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1
Davis, Alison Jane. "Kinase-anchoring proteins and the intracellular targeting of cyclic adenosine monophosphate (cAMP)-dependent protein kinase". Thesis, University of Leicester, 2007. http://hdl.handle.net/2381/29960.
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Kinase anchoring proteins (AKAPs) are a family of structurally diverse multidomain proteins classified solely by their ability to bind cAMP-dependent protein kinase/protein kinase A (PKA). We show that the prototypic AKAP, AKAP79, targets PKA to the plasma membrane. This subcellular localisation is mediated via interactions between basic regions on AKAP79 and acidic phospholipids in the plasma membrane. We show that while PKA and protein kinase C (PKC)-dependent phosphorylation of AKAP79 targeting residues has no effect on its subcellular localization, Gq-coupled receptor-driven depletion of the lipid anchor PtdIns(4,5)P2 causes release of AKAP79 from the membrane. Receptor-mediated regulation of AKAP membrane binding may be an important feedback mechanism controlling the degree of access that AKAP-bound enzymes have to membrane-associated substrates. Real-time confocal imaging failed to show significant redistribution of AKAP79 following Gq-coupled receptor activation, which may reflect only limited short-range movements.;In addition to regulated association with membrane, we also show through co-immunoprecipitation studies a direct interaction between AKAP79 and all three members of the inwardly rectifying potassium channel Kir2 family. Using a combination of deletion analysis and structural modelling we show that AKAP79 binds the Kir2 family via a novel binding motif located on the C terminus of the ion channel and not via a leucine zipper as has been reported for other AKAP-channel interactions. Interestingly, we also show using a PKA regulatory subunit overlay assays that this region of Kir2.1 is capable of isolating other, potentially novel, AKAPs from rat brain and heart homogenates.;Finally, we use whole-cell patch clamp analysis to demonstrate that cAMP-dependent modulation of Kir2.1 channel activity is significantly enhanced in the presence of AKAP79, thus confirming an important role for AKAP79 in targeting PKA to key channel phosphorylation sites.
2
Yang, Zhaogang. "ADENOSINE RECEPTOR MEDIATED PROTEIN KINASE C ACTIVATION IN THE HEART". The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338320892.
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3
Kong, Cheng-Te. "Mechanism of the Adenosine 3',5'-Monophosphate Dependent Protein Kinase". Thesis, North Texas State University, 1988. https://digital.library.unt.edu/ark:/67531/metadc330934/.
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Isotope partitioning experiments were carried out with the adenosine 3',5'-monophosphate-dependent protein kinase catalytic subunit (cAPK) from bovine hearts to obtain information on the order of addition of reactants and the relative rates of reactant release from enzyme compared to the catalytic step(s). A value of 100% trapping for both ErMgATP-[γ-32P] and E:3H-Serpeptide at low Mgf indicates that MgATP and Serpeptide dissociate slowly from the enzyme compared to the catalytic step(s). The K_Serpeptide for MgATP trapping is 17 μM, while the K_MgATP for Serpeptide trapping is 0.58 mM. The latter data indicate that the off-rate for MgATP from the E:MgATP complex is 14 s^-1 while that for Serpeptide from the E: Serpeptide complex is 64 s^-1. At high Mg^, 100% trapping is obtained for the E:MgATP-[γ-32P] complex but only 40% is obtained for the E:Serpeptide complex. Thus, the off-rate for Serpeptide from the E:MgATP:Serpeptide complex becomes significant at high Mg_f. Data suggest a random mechanism in which MgATP is sticky. The V for the cAPK reaction increases 1.5-1.7 fold in the presence of the R_II in the presence of saturating cAMP at a stoichiometry of R:C of 1:1. No change is obtained with the type-I complex under these conditions. At higher ratio of R:C (up to 100) no further change is observed with the type-II complex but inhibition by the type-I R_2(cAMP)_4 complex competitive vs. Serpeptide is observed. The activiation observed in the presence type-II R_2(cAMP)_4 effects neither the K_m for Serpeptide nor the K_m for MgATP. Both the activating affect of the type-II complex and the inhibitory effect of the type-I complex are dependent on the Mg_f with more type-II activation obtained the higher the Mg_f and more type-I complex required for inhibition the higher the Mg_f. The activation and inhibition are discussed in terms of the mechanism of the protein kinase.
4
Dahlstedt, Anders. "Intracellular mechanisms of skeletal muscle fatigue : role of creatine kinase /". Stockholm : [A. Dahlstedt], 2001. http://diss.kib.ki.se/2001/91-631-1673-1/.
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Wang, Hui. "Geminivirus AL2 and L2 proteins interact with and inactivate adenosine kinase". Connect to this title online, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1078773653.
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Thesis (Ph. D.)--Ohio State University, 2004.Title from first page of PDF file. Document formatted into pages; contains xvii, 201 p.; also includes graphics (some col.) Includes bibliographical references (p. 171-201). Available online via OhioLINK's ETD Center
6
Willems, Laura E., i n/a. "Adenosine and Ischaemia in Young To Aged Hearts". Griffith University. School of Medical Science, 2006. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20061011.163451.
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Ischaemic heart disease is a major contributor to premature death and heart failure in the Westernised world. Ischaemic injury within the heart may be beneficially modulated by the nucleoside adenosine. Derived from catabolism of ATP, adenosine was initially known as a potent bradycardic and hypotensive agent. However, more recently the protective function of adenosine has been investigated. The protective effects of adenosine are mediated via activation of adenosine receptors: A1, A2A, A2B, and A3 receptors. Activation of these potentially protective (or retaliatory) adenosine receptors hinges upon accumulation of adenosine during ischaemia-reperfusion. This Thesis examines the role and mechanisms of adenosine mediated cardioprotection in young and aged hearts, exploring endogenous and exogenous adenosine receptor activation, genetic manipulation of A1 receptors and adenosine deaminase and pharmacological manipulation of adenosine metabolism. The effects of age on ischaemic responses and adenosine handling and protection are also assessed. The core approach to assess each of the above issues involved the Langendorff isolated mouse heart preparation. Experiments within Chapter 3 focuses on the contractile effects of adenosine receptors in normoxic hearts. This study indicates A2A receptors have no direct effect on contractility, while adenosine exerts positive inotropy independently of coronary flow and perfusion pressure (i.e. Independent of the Gregg phenomenon). In addition, investigations in genetically modified hearts hint at positive inotropy in response to A1 receptors. Since the latter is only evidenced in modified lines, it is possible A1-mediated inotropy may be abnormal or supraphysiological. In Chapter 4 the impact of genetic 'deletion' of A1ARs and/or adenosine deaminase (ADA) on intrinsic tolerance to ischaemia were studied. Data demonstrate that genetic deletion of A1 receptors significantly limits the ability of the mouse myocardium to withstand injury during ischaemic insult. Thus, providing strong support for a role of A1ARs in determining intrinsic tolerance to ischaemia-reperfusion. ADA KO mice confirm protection afforded by endogenous adenosine and the notion of adenosine metabolism modification as a protective strategy. Interestingly, effects of A1AR KO differ from A1AR overexpression or A1AR agonism in that the latter decrease contractile diastolic dysfunction while A1AR KO selectively increase systolic dysfunction and increase oncosis without altering diastolic injury. This challenges current dogma regarding the action of A1 adenosine receptors on ischaemic injury. In Chapter 5 the effects of adenosine metabolism inhibition (via adenosine deaminase (ADA) and adenosine kinase (AK) inhibitors) were studied. Inhibition of adenosine deaminase with the drug EHNA, and adenosine phosphorylation with iodotubercidin significantly protected mouse hearts from ischaemia-reperfusion, reducing contractile dysfunction and cardiac enzyme efflux. However, inhibitors failed to improve the outcome of the aged myocardium. 8-SPT and 5-HD reduced the protective effects of EHNA and iodotubercidin demonstrating thus; cardioprotection via ADA and AK appears to rely on adenosine receptor activation and involves a mitoK ATP dependent mechanism. Since aging is of considerable importance with regard to outcomes of ischaemic heart disease, experiments in Chapter 6 focused on effects of aging (and gender) on cardiovascular function and injury during ischaemia-reperfusion. In assessing post ischaemic outcomes in hearts from young adult (2-4 months), mature adult (8 months), middle aged (12 months), aged (18 months) and senescent (24-28 months) C57/BL/6J mice, data reveal a substantial age-related decline in ischaemic tolerance (which appears selective for myocardial vs. vascular injury). The decline in ischaemic tolerance is expressed primarily within the initial 12 months in both males and females with relatively little further decline with continued aging. There is evidence of a modest improvement in tolerance in senescence vs. aged hearts possibly reflecting selection of a protected phenotype in senescent populations. In addition, mature and middle-aged female hearts showed improved tolerance to ischaemia-reperfusion compared to males, supporting a role for hormonal changes. Effects of aging and purine metabolism were studied in Chapter 7. Data suggest impaired tolerance to ischaemia-reperfusion in older hearts may stem in part from shifts in myocardial purine catabolism. Data reveal reduced accumulation of salvageable and cardioprotective adenosine and enhanced accumulation of poorly salvaged (and potentially injurious) hypoxanthine and xanthine. These changes may potentially predispose the aged myocardium to ischaemic injury and radical generation via the xanthine oxidase reaction. The final data Chapter of this Thesis describes preliminary data regarding aging, signalling and adenosine mediated protection. It was found that protein kinase C (PKC) and A1 receptors mediate protection in young hearts and also that A1 receptors appear to mediate protection via a PKC LindependentLi signalling cascade. In addition, experiments in aged hearts (attempting to elucidate mechanisms behind impaired adenosinergic protection with age) suggest a PKC-independent A1-mediated protection path may be preserved with aging, since A1 receptors continue to offer some protection while PKC activation does not. It is possible the failure of exogenous adenosine to offer protection in aged hearts may result from a lesion at or downstream of PKC.
7
Willems, Laura E. "Adenosine and Ischaemia in Young To Aged Hearts". Thesis, Griffith University, 2006. http://hdl.handle.net/10072/365196.
