messenger molecule cyclic di-GMP. Downstream from the type 3 fimbrial operon a gene encoding a phosphodiesterase is present; the product of this gene breaks down cyclic di-GMP. In the absence of this gene the levels of type 3 fimbrial expression are increased. Also adjacent to the mrk operon is a two-gene operon containing the determinants we have named mrkH and mrkI. mrkH encodes a PilZ domain containing protein, which we have shown binds cyclic di-GMP. Using a transcriptional fusion we have shown that the mrk gene promoter is activated modestly in the presence of MrkH, but when MrkH and MrkI are both present the activity is increased 100-fold. This has lead to the hypothesis that MrkH and MrkI interact, which we have been able to demonstrate using copurification procedures. This interaction appears to occur in a cyclic di-GMP dependent manner with the resulting protein complex binding to the mrk promoter region and activating the expression of type 3 fimbriae.
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Fletcher, Madison Hill. "Synthesis of Non-Natural Cyclic Di-Nucleotides for the Investigation of Bacterial Signaling Pathways." Diss., Temple University Libraries, 2017. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/429284.
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ChemistryPh.D.
Humans navigate the world and interact with others through a complex series of communicative tools. We experience both internal and external stimuli, such as pangs of hunger or pain from an injury, and both verbal and nonverbal language. Bacteria also possess the ability to communicate, albeit in more discreet, yet no less complex ways. Bacteria rely on an incredibly diverse signaling system of triggers and responses in order to survive and to thrive. While we perceive language with our eyes and ears, bacteria employ a system of small molecules to relay both intra- and extracellular messages. They utilize this ability, known as quorum sensing to "talk" to their neighbors, express otherwise latent genetic characteristics, and to defend themselves against enemies. It has been suggested that this internal and external activity is linked, however, little is known about their interplay. This family of molecules, the cyclic di-nucleotides, which includes c-di-GMP and c-di-AMP, are critical to regulating bacterial processes such as motility, glucose remediation, and cell wall homeostasis. Their importance has spurred numerous investigations into their mechanism of action. Although found in very low concentrations within cells, they are capable of regulating a multitude of processes due to their ability to adopt variable conformations. To date, analog design by other groups has focused on the modification of the innate phosphate moiety as well as various substitutions or deletions at the 2'-position on the ribofuranose ring. However, these analogs have not been water soluble, limiting them to in vitro investigations only. We propose that by replacing the phosphate linkage entirely we can increase water solubility and have pursued a divergent total synthesis of various cyclic di-nucleotides featuring biomimetic linkages. Herein we address the methods we explored to optimize the synthesis of our three monomers, coupling strategies employed, the novel application of a Staudinger ligation to afford our abasic macrocycles and finally our progress towards implementing a bis-glycosylation strategy to install the desired nucleobase. We are able to efficiently provide large amounts of a di-amino, azide methyl ester, and N,O-substituted furanose monomers in no more than six steps from a common intermediate. These monomers are coupled and cyclized to form our four scaffolds, amide, carbamate, squaramide, and urea. Finally, we have begun to successfully implement our Brønsted acid mediated glycosylation strategy and understand its limitations. It is our goal to develop a general method to afford a diverse array of conformationally unique and water soluble cyclic di-nucleotide analogs with which to probe these essential bacterial signaling pathways.
Temple University--Theses
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Bedrunka, Patricia [Verfasser], and Peter L. [Akademischer Betreuer] Graumann. "The role of the second messenger cyclic di-GMP in Bacillus subtilis / Patricia Bedrunka ; Betreuer: Peter L. Graumann." Marburg : Philipps-Universität Marburg, 2018. http://d-nb.info/1153881454/34.
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Mallory, Katherine Louise. "Characterization of cyclic di-GMP binding by the sole Borrelia burgdorferi and Borrelia hermsii PilZ domain-containing proteins." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/532.
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Borrelia burgdorferi and Borrelia hermsii cause Lyme disease and relapsing fever, respectively. These spirochetes are maintained in an enzootic cycle, involving tick vectors and mammalian hosts. Differential gene expression is central in their survival in various environmental conditions. C-di-GMP has been demonstrated to be important in bacterial adaptation. Borrelia deletion mutant phenotypes have shown that c-di-GMP regulates motility, infectivity, and enzootic cycle progression. As the only known receptors encoded by Borrelia, PlzA and PlzC characterization is necessary in delineating c-di-GMP roles within the cell. In this study, biochemical, biophysical, and FRET methods demonstrated that these proteins exhibit a structural rearrangement when binding c-di-GMP likely significant to downstream activities. Substitution of a highly conserved residue within PlzA altered the structure and charge of the PilZ domain, leading to abolished binding. PlzA and PlzC functionality studies are vital to discover mechanisms of c-di-GMP-mediated regulation of motility and host invasion by the Borrelia.