Pełny tekst źródłaStreszczenie:
Ischaemic heart disease is a major contributor to premature death and heart failure in the Westernised world. Ischaemic injury within the heart may be beneficially modulated by the nucleoside adenosine. Derived from catabolism of ATP, adenosine was initially known as a potent bradycardic and hypotensive agent. However, more recently the protective function of adenosine has been investigated. The protective effects of adenosine are mediated via activation of adenosine receptors: A1, A2A, A2B, and A3 receptors. Activation of these potentially protective (or retaliatory) adenosine receptors hinges upon accumulation of adenosine during ischaemia-reperfusion. This Thesis examines the role and mechanisms of adenosine mediated cardioprotection in young and aged hearts, exploring endogenous and exogenous adenosine receptor activation, genetic manipulation of A1 receptors and adenosine deaminase and pharmacological manipulation of adenosine metabolism. The effects of age on ischaemic responses and adenosine handling and protection are also assessed. The core approach to assess each of the above issues involved the Langendorff isolated mouse heart preparation. Experiments within Chapter 3 focuses on the contractile effects of adenosine receptors in normoxic hearts. This study indicates A2A receptors have no direct effect on contractility, while adenosine exerts positive inotropy independently of coronary flow and perfusion pressure (i.e. Independent of the Gregg phenomenon). In addition, investigations in genetically modified hearts hint at positive inotropy in response to A1 receptors. Since the latter is only evidenced in modified lines, it is possible A1-mediated inotropy may be abnormal or supraphysiological. In Chapter 4 the impact of genetic 'deletion' of A1ARs and/or adenosine deaminase (ADA) on intrinsic tolerance to ischaemia were studied. Data demonstrate that genetic deletion of A1 receptors significantly limits the ability of the mouse myocardium to withstand injury during ischaemic insult. Thus, providing strong support for a role of A1ARs in determining intrinsic tolerance to ischaemia-reperfusion. ADA KO mice confirm protection afforded by endogenous adenosine and the notion of adenosine metabolism modification as a protective strategy. Interestingly, effects of A1AR KO differ from A1AR overexpression or A1AR agonism in that the latter decrease contractile diastolic dysfunction while A1AR KO selectively increase systolic dysfunction and increase oncosis without altering diastolic injury. This challenges current dogma regarding the action of A1 adenosine receptors on ischaemic injury. In Chapter 5 the effects of adenosine metabolism inhibition (via adenosine deaminase (ADA) and adenosine kinase (AK) inhibitors) were studied. Inhibition of adenosine deaminase with the drug EHNA, and adenosine phosphorylation with iodotubercidin significantly protected mouse hearts from ischaemia-reperfusion, reducing contractile dysfunction and cardiac enzyme efflux. However, inhibitors failed to improve the outcome of the aged myocardium. 8-SPT and 5-HD reduced the protective effects of EHNA and iodotubercidin demonstrating thus; cardioprotection via ADA and AK appears to rely on adenosine receptor activation and involves a mitoK ATP dependent mechanism. Since aging is of considerable importance with regard to outcomes of ischaemic heart disease, experiments in Chapter 6 focused on effects of aging (and gender) on cardiovascular function and injury during ischaemia-reperfusion. In assessing post ischaemic outcomes in hearts from young adult (2-4 months), mature adult (8 months), middle aged (12 months), aged (18 months) and senescent (24-28 months) C57/BL/6J mice, data reveal a substantial age-related decline in ischaemic tolerance (which appears selective for myocardial vs. vascular injury). The decline in ischaemic tolerance is expressed primarily within the initial 12 months in both males and females with relatively little further decline with continued aging. There is evidence of a modest improvement in tolerance in senescence vs. aged hearts possibly reflecting selection of a protected phenotype in senescent populations. In addition, mature and middle-aged female hearts showed improved tolerance to ischaemia-reperfusion compared to males, supporting a role for hormonal changes. Effects of aging and purine metabolism were studied in Chapter 7. Data suggest impaired tolerance to ischaemia-reperfusion in older hearts may stem in part from shifts in myocardial purine catabolism. Data reveal reduced accumulation of salvageable and cardioprotective adenosine and enhanced accumulation of poorly salvaged (and potentially injurious) hypoxanthine and xanthine. These changes may potentially predispose the aged myocardium to ischaemic injury and radical generation via the xanthine oxidase reaction. The final data Chapter of this Thesis describes preliminary data regarding aging, signalling and adenosine mediated protection. It was found that protein kinase C (PKC) and A1 receptors mediate protection in young hearts and also that A1 receptors appear to mediate protection via a PKC LindependentLi signalling cascade. In addition, experiments in aged hearts (attempting to elucidate mechanisms behind impaired adenosinergic protection with age) suggest a PKC-independent A1-mediated protection path may be preserved with aging, since A1 receptors continue to offer some protection while PKC activation does not. It is possible the failure of exogenous adenosine to offer protection in aged hearts may result from a lesion at or downstream of PKC.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Full Text
8
Gaskin, F. Spencer. "Mechanisms of adenosine monophosphate-activated protein kinase-induced preconditioning in ischemia/reperfusion". Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4805.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2007.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.
9
Fedele, Denise E. "Adenosine Kinase: a key regulator of hippocampal inhibition, seizure susceptibility, and neuronal development /". Zürich : ETH, 2006. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=16609.
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10
Shah, Mittal. "The role of 5' adenosine monophosphate-activated protein kinase (AMPK) in bone physiology". Thesis, Royal Veterinary College (University of London), 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.559073.
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Raslan, Zaher. "Characterisation of cyclic adenosine monophosphate/protein kinase A signalling networks in blood platelets". Thesis, University of Hull, 2012. http://hydra.hull.ac.uk/resources/hull:6431.
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Platelet activation is a critical physiological event, whose main role is to prevent excessive blood loss and repair vessel wall injuries. However, platelet activation must be controlled to prevent unwanted and exaggerated responses leading to the occlusion of the blood vessel. The endothelial-derived inhibitors prostacyclin (PGI2) and nitric oxide (NO) are known to play a critical role in the control of platelet activity, although the mechanism underlying their actions remains unclear beyond the triggering of cyclic nucleotides signaling pathways. The aim of this study was to improve our understanding of platelet regulation by cAMP signaling networks. We observed differences in cAMP signaling depending on the agonists used. Using phosphorylation of PKA substrates as a marker of PKA activity, it was observed that PKA substrates were phosphorylated and dephosphorylated at different time points in a unique temporal pattern. Consistent with this observation we found that individual PKA isoforms, PKA I and II, were localized in distinct subcellular compartments, with PKA I being identified as a lipid raft protein. Our experimental data suggest that the localization of PKA I to lipid rafts is mediated by interaction with A-kinase anchoring proteins (AKAPs). Additionally, PKA signaling events were reversed when potential PKA type I interactions with AKAPs were disrupted with competitive peptides. Using this approach we found that the redistribution of PKA I to lipid rafts facilitated the phosphorylation of GPIbβ and the inhibition of von-Willebrand factor-mediated aggregation. Our data also demonstrated for the first time that the chemical disruption of lipid rafts increased platelet sensitivity to PGI₂, through increased cAMP production and PKA activity. The mechanism by which this occurs may involve sequestering a population of adenylyl cyclase 5/6 to a location remote from Gαs. In conclusion, data presented in this thesis suggest differential roles of PKA subtypes in the regulation of platelet activity. This involves, at least in part, the localisation of PKA I into specific subcellular compartments through an interaction with AKAPs. The potential presence of PKAII-AKAP interactions and the identification of specific AKAPs will be the main aim of future work.
12
Nadal, Magriñà Laura. "Muscarinic, adenosine and tropomyosin-related kinase B receptors modulate the neuromuscular developmental synapse elimination process". Doctoral thesis, Universitat Rovira i Virgili, 2017. http://hdl.handle.net/10803/441749.
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El desenvolupament del sistema nerviós perifèric implica una inicial exuberant producció de neurones i, una posterior reducció dependent de l'activitat del nombre de sinapsis de les unions neuromusculars (NMJ). Aquest procés s’anomena eliminació sinàptica. Al final de la primera setmana postnatal, cada fibra muscular està innervada per una sola motoneurona. Els receptors muscarínics d’acetilcolina (mAChR), els receptors d’adenosina (AR) i el receptor cinasa de tropomiosina B (TrkB) podrien permetre la competició entre terminals nerviosos durant el procés d’eliminació sinàptica mitjançant la modulació de l’alliberament d’acetilcolina. En aquesta tesi s’ha investigat, mitjançant microscòpia confocal i un anàlisi morfològic quantitatiu, el paper dels receptors mAChRs (M1, M2 i M4), dels AR (A1 i A2A) i del receptor TrkB en el procés d’eliminació en el desenvolupament de la NMJ.
Els resultats mostren que els receptors mAChRs, AR i el receptor TrkB promouen una desconnexió axonal al principi de la segona setmana postnatal independentment de la maduració dels receptors d’acetilcolina postsinàptics. En resum, els receptors mAChRs, AR i el receptor TrkB endarrereixen el procés d’eliminació sinàptica a P7 però l’acceleren a P9. Pel que fa la cooperació d’aquests receptors, M4 produeix un efecte oclusiu sobre M1 i un efecte additiu sobre a P7. La cooperació entre els receptors M1, A1 i A2A promou la pèrdua axonal a P9, mentre que, l’efecte de M2 és independent dels altres receptors. El M1 i TrkB treballen junts per incrementar la pèrdua axonal a P9 independentment dels receptors M2 i TrkB.
En conclusió, l’eliminació sinàptica postnatal és regulada per un mecanisme que depèn de varis receptors, involucrant la cooperació dels diferents subtipus de receptors muscarínics, d’adenosina i el receptor TrkB, els quals garanteixen la monoinnervació de les sinapsis neuromusculars al final del procés.El desarrollo del sistema nervioso periférico implica una inicial exuberante producción de neuronas y, una posterior reducción dependiente de actividad del número de sinapsis en las uniones neuromusculares (NMJ). Este proceso se denomina eliminación sináptica. Al final de la segunda semana postnatal, cada fibra muscular esta inervadas por una solo motoneurona. Los receptores muscarínicos de acetilcolina (mAChR), los receptores de adenosina (AR) y el receptor quinasa de tropomiosina B (TrkB) podrían permitir la competición entre los terminales nerviosos durante el proceso de eliminación sináptica mediante la modulación en la liberación de acetilcolina. En esta tesis se ha investigado, mediante microscopía confocal y un análisis morfológico cuantitativo, el papel de los receptores mAChRs (M1, M2 y M4), de los receptores de adenosina (A1 y A2A) y del receptor TrkB en el del proceso de eliminación en el desarrollo de la NMJ. Los resultados muestran que los receptores mAChRs, AR y el receptor TrkB promueven una desconexión axonal al inicio de la segunda semana postnatal independientemente de la maduración de los receptores de acetilcolina postsinápticos. En resumen, los receptores mAChRs, AR y el receptor TrkB retrasan el proceso de eliminación sináptica en P7 pero lo aceleran en P9. En la cooperación de estos receptores, se ha demostrado que M4 produce un efecto oclusivo sobre M1 y aditivo sobre A1 en P7. La cooperación entre M1, A1 y A2A promueve la pérdida axonal en P9, mientras que M2 es independiente de los otros receptores. M1 y TrkB cooperan para incrementar la pérdida axonal en P9 independientemente de M2 y TrkB. En conclusión, la eliminación sináptica postnatal está regulada por un mecanismo que depende de varios receptores, involucrando la cooperación de diferentes subtipos de receptores muscarínicos, de adenosina y el receptor TrkB, los cuales garantizan la monoinnervación de las sinapsis neuromusculares al final del proceso. saludable.
The development of the peripheral nervous system involves an initially exuberant production of neurons and a subsequent activity-dependent reduction in the number of synapses at the neuromuscular junctions (NMJ). This process is called synaptic elimination. At the end of the first postnatal week, each muscle fiber is innervated by a single motoneuron. Muscarinic acetylcholine receptors (mAChR), adenosine receptors (AR) and the tropomyosin-related kinase B (TrkB) receptor may allow the direct competition between nerve endings during synapse elimination through the modulation of acetylcholine release. Here, it has been investigated by confocal microscopy and quantitative morphological analysis the involvement of the individual and synergic or oclusive effect of M1-, M2- and M4-subtypes of mAChRs, A1 and A2A of ARs and TrkB in the control of the axonal elimination in developing NMJ. The results show that mAChRs, ARs and TrkB promote axonal disconnection at the beginning of the second postnatal week without affecting the postsynaptic maturation of the nicotinic receptor cluster. In summary, mAChRs, ARs and TrkB delay axonal loss at P7 but accelerate it at P9. In terms of receptor cooperation, M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway at P7. The cooperation between M1, A1 and A2A receptors promotes axonal loss at P9, whereas the effect of M2 is independent of the other receptors. M1 and TrkB receptors work together to increase axonal loss rate at P9 but the effect of M2 is largely independent of the TrkB receptor. In conclusion, postnatal synapse elimination is a regulated multireceptor mechanism involving the cooperation of several muscarinic, adenosine and TrkB receptor subtypes that guarantees the monoinnervation of the neuromuscular synapses in the end of the process.
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Qiu, Jiehua [Verfasser], i Ulrich [Akademischer Betreuer] Pohl. "5’-adenosine monophosphate-activated protein kinase (AMPK) modulates myoendothelial junctions / Jiehua Qiu ; Betreuer: Ulrich Pohl". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/1155097319/34.
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Murphy, Cody. "Transregulation of Cardiac Ischaemic Tolerance and Stress Kinase Signalling by A1 Adenosine and ¿-Opioid Receptors". Thesis, Griffith University, 2018. http://hdl.handle.net/10072/382690.
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Protecting hearts from damage sustained during myocardial ischaemia and reperfusion remains an ongoing challenge. Despite successful findings with animal models, the task of trialling and effectively translating experimental findings from laboratory to patients has proven difficult, therefore treatments and clinical therapies are still urgently needed to protect the heart and improve cardiac functional outcome. Previous research has implicated adenosine and opioid receptor participation in the protective response preceding or following myocardial infarction, with evidence of potential cross-talk between receptors. This project aimed to investigate whether A1 adenosine (A1AR) and δ-opioid receptor (δ-OR) dependent cytoprotection and prosurvival kinase activation in murine hearts share common dependencies on „cross-talk‟ between both G-protein coupled receptor (GPCR) sub-types and whether these responses involve a common Matrix Metalloproteinase (MMP) and Epidermal Growth Factor Receptor (EGFR) dependent signalling pathway. This was achieved via four inter-related in vitro studies.
Study 1: Healthy mouse hearts were cannulated in a Langendorff mode enabling the coronary circulation to be perfused. Hearts were subjected to 25 minutes of global (zero-flow) ischaemia followed by 45 minutes of aerobic reperfusion. The groups investigated included untreated control hearts, hearts receiving the selective A1AR agonist CCPA (± DPCPX, a selective A1AR antagonist, or BNTX, a selective δ-OR antagonist) and hearts receiving the selective δ-OR agonist BW373U86 (± DPCPX, a selective A1AR antagonist, or BNTX, a selective δ-OR antagonist). Agonists were applied 5 minutes pre-ischaemia and the antagonists were administered 10 minutes prior to agonist treatment. Cardiac functional outcomes were assessed via changes in coronary flow, left ventricular (LV) end diastolic pressure and pressure development, and ±dP/dt (± differentials of pressure change with time – indexing lusitropic and inotropic state). Cell death outcomes were also assessed via lactate dehydrogenase (LDH) release.
Treatment with either the selective A1AR agonist CCPA, or the selective δ-OR agonist BW373U86, significantly reduced (p≤0.0001 vs. CTRL) LV end-diastolic pressure following ischaemia/reperfusion. Recovery of LV developed pressure (LVDP) was significantly increased following A1AR activation via CCPA (p≤0.0001 vs. CTRL) or δ-OR activation via BW373U86 (p≤0.001 vs. CTRL). Ventricular contractility
(+dP/dt) and relaxation (-dP/dt) were also significantly improved with both CCPA (p≤0.0001 vs. CTRL, p≤0.01 vs. CTRL) and BW373U86 (p≤0.001 vs. CTRL, p≤0.001 vs. CTRL). Treatment with CCPA, but not BW373U86, significantly improved the recovery of coronary flow rate at the termination of reperfusion (p≤0.01 vs. CTRL).
LDH release (corresponding to cell death) was significantly reduced by both CCPA and BW373U86 (p<0.05 vs. CTRL, p<0.05 vs. CTRL). A1AR or δ-OR inhibition, via the selective antagonists DPCPX and BNTX respectively (applied alone), did not significantly affect the recovery of functional outcomes or cell death relative to control. These results show that cardioprotection against ischaemic injury is induced with activation of A1ARs and δ-ORs, and that endogenous levels of receptor agonists may not be sufficient to induce this response. Protection with CCPA was abolished via cotreatment with either the selective A1AR antagonist DPCPX or the selective δ-OR antagonist BNTX. Conversely protection with BW373U86 administration was negated by co-treatment with either BNTX or DPCPX. This reveals that A1AR dependent cardioprotection is reliant on the activation of δ-ORs, and δ-OR mediated protection is dependent on A1AR activity, confirming essential cross-talk.
Study 2: Perfused hearts from study 1 were snap-frozen in liquid nitrogen following the termination of reperfusion. Hearts were homogenised and fractioned to yield cytosolic proteins. Total and phosphorylated levels of Erk1/2 and Akt were subsequently assessed via western immunoblot. Both A1AR and δ-OR stimulation via CCPA and BW373U86 (respectively) did not significantly influence Erk1/2 phosphorylation. Akt phosphorylation, on the other hand, was increased by both CCPA and BW373U86; although only the latter effect achieved statistical significance (p<0.01 vs. CTRL). The A1AR antagonist DPCPX had minimal effect on Erk1/2 and Akt phosphorylation when applied alone. Alternatively inhibition of the δ-OR via BNTX, applied alone, was found to increase both Akt and Erk1/2 phosphorylation, a response in conflict with the existing literature.
Co-treatment with the A1AR antagonist DPCPX or the δ-OR antagonist BNTX did not significantly influence Erk1/2 signalling compared to controls. Alternatively Akt phosphorylation was reduced by ~50% relative to control when hearts were co-treated with DPCPX or BNTX applied in conjunction with either the A1AR agonist CCPA or the δ-OR agonist BW373U86. These results imply that both A1ARs and δ-ORs together are necessary to induce protective Akt signalling during ischaemia/reperfusion with either receptor agonist.
Study 3: To assess the roles of EGFRs and MMPs in A1AR and δ-OR responses, agonist studies with CCPA and BW373U86 were repeated with co-treatment with the EGFR antagonist AG1478 or the MMP inhibitor GM6001. Functional and cytoprotective outcomes were assessed in perfused hearts subjected to ischaemiareperfusion. The protective response observed with either A1AR and δ-OR stimulation was negated via co-treatment with either AG1478 or GM6001. A1AR and δ-OR dependent recovery of end diastolic pressure, LV developed pressure, +dP/dt and -dP/dt were all repressed via EGFR or MMP inhibition. Moreover, the cytoprotective response conferred by δ-OR activation was completely abolished via co-treatment with AG1478 or GM6001, providing evidence that adenosinergic and opioidergic protection within the myocardium involves an EGFR and MMP dependent pathway.
Study 4: Western blot analysis was used to assess changes in Erk1/2 and Akt expression and phospho-regulation in hearts treated with CCPA or BW373U86 in the presence of AG1478 or GM6001. Due to time constraints, data collected previously in our lab was used in this research; therefore the effect of EGFR and MMP inhibition on A1AR dependent Erk1/2 and Akt signalling was assessed in whole heart rather than cytosolic fractions. In these hearts, administration of CCPA significantly elevated both
Erk1/2 and Akt phosphorylation, a response negated via co-treatment with either
AG1478 or GM6001 (p≤ 0.05 vs. CCPA). Infusion of the selective δ-OR agonist BW373U86 did not significantly alter Erk/1/2 expression or phosphorylation in cytosolic fractions. Despite this, co-treatment with AG1478 reduced Erk1/2 phosphorylation by ~50% compared to the agonist alone (p ≤ 0.05 vs. BW373U86), suggesting an EGFR dependent mechanism. Surprisingly co-treatment with GM6001 did not significantly influence δ-OR mediated Erk1/2 activity. Akt phosphorylation was increased by more than 60% with BW373U86 (p≤0.05 vs. CTRL) and this response was abolished via treatment with the selective EGFR antagonist AG1478 or the selective MMP inhibitor GM6001. This provides further evidence that adenosinergic and opioidergic protective signalling during ischaemia-reperfusion requires the activity of
EGFRs and MMPs.
Conclusions: As a whole, the present study confirms an essential interaction between ARs and ORs in the heart, with kinase signalling and tissue protection via either A1ARs or -ORs exhibiting common and essential dependencies on activity of both receptors. The basis of this intriguing response remains unclear, although we show that both receptors engage distal kinases (and cardioprotection) in an MMP/EGFR dependent manner, adding an additional level to this novel cross-talk. This research provides further insight into cardioprotective receptor interactions in the heart, potentially leading to the development of new pharmacotherapeutics and improved outcomes for cardiovascular disease patients. Further research is needed to clarify the mechanism behind adenosinergic and opioidergic cross-talk and cardioprotection, potentially exploring membrane signalling, temporal expression of kinases, existence of A1AR/δ-OR dimers/oligomers, and concepts such as dual agonism and potential signalling thresholds for protection.Thesis (Masters)
Master of Medical Research (MMedRes)
School of Medical Science
Griffith Health
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Ng, Fung-kei, i 吳鋒奇. "The influence of a protein kinase A inhibitor on interstitial adenosine of muscle at rest and during contraction". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B45830708.
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Goto, Ayumi. "Effects of acute heat stress on glucose metabolism and 5' adenosine monophosphate-activated protein kinase in skeletal muscle". Kyoto University, 2016. http://hdl.handle.net/2433/215632.
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Kyoto University (京都大学)0048
新制・課程博士
博士(人間・環境学)
甲第19806号
人博第777号
新制||人||187(附属図書館)
27||人博||777(吉田南総合図書館)
32842
京都大学大学院人間・環境学研究科共生人間学専攻
(主査)教授 林 達也, 教授 森谷 敏夫, 教授 石原 昭彦
学位規則第4条第1項該当
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Qamar, Raheel. "Dependence of the Kinetic Mechanism of Adenosine 3',5'-Monophosphate Dependent Protein Kinase Catalytic Subunit in the Direction of Magnesium Adenosine 5'-Diphosphate Phosphorylation on pH and the Concentration of Free Magnesium Ions". Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc277956/.
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To define the overall kinetic and chemical mechanism of adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit, the mechanism in the direction of MgADP phosphorylation was determined, using studies of initial velocity in the absence and presence of dead-end inhibitors. The kinetic mechanism was determined as a function of uncomplexed Mg^2+ (Mg_f) at pH 7.2 and as a function of pH at low (0.5 mM) Mg_f. At pH 7.2 data are consistent with a random kinetic mechanism in the direction of MgADP phosphorylation with both pathways allowed: the pathway in which MgADP binds to enzyme prior to phosphorylated peptide (PSP) and that in which PSP binds before MgADP. One or the other pathway predominates, depending on Mg_f concentration. At 0.5 mM Mg_f, the mechanism is steady-state ordered with the pathway where PSP binds first preferred; at 10 mM Mg_f, the mechanism is equilibrium ordered, and the pathway in which MgADP binds first preferred. This change in mechanism to equilibrium ordered is due to an increase in affinity of enzyme for MgADP and a decrease in affinity for PSP. There is also a pH-dependent change in mechanism at 0.5 mM Mg_f. At pH 6 the mechanism is equilibrium ordered with the pathway where PSP binds first preferred. At pH 7.6 the mechanism is ordered with MgADP binding first. The log V/E_t vs. pH profile is pH-independent, suggesting only the correctly protonated form of each substrate binds to enzyme. The log V/K_MgADP vs. PH profile gives a pK of 7, likely that of a general acid, which must be protonated for activity. The pK_iPSP vs. pH profile gives a pK of 6.5, likely reflecting the peptide phosphoryl group, which must be unprotonated for activity.
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Barnes, Brian R. "AMP-activated protein kinase : the connection between exercise and type II diabetes /". Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-165-2/.
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Rodrigues, Fábio Henrique dos Santos 1986. "Derivados de quinazolinas na inibição da adenosina quinase". [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/248424.
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Orientador: Ljubica TasicDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química
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Resumo: A Adenosina Quinase (ADK) é uma enzima importante (EC 2.7.1.20), cuja ação pode estar relacionada a diversas doenças, tais como inflamações, derrame, infarto, entre outras. Desse modo, a inibição de sua atividade é de grande importância, e desperta interesse científico. Na tentativa de inibir a ação da ADK, houve busca por compostos orgânicos cuja capacidade inibitória seja superior comparando-se com inibidores da ADK existentes. Desse modo, derivados de 4-anilinoquinazolinas mostraram-se alvos interessantes. Foi sintetizada uma série de 22 derivados de 8-metóxi-4-anilinoquinazolinas, substituídas nas posições 3'e 4'do anel anilínico. Os compostos sintetizados foram caracterizados e testados frente à ADK, de forma a verificar seu potencial inibitório, principalmente através da técnica de fluorescência de emissão. Da série de compostos, seis apresentaram-se promissores na inibição da ADK. Ensaios in silico também foram realizados, buscando-se uma melhor compreensão do mecanismo de inibição do sistema compostos/ADK
Abstract: The Adenosine Kinase (ADK) is an important enzyme (EC 2.7.1.20) that might be related to several diseases, such as inflammation, stroke and infarct, and many others. Therefore, its activity inhibition is of great importance, arising significant scientific interest. Aiming ADKs inhibition, a search for suitable organic species was realized, in such way that 4-anilinoquinazoline derivatives showed themselves interesting targets. A serie of 22 8-methoxy-4-anilinequinazoline derivatives, substituted on the aniline ring at 3'and 4'positions, was synthesized. The compounds were characterized and tested in in vitro ADKs inhibition, in order to verify their inhibitory potentials, mainly applying emission fluorescence technique. Six compounds of this serie presented promising properties in ADKs inhibition. In silico assays were also conducted, in order to better explain the inhibitory mechanism of the system compounds/ADK
Mestrado
Quimica Organica
Mestre em Química
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Long, Yun Chau. "Skeletal muscle metabolic flexibility: the roles of AMP-activated protein kinase and calcineurin /". Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-152-4/.
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Melemedjian, Ohannes, Marina Asiedu, Dipti Tillu, Raul Sanoja, Jin Yan, Arianna Lark, Arkady Khoutorsky i in. "Targeting adenosine monophosphate-activated protein kinase (AMPK) in preclinical models reveals a potential mechanism for the treatment of neuropathic pain". BioMed Central, 2011. http://hdl.handle.net/10150/610214.
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Neuropathic pain is a debilitating clinical condition with few efficacious treatments, warranting development of novel therapeutics. We hypothesized that dysregulated translation regulation pathways may underlie neuropathic pain. Peripheral nerve injury induced reorganization of translation machinery in the peripheral nervous system of rats and mice, including enhanced mTOR and ERK activity, increased phosphorylation of mTOR and ERK downstream targets, augmented eIF4F complex formation and enhanced nascent protein synthesis. The AMP activated protein kinase (AMPK) activators, metformin and A769662, inhibited translation regulation signaling pathways, eIF4F complex formation, nascent protein synthesis in injured nerves and sodium channel-dependent excitability of sensory neurons resulting in a resolution of neuropathic allodynia. Therefore, injury-induced dysregulation of translation control underlies pathology leading to neuropathic pain and reveals AMPK as a novel therapeutic target for the potential treatment of neuropathic pain.
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Fattori, Ana Carolina Maragno. "Efeitos da imunização com Adenosina Quinase (AK) e Hipoxantina-Guanina Fosforibosiltransferase (HGPRT) recombinantes de Schistosoma mansoni : controle da infecção murina". Universidade Federal de São Carlos, 2016. https://repositorio.ufscar.br/handle/ufscar/7924.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
The mansoni schistosomiasis is the most important of human helminthiasis. Despite advances in its control this disease continues to spread to new geographical areas. It currently affects more than 250 million people. However, limited options are available for and Praziquantel is the drug of choice. Various authors have been searching new drugs and vaccines to control schistosomiasis. This study aimed to evaluate the effects of a prior immunization with recombinant enzymes of Schistosoma mansoni: Adenosine Kinase (AK) and Hypoxanthine-guanine Phosphoribosyltransferase (HGPRT), which are important for parasite purine metabolism, as well as a MIX of these enzymes, and subsequent challenge with cercariae of the parasite in the control of murine infection. Female Balb/c mice were divided into 5 groups. The groups were enzyme-immunized in three doses and 15 days after the last immunization, animals were infected with S. mansoni. After infection in the 47º day egg count were carried in mice faeces and in the 48º day mice were sacrificed for evaluation of leukocyte numbers (blood and peritoneal cavity), worm burden, antibodies production, cytokines quantification and histopathological analysis of the liver of these animals. Our results strongly suggest that, immunization with a MIX originated in these animals reduction in the number of eggs in faeces by 46% when compared with the animals of the infected group. Animals of the groups immunized with AK, HGPRT and/or MIX seem to induce a reduction in the number of eosinophils in the peritoneal cavity when compared to the animals of the infected group. Concerning worm burden, the animals of the MIX group presented greater reduction (31.27%) when compared to the animals of the infected group. The animals of the immunized groups, AK, HGPRT and/or MIX were capable of producing IgG1 antibodies and IgE anti the enzymes and anti the parasite proteins. The animals of the immunized group MIX showed a slight increase in IL-4 production and observed reduction of IL-10, and in the HGPRT group induced a slight increase on IFN-γ production when in compared with the infected group. In addition, the animals of the AK group showed a decrease in the number of hepatic granulomas in tissue (44,55%) and the eggs present in liver (42,31%). Therefore, it suggests that immunization with these enzymes can contributes to schistosomiasis control, as well as it might helps to modulate experimental infection inducing reduction of physiopathology of this parasitosis.
A esquistossomose mansônica é a mais importante das helmintíases humanas. Apesar dos avanços no seu controle continua se espalhando para novas áreas geográficas. Atualmente afeta mais de 250 milhões de pessoas. Entretanto, opções limitadas estão disponíveis para o tratamento da doença e o único fármaco de escolha é o Praziquantel. Assim, vários estudos têm sido propostos para encontrar novos fármacos e vacinas para combater a esquistossomose. Dessa forma, o presente estudo teve como proposta avaliar os efeitos da imunização prévia com as enzimas recombinantes de Schistosoma mansoni Adenosina Quinase (AK) e Hipoxantina-Guanina Fosforibosiltransferase (HGPRT), que participam do metabolismo de purinas do parasito, bem como com o MIX das duas enzimas, e posterior desafio com cercárias do parasito, para o controle da infecção murina. Camundongos fêmea Balb/c foram divididos em 5 grupos. Os grupos imunizados receberam três doses das enzimas e após 15 dias da última imunização, os animais foram infectados com S. mansoni. Após a infecção, no 47° dia foi realizada a contagem de ovos nas fezes e no 48° dia foi realizada a eutanásia dos animais para avaliação de resposta leucocitária (sangue e lavado da cavidade peritoneal), carga parasitária, produção de anticorpos, quantificação de citocinas e análise histopatológica do fígado desses animais. Os resultados demonstraram que, a imunização com o MIX promoveu nesses animais redução do número de ovos nas fezes de 46% quando comparado com os animais do grupo somente infectado. Os animais dos grupos imunizados com AK, HGPRT e/ou MIX apresentaram diminuição na quantidade de eosinófilos na cavidade peritoneal quando comparados com os animais do grupo somente infectado. Em relação à carga parasitária, os animais do grupo imunizado com o MIX apresentaram maior redução (31,27%) quando comparados aos animais do grupo somente infectado. Os animais dos grupos imunizados com AK, HGPRT e/ou MIX foram capazes de produzir anticorpos IgG1 e IgE anti as enzimas e anti as proteínas do parasito. Os animais do grupo imunizado com o MIX apresentaram aumento discreto de IL-4 e foi observada redução de IL-10, e no grupo imunizado com HGPRT houve aumento discreto de IFN-γ, quando comparados com os animais do grupo somente infectado. Além disso, os animais do grupo imunizado com AK apresentaram redução do número de granulomas hepáticos (44,55%) e de ovos no fígado (42,31%), quando comparados com o grupo somente infectado. Assim, sugere-se que a imunização com essas enzimas pode contribuir para o controle da esquistossomose, bem como auxiliar na modulação da infecção experimental, induzindo redução da fisiopatologia desta parasitose.
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Robinson, Alexander John. "Stimulation of the mitogen-activated protein kinase (MAPK) pathway in DDTâ‚MF-2 cells by adenosine Aâ‚ receptors and histamine Hâ‚ receptors". Thesis, Nottingham Trent University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252331.
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Sanders, Charles Ray. "Examination of the relationship of substrate dynamics to enzymic structure, binding energy, and catalysis: NMR studies of adenosine 5'-triphosphate and adenylate kinase /". The Ohio State University, 1988. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487597424137198.
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Ahmed, Ishtiaq. "Structural studies of Vps4 protein complexes". Thesis, Griffith University, 2022. http://hdl.handle.net/10072/412412.
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ESCRT (Endosomal sorting complex required for transport) machinery drives different cellular processes such as endosomal sorting, organelle biogenesis, vesicular trafficking, maintenance of plasma membrane integrity, membrane fission during cytokinesis and enveloped virus budding. The normal cycle of assembly and disassembly of some ESCRT complexes at the membrane requires the AAA-ATPase vacuolar protein sorting 4 (Vps4). A number of ESCRT proteins are hijacked by clinically significant enveloped viruses including Ebola, and Human Immunodeficiency Virus (HIV) to enable enveloped virus budding and Vps4p provides energy for the disassembly and recycling of these ESCRT proteins. Vps4, a member of the AAA-family (ATPase Associated with a variety of cellular Activities) of proteins is present in all eukaryotes where it mediates endosomal membrane trafficking. Comprehensive evaluation of genomic databases validated the existence of three isoforms in animals, Vps4 -a, -b, and -c. However, yeast has only one Vps4 representative, while two isoforms Vps4a and Vps4b exist in mammals. Of particular interest to this study was the protein Vps4 structure and its binding properties. Vps4 protein possesses an MIT (Microtubule-Interacting and Trafficking) domain at the N-terminus, separated by a long linker from a central AAA-domain, which includes the ATPase catalytic site, and then a small domain comprising beta-sheet (β-domain). At the C-terminus of Vps4 is an α-helix (C-terminal helix). The various domains of Vps4 protein play vital roles by mediating the interaction of Vps4 with a number of key partner proteins. Vps4p has been proposed to use the energy of ATP hydrolysis to break protein-protein interactions on the surface of endosomes. The N-terminal coiled-coil domain of Vps4p appeared to be responsible for binding substrate endosomal coiled-coil domain proteins while ATP hydrolysis by the AAA domain supplies energy to disrupt coiled-coil interactions. The present project yielded single particle analysis of the Vps4p-Vps2p co-complex as the foundation of future structural biology studies to obtain greater resolution which is critical to the future design of low molecular weight compounds that will fit into this interaction interface and perturb Vps4p-Vps2p interaction. Thus, the drugs that perturb Vps4p-Vps2p interaction would be anticipated to interfere with Vps4 function and to have a broad-spectrum antiviral activity. Saccharomyces cerevisiae was used as an alternative to the high usage of animals and animal cell lines in antiviral drug development. This study was aimed to identify the structure of the Vps4p-Vps2p interacting complex using Transmission Electron Microscopy (TEM). In this study, we also investigated the functional role of the novel Vps4p-interacting proteins γ-glutamyl kinase (Pro1p) and a serine/threonine-specific protein kinase (Sak1p). Both Pro1p and Sak1p interacted in the yeast two-hybrid system with the N-terminal coiled-coil domain of Vps4p and form complexes with Vps4p in vitro. The interaction of Pro1p with Vps4p is direct and is broken upon hydrolysis of adenosine triphosphate (ATP). In contrast the interaction of Sak1p with Vps4p appears to require other factors. We identified two highly conserved sequence motifs within the N-terminal domain of Vps4p which are essential for interaction of Vps4p with both Pro1p and Sak1p. The current technical advancements in microscopes are driving visualisation of biological samples at the micro- and nano-scales. The single particle analysis of macromolecular complexes in combination with negatively-stained biological samples were elucidated by the use of the TEM. The negatively-stained samples typically result in lower resolution images and that limited visualisation of 3D structural details. We believe that an understanding of the structure of Vps4 interacting with its binding partners will lead to clear targets for future antiviral treatments.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Pharmacy & Med Sci
Griffith Health
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Marais, Erna. "Role of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and p38 mitogen activated protein kinase (p38 MAPK) in preconditioning of the ischaemic myocardium". Thesis, Stellenbosch : Stellenbosch University, 2002. http://hdl.handle.net/10019.1/53039.
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Thesis (PhD)--University of Stellenbosch, 2002.ENGLISH ABSTRACT: Ischaemic preconditioning (PC) is the phenomenon whereby a short episode of coronary occlusion followed by reperfusion protects the myocardium against a subsequent period of prolonged (also called index or sustained) ischaemia. Even though the exact mechanism of PC remains to be established, it implies that the heart has an endogenous protective mechanism against ischaemia which, if identified, may have important clinical implications. The importance of establishing the mechanism of PC lies in the potential to convert this biological phenomenon into a therapeutic modality to be used clinically. If mediated by certain components of a signal transduction pathway, such a goal will be achievable. Several triggers and signal transduction pathways have been implicated in the mechanism of protection induced by PC: for example, receptor-dependent endogenous triggers (such as adenosine and opioids) and receptor-independent endogenous triggers (such as free radicals and calcium). However, the involvement of both the ~-adrenergic signalling pathway as well as nitric oxide (NO) in PC has not been defined. It has been suggested that all triggers are linked to a common final pathway, for example, activation of protein kinase C (PKC) and/or the mitogen-activated kinases (MAPKs), in particular p38 MAPK. However, the role of the latter is still controversial. The aim of this study was to: (A) characterize changes in the cyclic nucleotides, cAMP and cGMP, and p38 MAPK occurring during the entire experimental procedure in an attempt to gain insights into the possible mechanisms involved in ischaemie PC (Chapter 3); (8) establish the significance of the changes observed in cAMP and cGMP by pharmacological manipulation of their respective pathways (Chapters 4 and 5); (C) establish the role of p38 MAPK in ischaemie PC: trigger or mediator involvement (Chapter 6). Isolated perfused working rat hearts were preconditioned by 3 x 5 min global ischaemia, interspersed by 5 min reperfusion, followed by 25 min global ischaemia and 30 min reperfusion. Functional recovery during reperfusion was used as end-point. Hearts were freeze-clamped at different times during the PC protocol, sustained ischaemia, as well as during reperfusion. Tissue cyclic nucleotides (cAMP and cGMP), cyclic nucleotide phosphodiesterase (cAMP- and cGMP-PDE) activities, adenylyl cyclase and protein kinase A activities and p-adrenergic receptor characteristics were determined. p38 MAPK activation was also assessed by Western blotting, using dual phospho-p38 MAPK (Thr180ITyr182) antibody as well as activating transcription factor 2 (ATF2) activation. In addition, to evaluate the role of p38 MAPK in PC protection, the effect of inhibition of p38 MAPK activation, by 8B203580, was determined in adult isolated rat cardiomyocytes as well as in isolated perfused rat hearts. Based on the results obtained, it is proposed that during a multi-cycle ischaemie PC protocol triggers (presumably endogenous catecholamines and NO) are released which induce cyclic changes in cyclic nucleotides, cAMP and cGMP. Both these cyclic nucleotides transiently activate the downstream stress kinase, p38 MAPK, which may trigger further downstream adaptive processes. Furthermore, the sustained ischaemic period of PC hearts was characterized by attenuated cAMP and elevated cGMP levels, as well as attenuated activation of p38 MAPK, which was associated with cardioprotection. In addition, pharmacological attenuation of p38 MAPK activation during sustained ischaemia led to functional recovery. It is concluded that the cardioprotection of PC is due to attenuation of ischaemia-induced p38 MAPK activation. Pharmacological manipulation of this kinase should be considered as a therapeutic modality in the future.
AFRIKAANSE OPSOMMING: Isgemiese prekondisionering (PK) verwys na die verskynsel waardeur 'n kort, verbygaande episode van isgemie gevolg deur herperfusie, die miokardium teen 'n daaropvolgende langdurige periode van isgemie beskerm. Die presiese meganisme van beskerming van PK moet nog opgeklaar word, maar dit impliseer dat die hart oor 'n endogene beskermingsmeganisme beskik wat, indien geïdentifiseer, belangrike kliniese implikasies mag hê. Die belang van opklaring van die meganisme van PK lê daarin dat 'n biologiese verskynsel in 'n terapeutiese modaliteit vir kliniese gebruik, omgeskakel kan word. Sou dit deur bepaalde komponente van 'n seintransduksiepad gemedieër word, is so 'n doel bereikbaar. Verskeie stimuli en seintransduksiepaaie is in PK betrokke: byvoorbeeld, reseptorafhanklike endogene stimuli (soos adenosien en opioïde), asook reseptor-onafhanklike endogene stimuli (soos vrye radikale en kalsium). Die betrokkenheid van die padrenerge seintransduksiepad asook stikstofoksied (NO) in PK egter nog nie behoorlik evalueer nie. Dit is voorgestel dat alle stimuli op 'n finale algemene pad uitloop, soos byvoorbeeld die aktivering van protein kinase C (PKC) en/of die mitogeen-geaktiveerde kinases (MAPKs), spesifiek die p38 MAPKs. Laasgenoemde se rol in PK is steeds kontroversieël. Die doel van die studie was dus: (A) karakterisering van die veranderinge in die sikliese nukleotiede, cAMP en cGMP, en p38 MAPK wat tydens die hele eksperimentele prosedure plaasvind, in 'n poging om meer insig te verkry aangaande moontlike meganismes betrokke in isgemiese PK (Hoofstuk 3); (8) bepaling van die belang van die waargenome veranderinge in cAMP en cGMP deur hulonderskeie paaie farmakologies te manipuleer (Hoofstukke 4 en 5); (C) bepaling van die rol van p38 MAPK in PK: betrokkenheid as stimulus of mediator (Hoofstuk 6). Geïsoleerde, geperfuseerde werkende rotharte is geprekondisioneer deur blootstelling aan 3 x 5 min globale isgemie, afgewissel met 5 min herperfusie, gevolg deur 25 min globale isgemie en 30 min herperfusie. Funksionele herstel tydens herperfusie is as eindpunt gebruik. Harte is op verskillende tye tydens die PK protokol, volgehoue isgemie, asook herperfusie gevriesklamp. Weefsel sikliese nukleotiede (cAMP en cGMP), die aktiwiteit van sikliese nukleotied fosfodiesterases (cAMP- en cGMP-PDE), adeniel siklase en protein kinase A (PKA) asook die eienskappe van die p-adrenerge reseptor is gemeet. p38 MAPK aktivering is met Westerse oordragtegnieke bepaal, deur van dubbel gefosforileerde p38 MAPK (Thr180fTyr182) antiliggame asook geaktiveerde transkripsie faktor 2 (ATF2) gebruik te maak. Die rol van p38 MAPK in PK beskerming is evalueer deur die effek van inhibisie van p38 MAPK aktivering met SB 203580, in volwasse geïsoleerde rot kardiomiosiete asook in geïsoleerde geperfuseerde rotharte, te bepaal. Na aanleiding van die resultate, is voorgestel dat, tydens 'n multi-siklus isgemie PK protokol, stimuli (moontlik endogene katekolamiene en NO) vrygestel word wat die sikliese veranderinge in sikliese nukleotiede, cAMP en cGMP, veroorsaak. Beide hierdie sikliese nukleotiede aktiveer die distale stres kinase, p38 MAPK, op 'n betekenisvolle, maar verbygaande manier. Hierdie kinase mag verdere distale aanpassingsprosesse stimuleer. Die volgehoue isgemiese periode van PK harte is gekenmerk deur verminderde cAMP en verhoogde cGMP vlakke, asook verminderde aktivering van p38 MAPK. Hierdie veranderinge is met beskerming van die hart teen isgemie geassosieer. Daarbenewens, farmakologiese vermindering van p38 MAPK aktivering tydens volgehoue isgemie het tot verbeterde funksionele herstel gelei. Die gevolgtrekking is gemaak dat die beskermende effek van PK die gevolg is van verminderde aktivering van isgemies-geïnduseerde p38 MAPK. Farmakologiese manipulasie van hierdie kinase moet in die toekoms as terapeutiese modaliteit oorweeg word.
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Goaillard, Jean-Marc. "Étude de la modulation de conductances intrinsèques par la voie de l'adenosine monophosphate cyclique dans deux types de cellules excitables : action du récepteur beta2-adrénergique dans les cardiomyocytes ventriculaires de grenouille et du récepteur 5-HT7 dans les neurones intralaminaires thalamiques de rat". Paris 6, 2002. http://www.theses.fr/2002PA066159.
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Santos, Stephanie Cristine Carvalho dos. "Efeitos da 5-iodotubercidina em linhagens de melanoma humano parentais e resistentes ao inibidor de BRAF". Universidade de São Paulo, 2019. http://www.teses.usp.br/teses/disponiveis/9/9142/tde-18032019-150858/.
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O melanoma é responsável por menos de 5% dos cânceres de pele, porém, 95% das mortes ocorrem devido a ocorrência de metástases. O melanoma metastático é refratário às terapias convencionais e rapidamente adquire resistência às terapias como as oncogene-dirigidas, como o inibidor de BRAF, da via de MAPK. Estudos prévios de screening in silico do nosso grupo, onde se utilizou as bases de dados TCGA e GEO, identificaram o gene adenosina quinase (ADK) como sendo diferencialmente expresso entre o melanoma invasivo e os nevus. A 5-iodotubercidina (5-ITu) é um potente inibidor farmacológico da ADK que dentre os diversos efeitos relatados na literatura destaca-se pelo potencial genotóxico. Os danos no DNA são os principais ativadores de checkpoint do ciclo celular, que levam a parada do ciclo celular transitória ou permanente, além de induzir morte celular, levando a hipótese de que ADK possa ser potencial agente anti-melanoma. Este trabalho objetivou avaliar a expressão do gene ADK em melanomas humanos e quimiorresistentes ao inibidor de BRAF (iBRAF), avaliou os impactos de 5-ITu sobre a proliferação, progressão do ciclo celular e morte celular e por fim avaliamos sua capacidade de aumentar a sensibilidade das células. Foi realizado PCR em tempo real para avaliar os níveis de expressão de mRNA de ADK em linhagens de melanoma e na cultura primária de melanócitos; a fim de avaliar a citotoxicidade de 5-ITu foram realizados os ensaios de exclusão por azul de tripan e de apoptose - Anexina V e PI e em modelo de esferoide, usando live/dead; também foi avaliada a influência de 5-ITu sobre a capacidade clonogênica e seus efeitos sobre a proliferação celular, a partir dos ensaios de ciclo celular e avaliação de marcadores de proliferação por imunofluorescência; as linhagens foram submetidas a diferentes regimes de tratamento com 5-ITu e o iBRAF, a fim de avaliar a curva de crescimento e a sensibilidade ao iBRAF por MTT níveis de expressão de mRNA de ADK maiores nas linhagens tumorais em relação aos melanócitos. 5-ITu mostrou-se capaz de inibir a proliferação (IC50) das linhagens de melanoma em concentrações de 1,9 a 3,5 µM. 5-ITu não foi capaz de induzir inviabilidade celular, apesar de reduzir a quantidade de células viáveis em todas as condições de tratamento, também não foi capaz de induzir aumento significativo de células apoptóticas, nem mesmo necróticas. No entanto, o tratamento com 5-ITu reduziu a capacidade clonogênica de linhagens de melanoma e promoveu parada de ciclo celular nas fases G1 e G2/M, levou ao aumento da população subG1. O tratamento com 5-ITu promoveu a redução da expressão de marcadores de proliferação, como ki67, e a combinação de tratamentos 5-ITu e iBraf foi capaz de aumentar o tempo de dobramento das linhagens de melanoma, embora tenha se mostrado incapaz de sensibilizar as células de melanoma ao tratamento com iBRAF. Desse modo, pode-se concluir que 5-ITu induz o efeito citostático e se mostra um potente agente antiproliferativo para melanoma parental e resistente.Melanoma accounts for less than 5% of skin cancers, but 95% of deaths occur due to metastases. Metastatic melanoma is refractory to conventional therapies and rapidly acquires resistance to therapies such as oncogene-directed, such as the BRAF inhibitor, of the MAPK pathway. Previous studies of screening in silico of our group, using the databases TCGA and GEO, identified the adenosine kinase gene (ADK) as differentially expressed between invasive melanoma and nevus. 5-iodotubercidin (5-ITu) is a potent pharmacological inhibitor of ADK that among the several effects reported in the literature stands out for the genotoxic potential. DNA damage is the main activator of the cell cycle checkpoint, which leads to transient or permanent cell cycle arrest, in addition to inducing cell death, leading to the hypothesis that ADK may be a potential anti-melanoma agent. This work aimed to evaluate the expression of the ADK gene in human melanomas and chemoresistants to the BRAF inhibitor (iBRAF), evaluated the impacts of 5-ITu on proliferation, cell cycle progression and cell death and finally we evaluated its ability to increase the sensitivity of cells. Real-time PCR was performed to assess the levels of ADK mRNA expression in melanoma lines and primary melanocyte culture; in order to evaluate the cytotoxicity of 5-ITu, the trypan blue and apoptosis - Annexin V and PI exclusion and blue spheroid models were performed using live / dead; the influence of 5-ITu on the clonogenic capacity and its effects on cell proliferation, from the cell cycle assays and the evaluation of proliferation markers by immunofluorescence; the cell lines were submitted to different treatment regimens with 5-ITu and iBRAF in order to evaluate the growth curve and the sensitivity to iBRAF by MTT levels of mRNA expression of ADK higher in the tumor lines in relation to the melanocytes. 5-ITu was able to inhibit the proliferation (IC 50) of melanoma lines at concentrations of 1.9 to 3.5 181;M. 5-ITu was not able to induce cell non-viability, although it reduced the amount of viable cells in all treatment conditions, nor was it able to induce a significant increase in apoptotic or even necrotic cells. However, treatment with 5-ITu reduced the clonogenic capacity of melanoma cells and promoted cell cycle arrest in the G1 and G2 / M phases, leading to an increase in the subG1 population. Treatment with 5-ITu promotes the reduction of expression of proliferation markers, such as ki67, and the combination of 5-ITu and iBRAF treatments was able to increase the doubling time of melanoma cells, although it has been shown to be unable to sensitize melanoma cells to treatment with iBRAF. Thus, it can be concluded that 5-ITu induces the cytostatic effect and shows a potent antiproliferative agent for parental and resistant melanoma.
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Matsumoto, João Paulo de Pontes. "Receptor A2a de adenosina: estudo da modulação da liberação de neurotransmissores em modelo in vitro". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/41/41135/tde-03042013-115748/.
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A transmissão sináptica é essencial para o funcionamento do sistema nervoso. A neuromodulação permite regular esse processo de forma precisa. Um desses mecanismos modulatórios é a regulação da liberação de neurotransmissores. A adenosina é um importante modulador da transmissão sináptica. Além disso, a ativação do subtipo A2a dos receptores para adenosina está envolvida com a facilitação da liberação de neurotransmissores no sistema nervoso central. O presente trabalho teve como objetivo avaliar os efeitos modulatórios da ativação do receptor A2a de adenosina sobre a liberação de neurotransmissores e sua via de sinalização intracelular em modelo in vitro. Além disso, a tese contempla a construção histórica dos conceitos abordados no trabalho permitindo uma visão clara de sua evolução. Esse projeto foi o pioneiro no Brasil a utilizar o sensor biossintético fluorescente de liberação de vesículas sinápticas (supereclipse sinapto-pHluorina), o qual foi gentilmente cedido pelo professor Gero Miensenboeck do Sloan-Kettering Institute for Cancer Research. Nossos resultados demonstraram que o tratamento com o agonista do receptor A A2a de adenosina aumentou a fluorescência do supereclipse sinapto-pHluorina, assim como os níveis de glutamato e noradrenalina. Além disso, foi demonstrado que o inibidor da proteína cinase dependente de AMPc aboliu o aumento nos níveis do glutamato e noradrenalina, tal como a fosforilação da proteína sináptica sinapsina I evocado pelo agonista do receptor A2a de adenosina. Desta forma, nossos dados sugerem que a ativação do receptor A2a de adenosina em cultura de células do bulbo de ratos Wistar modula a liberação de neurotransmissores e a fosforilação da sinapsina I, assim como a proteína cinase dependente do AMPc pode ser o modus operandi desse fenômeno modulatórioSynaptic transmission is a sine qua non process for nervous system physiology. Such precise process is accomplished in part due to modulation of neurotransmitter release. Adenosine is a putative synaptic transmission modulator. Moreover, adenosine A2a receptor facilitates neurotransmitter release in the Central Nervous System. The present study focuses on the modulation of neurotransmission by adenosine A2a receptor and its intracellular signaling pathway in in vitro model. Here, we provided evidence that adenosine A2a receptor agonist increases an optical biosynthetic sensor of synaptic vesicle release (supereclipct synapto-pHluorin), as well as glutamate and noradrenaline. Furthermore, it was demonstrated that cAMP-dependent protein kinase inhibitor abolished glutamate and norepinephrin increase, as well as synapsin I phosphorylation evoked by adenosine A2a receptor agonist. Therefore, our data suggest that adenosine A2a receptor activation modulates neurotransmitter release and synapsin I phosphorylation in cultured cells from medulla oblongata of Wistar rats, as well as cAMP-dependent protein kinase might be the modus operandi of this modulatory phenomenon
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Choe, Jungwoo. "Structural and biochemical studies of trypanosomatid drug target proteins /". Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/9197.
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Vodnala, Munender. "Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei". Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-80904.
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Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs. T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease. It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice. The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open reading frame. The recombinant T. brucei TK, which belongs to the TK1 family, showed broad substrate specificity. The enzyme phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine, as well as the purine nucleosides deoxyinosine and deoxyguanosine. When the repeated sequences of the tandem TbTK were expressed individually as domains, only domain 2 was active. However, the protein could not dimerize and had a 5-fold reduced affinity to its pyrimidine substrates but a similar turnover number as the full-length enzyme. The expressed domain 1 was inactive and sequence analysis revealed that some active residues, which are needed for substrate binding and catalysis, are absent. Generally, the TK1 family enzymes form dimers or tetramers and the quaternary structure is linked to the affinity for the substrates. The covalently linked inactive domain-1 helps domain-2 to form a pseudodimer for the efficient binding of substrates. In addition, we discovered a repetition of an 89-bp sequence in both domain 1 and domain 2, which suggests a genetic exchange between the two domains. T. brucei is very dependent on de novo synthesis via ribonucleotide reductase (RNR) for the production of dNTPs. Even though T. brucei RNR belongs to the class Ia RNR family and contains an ATP-binding cone, it lacks inhibition by dATP. The mechanism behind the RNR activation by ATP and inactivation by dATP was a puzzle for a long time in the ~50 years of RNR research. We carried out oligomerization studies on mouse and E. coli RNRs, which belongs to the same family as T. brucei, to get an understanding of the molecular mechanism behind overall activity regulation. We found that the oligomerization status of RNRs and overall activity mechanism are interlinked with each other.Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.
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Thomas, Julia Dominique Janine. "The characterisation of growth hormone-related cardiac disease with magnetic resonance imaging & The effects of growth hormone dysregulation on adenosine monophosphate-activated protein kinase in cardiac tissue". Thesis, Queen Mary, University of London, 2012. http://qmro.qmul.ac.uk/xmlui/handle/123456789/5391.
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Chronic growth hormone (GH) excess, acromegaly, causes a specific cardiomyopathy, which remains poorly understood. The pattern of hypertrophy is distinct from other forms of cardiac disease and begins to appear before hypertension or diabetes. GH deficiency (GHD) also causes cardiovascular problems, with reduced ability to mount a cardiovascular response to exercise. Acromegaly and GHD patients have increased cardiac mortality. Cardiac magnetic resonance imaging (CMR) is the gold standard for assessment of cardiac mass and provides data on cardiac function, fibrosis, valve function and ischaemia. This study used CMR to assess 23 patients with acromegaly or GHD, before and after treatment of their GH disorder, and 23 healthy controls. Patients with acromegaly demonstrated increased left ventricular mass index (LVMi), end diastolic volume index, stroke volume index and cardiac index, which persisted at one year, despite treatment of underlying disease. Patients with GHD demonstrated LVMi at the bottom (males) or beneath (females) published normal references ranges, which increased with one year of GH replacement. The mechanisms by which GH influences cardiac tissue are poorly understood. Adenosine monophosphate-activated protein kinase (AMPK) is an energy-regulator enzyme, which interacts with several metabolic hormones. Mutations in AMPK cause arrhythmias and cardiac hypertrophy. AMPK activation may be a mechanism by which GH causes some of its cardiac effects. This study used primary cardiomyocytes and mouse and rat models of GH excess and deficiency to study the effects of GH on cardiac AMPK. Acute GH treatment increased AMPK activity in both in vivo and in vitro studies; acute IGF-I treatment had the opposite effect. In 2 and 8 month old bovine GH-overexpressing (bGH) and GH receptor knock out (GHRKO) mice, functional AMPK assay did not demonstrate any difference in cardiac AMPK activity between transgenics and controls. However, Western blotting for Threonine-172 phospho (p)AMPK levels, a marker of AMPK activity, demonstrated increased cardiac pAMPK in 2 month old bGH mice and a reduction in cardiac pAMPK levels in 8 month old animals. A trend towards the same findings was seen in GHRKO mice. This indicates that both GH and IGF-I interact with myocardial AMPK, apparently via different mechanisms.
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Rofougaran, Reza. "DNA precursor biosynthesis-allosteric regulation and medical applications". Doctoral thesis, Umeå : Univ, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1678.
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Fijolek, Artur. "Salvage and de novo synthesis of nucleotides in Trypanosoma brucei and mammalian cells". Doctoral thesis, Umeå : Umeå University, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1850.
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Shi, Yun. "KATP Channel Phosphorylation: Mechanisms and Contribution to Vascular Tone Regulation by Vasodilating and Vasoconstricting Hormones and Neurotransmitters". unrestricted, 2007. http://etd.gsu.edu/theses/available/etd-11302007-150810/.
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Thesis (Ph. D.)--Georgia State University, 2007.Title from file title page. Chun Jiang, committee chair; Teryl Frey, Deborah Baro, Delon Barfuss, committee members. Electronic text (168 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Jan. 30, 2008. Includes bibliographical references (p. 146-151).
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Bertran-Gonzalez, Jesus. "Study of segregated signaling responses of striatonigral and striatopallidal neurons in BAC transgenic mice". Paris 6, 2009. http://www.theses.fr/2009PA066348.
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Le striatum reçoit et intègre des informations arrivant de régions corticales et thalamiques et de neurones dopaminergiques du mésencéphale, formant ainsi la station d'entrée principale au circuit des ganglions de la base (GB). Il est principalement composé de deux sous-types de neurones GABAergiques épineux de taille moyenne (MSNs), qui projettent vers différents noyaux. La voie directe est constituée par les neurones striatonigraux, qui projettent directement vers la substance noire pars reticulé (SNr), la station de sortie des GB. La voie indirecte lie le striatum et la SNr par des relais intermédiaires, dont les premières projections sont ceux des neurones striatopallidaux. Etant donné leurs influences opposées au niveau de la SNr, les informations transmises par les neurones striatonigraux et striatopallidaux à travers des circuits thalamocorticaux sont cruciales pour la sortie finale des GB. La dopamine (DA) module les informations corticostriatales et thalamostriatales au travers des différents types de récepteurs de la DA exprimés dans les MSNs. Les récepteurs de type D1 (D1R) sont connus pour activer des programmes importants de la signalisation moléculaire tels que les modules cAMP/PKA/DARPP-32 et MAPK/ERK, tous deux usant de réponses cytoplasmiques et nucléaires essentielles. D'autre part, les récepteurs de type D2 (D2R) sont négativement associés à la formation de l’AMPc, et sont fonctionnellement couplés aux récepteurs d'adénosine A2A (A2AR) dans un jeu opposé qui régule des réactions intracellulaires et au niveau de la membrane. À la lumière de la controverse existant encore sur la distribution ségrégée vs superposée des sous-types de récepteurs de la DA dans les neurones striatonigraux et striatopallidaux, nous avons entrepris une série d'études neuro-anatomiques et histologiques pour la caractérisation des souris transgéniques BAC drd1a-EGFP et drd2-EGFP, qui marquent les cellules exprimant D1R et D2R, respectivement. Nous avons constaté que les D1R et D2R sont respectivement distribués dans les neurones striatonigraux et striatopallidaux de manière très ségrégée, et nous avons estimé les proportions de neurones exprimant D1R, D2R ou les deux dans différentes régions du striatum. Nos résultats appuient l'organisation des GB dans les voies directe et indirecte, mais nous avons détecté la présence de certains terminaux des D1R-MSNs au niveau du globus pallidus latéral. Surtout, nous avons clairement démontré que, après des traitements de cocaïne aigus et chroniques, à la fois des réponses cytoplasmiques -révélées par l'activation de ERK- et réponses nucléaires -révélées par l'activation de MSK1 et la phosphorylation de l'histone H3- se produisent exclusivement dans les neurones striatonigraux, une distribution qui est largement suivie plus tard par l'induction des gènes immédiats précoces c-fos et zif268. Nous démontrons également que l’antipsychotique typique halopéridol, ainsi que des inhibiteurs spécifiques des D2R, activent toutes ces réponses de signalisation sélectivement dans les neurones striatopallidaux, par un mécanisme de dérépression des A2AR. En parallèle, nous révélons des régulations moléculaires exclusives des réponses nucléosomales dans ces cellules. Dans l'ensemble, les résultats présentés dans cette thèse mettent en évidence une forte ségrégation histologique et fonctionnelle des neurones du striatum, ce qui est essentiel pour la compréhension de la modulation des GB exercée par la DA dans le striatum.
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Nakano, Masako. "α2 isoform-specific activation of 5' adenosine monophosphate-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside at a physiological level activates glucose transport and increases glucose transporter 4 in mouse skeletal muscle". Kyoto University, 2008. http://hdl.handle.net/2433/135919.
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Liu, Huaize [Verfasser], Thorsten Gutachter] Pfirrmann, Regine [Gutachter] [Heller i Rüdiger [Gutachter] Horstkorte. "The GID-complex is a novel ubiquitin ligase involved in the regulation of adenosine monophosphate-activated protein kinase (AMPK) and the function of the primary cilium : [kumulative Dissertation] / Huaize Liu ; Gutachter: Thorsten Pfirrmann, Regine Heller, Rüdiger Horstkorte". Halle (Saale) : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2020. http://d-nb.info/1218530421/34.
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Croset, Amélie. "Identification et caractérisation des mécanismes d'action des molécules appats, les SiDNA, dans l'inhibition des voies de réparation des cassures simple-brin". Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T018.
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La plupart des traitements anticancéreux, comme la chimiothérapie ou la radiothérapie, sont cytotoxiques et causent des dommages à l'ADN dans le but d’induire la mort des cellules tumorales. Cependant, l’efficacité d’activité de réparation de l'ADN des tumeurs entraine des résistances intrinsèques et acquises aux traitements. L'une des étapes précoces de la réparation de l’ADN est le recrutement de protéines au niveau du site de dommage. Ce recrutement est coordonné par une cascade de modifications et est contrôlé par des protéines senseurs telles que la protéine kinase ADN dépendante (DNA-PK) et / ou la poly (ADP- ribose) polymérase (PARP). Dans ce manuscrit, nous avons identifié et caractérisé le mécanisme d'action de petites molécules d'ADN (les siDNA), mimant des cassures double brin (appelé Dbait) ou simple brin (appelé Pbait), dans l’inhibition des voies de réparation des cassures simple brin (SSBR/BER). Nous démontrons que les molécules Dbait recrutent et activent à la fois PARP et DNA-PK, contrairement aux molécules Pbait qui ne recrutent que la PARP. L'étude comparative de ces deux molécules permet d'analyser les rôles respectifs des deux voies de signalisation: les deux molécules recrutent les protéines impliquées dans la voie de réparation des cassures simple brin (comme PARP, PCNA et XRCC1) et empêchent leurs recrutements aux niveaux des lésions chromosomiques. Les molécules Dbait inhibent par ailleurs le recrutement des protéines impliquées dans la voie de réparation des cassures double brin (NHEJ et HR). Pbait et Dbait désorganisent la réparation de l’ADN et sensibilisent les cellules tumorales aux traitements. L’inhibition de la réparation des cassures simple brin semble dépendre d’un piégeage des protéines directement sur les siDNA ou indirectement sur les polymères PAR. L’inhibition des voies de réparation des cassures double brin (DSB) semble par contre se faire de façon indirecte ; cette inhibition résulterait plutôt de la phosphorylation des protéines de réparation des DSB de part l’activation de DNA-PK. Les molécules Dbait et Pbait induisent un effet de létalité synthétique des cellules tumorales BRCA mutées. Cependant, la mutation BRCA semble être suffisante mais non nécessaire pour induire la sensibilité des cellules tumorales aux traitements Dbait. En effet, nous avons démontré que les molécules Dbait peuvent aussi sensibiliser les cellules ne présentant pas de mutation BRCA mais ayant toutefois une forte instabilité génétique. Nous avons trouvé une corrélation entre le niveau basal de protéines de réparation de l'ADN (ɣH2AX, PARP et PAR), le taux basal de cassures à l’ADN, la présence de micronoyaux (MN) et la sensibilité des cellules tumorales au traitement Dbait. Nous avons émis l’hypothèse que cette instabilité génétique, déterminé par la quantification de MN dans des biopsies tumorales, pourrait être un biomarqueur prédictif de l’effet du Dbait, non seulement dans les cancers du sein, mais aussi dans les glioblastomes, les mélanomes, les mélanomes uvéaux et les cancers du côlonMost conventional cancer treatments, such as chemotherapy or radiotherapy, are cytotoxic and cause DNA damages in the tumoral treated cells, which ultimately lead to their death. However, several intrinsic and acquired resistances of tumors to these treatments are due to the tumor efficient DNA repair activities. One of the major early steps of DNA repair is the recruitment of repair proteins at the damage site and this is coordinated by a cascade of modifications controlled by sensor proteins such as DNA-dependent protein kinase (DNA-PK) and/or poly (ADP-ribose) polymerase (PARP). In this manuscript, we identify and characterize the mechanism of action of short interfering DNA molecules (siDNA), mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) in Single Strand Break Repair pathway (SSBR/BER) inhibition. We demonstrate that Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. The comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both molecules recruit proteins involved in single-strand break repair (such as PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break (DSB) repair. By these ways, Pbait and Dbait disorganized DNA repair, thereby sensitizing cells to treatments. SSB repair inhibition depends upon a direct trapping of the main proteins on both molecules and an indirect trapping in PAR polymers. DSB repair inhibition may be indirect, resulting from the phosphorylation of DSB repair proteins by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations tumoral cell lines. However, BRCA mutation could be sufficient but not necessary to induce breast cancer cell lines and tumors sensitivity to Dbait treatment. In fact, we demonstrate that Dbait molecules could also have a stand-alone effect in BRCA wild type cells with a high genetic instability. We found a correlation between DNA repair proteins basal level (ɣH2AX, PARP and PAR), DNA break basal level, presence of micronucleus (MN) and tumoral cell lines sensitivity to Dbait treatment. We hypothesis that this genetic instability, determined by MN in tumor biopsies, could be a predictive biomarker of Dbait stand-alone effect, not only in breast cancer treatment, but also in glioblastoma, melanoma, uveal melanoma and colon cancer treatment
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Welshhans, Kristy. "Neuronal growth cone dynamics are regulated by a nitric oxide-initiated second messenger pathway". unrestricted, 2007. http://etd.gsu.edu/theses/available/etd-09282007-114034/.
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Thesis (Ph. D.)--Georgia State University, 2007.Vincent Rehder, committee chair; Sarah Pallas, Walter William Walthall, committee members. Electronic text (248 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Jan. 28, 2008; title from file title page. Includes bibliographical references (p. 218-248).
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Romanello, Larissa. "Estudos das enzimas adenosina kinase e hipoxantinaguanina fosforibosiltransferase de Schistosoma mansoni". Universidade Federal de São Carlos, 2011. https://repositorio.ufscar.br/handle/ufscar/6981.
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Previous issue date: 2011-07-22Universidade Federal de Minas Gerais
Schistosoma mansoni is the parasite responsible for schistosomiasis mansonica, a disease that affects about 207 million people worldwide, and does not have the purine de novo sinthetic pathway, depending entirely on the purine salvage pathway to supply its demands on purines. Adenosine kinase (AK) and Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are important enzymes of the purine salvage, the AK directly phosphorylates adenosine into adenosine monophosphate (AMP) and HGPRT is responsible for the reversible phosphorybosylation of hypoxanthine or guanine into IMP or GMP. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. The nucleotide coding region of the isoform 2 of AK enzyme was amplified and cloned into pGEM vector and pET28a, the recombinant protein was expressed in E.coli BL21 (DE3), purified in a his-tag nickel-affinity resin and AMP-agarose resin, tested for its activity and crystallized. Two data sets were obtained by X-ray diffraction: a ternary complex of AK2-AMP-adenosine in the MX2 light line of the Synchrotron Light National Laboratory and a binary complex AK2-tubercidin in the rotatory anode X-ray source of the Institute of Physics at Sao Carlos - USP, both at 2.3A of resolution. The nucleotide coding region of the enzyme HGPRT was also amplified, cloned into pGEM and pET28a, which heterologous expression was done in E.coli BL21 (DE3) cells at 18°C and purified on a cobalt his-tag affinitiy resin.
Schistosoma mansoni e o parasita responsavel pela esquistossomose mansonica, doenca que afeta cerca de 207 milhoes de pessoas em todo mundo, e nao possui a via de sintese de novo de purinas, dependendo integralmente da via de salvacao para seu suprimento de purinas. A adenosina kinase (AK) e a Hipoxantina-guanina fosforibosiltransferase (HGPRT) sao importantes enzimas desta via, sendo a AK responsavel pela fosforilacao direta de adenosina para adenosina monofosfato (AMP) e a HGPRT pela fosforibosilacao reversivel de hipoxantina ou guanina para IMP ou GMP respectivamente. Essa via tem sido citada como alvo potencial para o desenvolvimento de novos farmacos contra a esquistossomose. A regiao nucleotidica codificadora da enzima AK, isoforma 2, foi amplificada e clonada em vetor pGEM e pET28a, a proteina recombinante foi expressa em E.coli BL21 (DE3), purificada em coluna de niquel e AMP-agarose, submetida a ensaios de atividade e cristalizada. Dois conjuntos de dados foram obtidos por difracao de raio-X: um complexo ternario de AK2- AMP-adenosina na linha de luz MX2 do Laboratorio Nacional de Luz Sincrotron e um complexo binario de AK2-tubercidina no raio-X do Instituto de Fisica de Sao Carlos USP anodo rotatorio, ambos a 2.3A de resolucao. A regiao nucleotidica codificadora da enzima HGPRT tambem foi amplificada, clonada em pGEM e pET28a, sendo a proteina recombinante expressa em E.coli BL21 (DE3) a 18°C e purificada em coluna de cobalto.
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Schulte, Gunnar. "Adenosine receptor signaling and the activation of mitogen-activated protein kinases /". Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-299-x/.
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Jackel, Jamie Nicole. "GEMINIVIRUSES AS MODELS TO STUDY THE ESTABLISHMENT AND MAINTENANCE OF DNA METHYLATION". The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1367494030.
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Mundell, Stuart James. "Role of G protein-coupled receptor kinases in the desensitization of Aâ†2 adenosine receptor responses". Thesis, University of Bristol, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262810.
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Bordes, Pastor Isabel. "Theoretical Studies of the Catalytic Mechanism of the Dihydroxyacetone Kinase". Doctoral thesis, Universitat Jaume I, 2017. http://hdl.handle.net/10803/436901.
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Dihydroxyacetone kinases (DHAKs) catalyse the transfer of the phosphoryl group from adenosine triphosphate (ATP) to dihydroxyacetone (Dha) generating Dha phosphate (Dha-P), a very important specie for C-C bond formation in nature. Kinases are present in humans and they are involved in cancer progression, inflammation and autoimmune disorders. In this Doctoral Thesis, it has been studied the molecular mechanism of the phosphoryl transfer from ATP to Dha in aqueous solution and in the DHAK of Escherichia coli employing the quantum mechanical/molecular mechanical (QM/MM) hybrid methodology. In addition, in collaboration with a experimental group of Madrid, it has been performed the tuning of the phosphoryl donor specificity in the DHAK from Citrobacter freundii, from ATP to inorganic polyphosphate, a very advantageous compound. It has been analyzed the binding effects of this compound with the enzyme and also the chemical reaction with the Dha.Las dihidroxiacetona quinasas (DHAKs) catalizan la transferencia del grupo fosfato desde el adenosin trifosfato (ATP) a la dihidroxiacetona (Dha), generando Dha fosfato (Dha-P), una especie importante para la formación de enlaces C-C en la naturaleza. Las quinasas están presentes en el cuerpo humano e involucradas en el cáncer, la inflamación y los desórdenes autoinmunes. En esta Tesis Doctoral, se ha estudiado el mecanismo de reacción de transferencia de fosfato desde el ATP a la Dha en disolución acuosa y en la DHAK de Escherichia coli empleando la metodología híbrida mecánica cuántica/mecánica molecular (QM/MM). Además, en colaboración con un grupo experimental de Madrid, se ha estudiado la modificación de la especificidad en la DHAK de Citrobacter freundii, desde el ATP al polifosfato inorgánico como dador de fosfatos, un compuesto con grandes ventajas. Se han analizado los efectos de unión de este compuesto con dicha enzima, además de estudiar la reacción química con la Dha.
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Noriega, Esteban Núria. "The Rtg1 and Rtg3 proteins are novel transcription factors regulated by the yeast hog1 mapk upon osmotic stress". Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7158.
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La adaptación de la levadura Saccharomyces cerevisiae a condiciones de alta osmolaridad está mediada por la vía de HOG ((high-osmolarity glycerol). La activación de esta vía induce una serie de respuestas que van a permitir la supervivencia celular en respuesta a estrés. La regulación génica constituye una respuesta clave para dicha supervivencia. Se han descrito cinco factores de transcripción regulados por Hog1 en respuesta a estrés osmótico. Sin embargo, éstos no pueden explicar la totalidad de los genes regulados por la MAPK Hog1. En el presente trabajo describimos cómo el complejo transcripcional formado por las proteínas Rtg1 y Rtg3 regula, a través de la quinasa Hog1, la expresión de un conjunto específico de genes. Hog1 fosforila Rtg1 y Rtg3, aunque ninguna de estas fosforilaciones son esenciales para regulación transcripcional en respuesta a estrés. Este trabajo también muestra cómo la deleción de proteínas RTG provoca osmosensibilidad celular, lo que indica que la integridad de la vía de RTG es esencial para la supervivencia celular frente a un estrés osmótico.In Saccharomyces cerevisiae the adaptation to high osmolarity is mediated by the HOG (high-osmolarity glycerol) pathway, which elicits different cellular responses required for cell survival upon osmostress. Regulation of gene expression is a major adaptative response required for cell survival in response to osmotic stress. At least five transcription factors have been reported to be controlled by the Hog1 MAPK. However, they cannot account for the regulation of all of the genes under the control of the Hog1 MAPK. Here we show that the Rtg1/3 transcriptional complex regulates the expression of specific genes upon osmostress in a Hog1-dependent manner. Hog1 phosphorylates both Rtg1 and Rtg3 proteins. However, none of these phosphorylations are essential for the transcriptional regulation upon osmostress. Here we also show that the deletion of RTG proteins leads to osmosensitivity at high osmolarity, suggesting that the RTG-pathway integrity is essential for cell survival upon stress.
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Fernandes, Maria Fernanda de Andrade. "Modulação da AMP-activated protein kinase (AMPK) em hipotalamo de ratos wistar submetidos ao exercicio". [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309954.
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Orientador: Jose Barreto Campello CarvalheiraDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: As proteínas AMPK e mTOR são as principais moduladoras do balanço energético intracelular, e exercem influência decisiva sobre a ação hipotalâmica da leptina. O exercício agudo, através da produção de IL-6, está associado ao aumento da sensibilidade à ação anorexigênica da leptina. Visando investigar a possível influência das vias AMPK e mTOR, neste aumento da sensibilidade hipotalâmica à leptina induzido pelo exercício agudo, ratos Wistar foram submetidos à natação e posteriormente receberam injeção intracerebroventricular de IL-6, ativadores e/ou inibidores da AMPK e mTOR. A IL-6 reduziu a ingestão alimentar dos animais não exercitados; no entanto, o pré-tratamento com ativador da AMPK ou inibidor da mTOR bloqueou esta ação da IL-6. Ativadores da AMPK aumentaram a ingestão alimentar de forma mais significativa nos animais não exercitados. Inibidores da AMPK reduziram a ingestão mais expressivamente nos animais exercitados. A injeção central de leptina reduziu a ingestão de ratos exercitados mais expressivamente do que foi observado nos animais controle. Tanto o pré-tratamento com inibidor da IL-6, como com ativador da AMPK ou inibidor da mTOR, reverteram esta ação da leptina. O exercício também está associado à redução da fosforilação da via da AMPK e à maior fosforilação da via da mTOR, no hipotálamo. A da resposta das vias em questão ao estímulo com leptina provavelmente seja um dos principais determinantes da modulação do set point hipotalâmico pelo exercício agudo
Abstract: AMP-activated protein kinase (AMPK) and mammalian Target of Rapamycin (mTOR) are key regulators of cellular energy balance and of the effects of leptin on food intake. Acute exerci se is associated with increased sensitivity to the effects of leptin on food intake in an IL-6-dependent manner. To determine whether exerci se ameliorates the AMPK and mTOR response to leptin in the hypothalamus in an IL-6-dependent manner, rats performed two 3-h exercise bouts, separated by one 45-min rest períod. Intracerebroventrícular IL-6 infusion reduced food intake and pretreatment with AMPK activators and mTOR inhibitors prevented IL-6-induced anorexia. Activators of AMPK increased food intake in control rats to a greater extent than that observed in exercised ones, whereas inhibitors of AMPK had the opposite effect. Exercise was associated with both reduced phosphorylation of the AMPK/ ACC signaling pathway and increased phosphorylation of proteins involved in mTOR signal transduction Ín the hypothalamus. The regulatory role of IL-6 in mediating the modulation in AMPK and mTOR pathways in the hypothalamus was also investigated. Treatment with leptin reduced food intake in exercised rats that were pretreated with vehicle, although no increase in sensitivity to leptin-induced anorexia after pretreatment with anti-IL6 antibody, AICAR or Rapamycin was detected. Improved responses of AMPK and mTOR to leptin may contribute to the appetite suppressive actions of exercise
Mestrado
Ciencias Basicas
Mestre em Clinica Medica
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Romanello, Larissa. "Estudos das enzimas adenosina kinase isoforma 1, hipoxantina-guanina fosforibosiltransferase isoformas 1, 2 e 3, adenilsuccinato liase, adenilsuccinato sintetase de Schistosoma mansoni". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-27102016-102455/.
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O Schistosoma mansoni, parasita responsável pela esquistossomose (barriga dágua), doença que afeta cerca de 300 milhões de pessoas em todo mundo, não possui a via de síntese de purinas, dependendo integralmente da via de salvação de purinas para seu suprimento dessas bases. Uma vez que a terapia se resume a administração de um único fármaco, o praziquantel, diversos casos de resistência do parasita a esse medicamento foram reportadas, sendo assim esta via tem sido citada como alvo potencial para o desenvolvimento de novos fármacos contra a doença. As enzimas adenosina kinase (AK), hipoxantina-guanina fosforibosiltransferase (HGPRT), adenilsuccinato liase (ADSL) e adenilsuccinato sintetase (ADSS) são enzimas chave desta via. Este trabalho faz parte de um projeto maior que visa a obtenção de todas as estruturas das enzimas envolvidas na via de salvação de purinas de Schistosoma mansoni. O cDNA correspondente às enzimas foi amplificado e clonado no vetor de expressão pOPIN; as enzimas AK isoforma 1, HGPRT isoforma 1 e ADSL foram expressas em E. coli Lemo21(DE3) e HGPRT isoforma 3 em E. coli B834(DE3); purificadas em coluna de cobalto agarose por afinidade, concentradas e cristalizadas no kit de cristalização Morpheus (Molecular Dimensions) no Oxford Protein Production Facility (OPPF) em Harwell UK. As coletas de dados por difração de raio-X foram realizadas no Síncrotron Diamond Light Source (DLS) - UK. Foram coletadas duas estruturas de ADSL, a 2.36Å de resolução em complexo com AMP e 2.14Å na forma Apo. A análise das estruturas revelou uma estrutura tetramérica bastante conservada entre as ADSLs, sendo este estado de oligomerização requerido, uma vez que resíduos de três das quatro subunidades compõem o sítio ativo. Apesar do sítio ativo ser altamente conservado entre SmADSL e ADSL humana, a interface dimérica dessas enzimas tem se apresentado suficientemente distintas, o que pode representar um potencial alvo para o desenvolvimento de um inibidor. O ensaio de atividade enzimática de ADSL revelou uma reação endotérmica, indicando que a contribuição da entropia relacionada a grande quantidade de moléculas de água presentes no sítio ativo é importante para a reação cinética. Após diversos experimentos de otimização dos cristais de HGPRT1 e aproximadamente 200 cristais testados, foi obtida uma estrutura em complexo com IMP a 2.8Å de resolução. A análise da estrutura revelou uma estrutura tetramérica. Apesar das subunidades não compartilharem o sítio ativo, este estado de oligomerização é requerido, uma vez que resíduos que compõem o sítio ativo também estão envolvidos em interações na interface dimérica, orientando o resíduo invariável Arg206 na direção do sítio ativo. Foram identificadas quatro mutações na região do sítio ativo entre SmHGPRT e HGPRT humana: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. Desta forma, a obtenção das estruturas contribui para o entendimento bioquímico desta via essencial para o parasita e de como este pode ser seletivamente privado de recursos.Schistosoma mansoni is the parasite responsible for schistosomiasis, disease that affects about 300 million people worldwide, and does not have the purine de novo pathway, depending entirely on the purine salvage pathway to supply its demands on purines. Currently, both direct treatment and most disease control initiatives, rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, has stimulated efforts to develop new drugs for the treatment of schistosomiasis. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenosine kinase (AK), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenylosuccinate lyase (ADSL), adenylosuccinate synthetase (ADSS) are key enzymes in this pathway. This work is part of a larger project aimed at obtaining all the structures of enzymes involved in purine salvage pathway of Schistosoma mansoni. The cDNA corresponding to the enzymes was amplified and cloned in vector pOPIN, AK isoform 1, HGPRT isoform 1 and ADSL were expressed in E. coli Lemo 21 (DE3) and HGPRT isoform 3 in E. coli B834(DE3); purified in cobalt agarose column, concentrated and crystallized in several conditions of the Morpheus (Molecular Dimensions) crystallization kit at the Oxford Protein Production Facility (OPPF) in Harwell UK. The data collection by xray diffraction were performed at Diamond Light Source UK. Two ADSL structures were obtained, ADSL in complex with AMP at 2.36Å resolution and ADSL Apo form at 2.14Å The analysis revealed a tetrameric structure highly conserved between ADSLs, and this oligomerization state is required since residues three of the four subunits comprise the active site. Despite the active site being highly conserved between human ADSL and SmADSL, the dimeric interface of these enzymes it has been shown sufficiently distinct, which may represent a potential target for the development of an inhibitor. The ADSL enzymatic activity assay showed an endothermic reaction, indicating the contribution of the entropy related to the large quantity of water molecules present in the active site is important for the reaction kinetics. After several optimization experiments of HGPRT1 crystals and about 200 crystals tested was obtained a structure in complex with IMP at 2.8Å resolution. The structure analysis revealed a tetrameric structure. Despite the subunits do not share the active site, this oligomerization state is required, since residues that make up the active site are also involved in interactions in dimeric interface, guiding the invariable residue Arg206 toward the active site. Four mutations were identified in the region of the active site between SmHGPRT and human HGPRT: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. These structures increase the important structural information available about the Schistosoma mansoni purine salvage pathway and how it can be selectively private resources.
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FANG, YING. "SIGNALING PATHWAY FROM THE A2B ADENOSINE RECEPTOR TO EXTRACELLULAR SIGNAL REGULATED KINASES (ERK1/2) IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) AND ITS ROLE IN HUVEC PROLIFERATION". University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1148392082.
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Pursell, Natalie W. "Hsp90-Mediated Maturation of Kinases and Nuclear Steroid Hormone Receptors: A Dissertation". eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/535.
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Among heat shock proteins, Hsp90 is unusual because it is not required for the proper folding of most cellular proteins but rather is disproportionally linked to the activation of signal transduction proteins including over forty kinases and many steroid hormone receptors. Mutated forms of many Hsp90 clients are causative agents in cancer, making Hsp90 a promising pharmacological target. Many small molecular inhibitors have been identified that competitively bind to the ATP binding site of Hsp90, some of which are in clinical trials as anticancer agents. Although the activation of kinase and hormone receptor clients by Hsp90 and its co-chaperones has been extensively studied, the molecular mechanism of client protein activation is poorly understood.
Hsp90 is a dimeric chaperone containing three domains: the N-terminal (N) and middle (M) domains contribute directly to ATP binding and hydrolysis and the C-terminal (C) domain mediates dimerization. At physiological concentration, Hsp90 predominantly forms dimers, but the possibility that full-length monomers might also function in cells has not been tested. In Chapter 3, we used a single-chain strategy to design a full-length Hsp90 monomer (NMCC). The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. NMCC Hsp90 was also defective at ATP hydrolysis and the activation of kinase and steroid hormone receptor clients in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric.
After finding that a full-length Hsp90 monomer containing only one ATPase site was unable to support yeast viability or activate Hsp90 clients, we set out to further explore the role of ATPase activity in client protein activation. Approximately 10 % of the yeast proteome binds to Hsp90 making it important to study Hsp90 function in the cellular environment where all binding partners are present. In Chapter 4, we observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered super-stabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. We utilized these super-stabilized dimers to analyze the ability of ATPase mutant homodimers to activate known Hsp90 client proteins in yeast cells. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of the glucocorticoid hormone receptor (GR) and v-src confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo.
In addition to its role in the activation of signal transduction client proteins, Hsp90 has been shown to suppress the in vitro aggregation of numerous hard-to-fold proteins. In Chapter 5, we examine the role of charge in Hsp90 anti-aggregation activity. The charge on Hsp90 is largely concentrated in two highly acidic regions. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. Addition of an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct rescued the anti-aggregation activity of Hsp90 indicating that the net charge contributes to its anti-aggregation activity.
The in vitro anti-aggregation activity of Hsp90 studied in Chapter 5 occurs in the absence of ATP. However, all of the biologically important functions of Hsp90 in cells identified to date, including the maturation of kinases and nuclear steroid hormone receptors, clearly require ATP hydrolysis. Why does Hsp90 robustly hinder the aggregation of hard-to-fold proteins without ATP in vitro, but in vivo uses ATP hydrolysis for all of its essential functions? By utilizing separation of function Hsp90 variants (that specifically lack in vitro anti-aggregation activity) we have begun to address this question. We find that anti-aggregation deficient Hsp90 is unable to support yeast growth under stressful conditions, potentially due to reduced cellular expression. Interestingly, the ATP-independent anti-aggregation activity of Hsp90 has no measureable impact on cellular function. Thus, hindering the aggregation of most hard-to- fold proteins by Hsp90 (independent of ATP hydrolysis) does not appear to be important for cell function. These results suggest a cellular model where the Hsp40/60/70 machinery is responsible for hindering the aggregation of most hard-to-fold proteins while Hsp90 assists in the maturation of a select set of clients in an ATP-dependent fashion, potentially aided by its inherent anti-aggregation properties.