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Physiologie und Biophysik der pflanzlichen GlutamatrezeptorenAnschütz, Uta January 2008 (has links)
Würzburg, Univ., Diss., 2008.
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Der Membranteil von H+-ATPasen Struktur des CF0 aus Spinatchloroplasten, Funktion des EF0 aus E.coli /Eisfeld, Jochen. January 1900 (has links) (PDF)
Freiburg (Breisgau), Univ., Diss., 2003. / Computerdatei im Fernzugriff.
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The osteoclast H⁺-ATPase isolation and initial characterization /Mattsson, Jan P. January 1995 (has links)
Thesis (doctoral)--University of Göteborg, 1995. / Includes bibliographical references.
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Isolation and Functional Studies of The F-type ATP Synthase from Spinach Chloroplasts and Heliobacterium modesticaldumJanuary 2015 (has links)
abstract: Adenosine triphosphate (ATP) is the universal chemical energy currency in most living cells, used to power many cellular reactions and generated by an enzyme supercomplex known as the ATP synthase, consisting of a hydrophilic F1 subcomplex and a membrane-bound FO subcomplex. Driven by the electrochemical gradient generated by the respiratory or photosynthetic electron transport chain, the rotation of the FO domain drives movements of the central stalk in response to conformational changes in the F1 domain, in which the physical energy is converted into chemical energy through the condensation of ADP and Pi to ATP. The exact mechanism how ATP synthesis is coupled to proton translocation is not known as no structure of the intact ATP-synthase nor the intact FO subcomplex has been determined to date. Structural information may shed light on these mechanisms and aid in understanding how structural changed relate to its coupling to ATP synthesis. The work in this thesis has successful established a defined large-scale CF1FO isolation procedure resulting in high purity and high yield of this complex from spinach thylakoid membranes by incorporating a unique combination of biochemical methods will form the basis for the subsequent structural determination of this complex. Isolation began from the isolation of intact chloroplasts and the separation of intact thylakoid membranes. Both native and denaturing electrophoresis analyses clearly demonstrated that the purified CF1FO retains its quaternary structure consisting of the CF1 and CFO subcomplexes and nine subunits (five F1 subunits: α, β, γ, δ and ε, and four FO subunits: a, b, b' and c). Moreover, both ATP synthesis and hydrolysis activities were successfully detected using protein reconstitution in combination with acid-base incubation and in-gel ATPase assays, respectively. Furthermore, the ATP-synthase of H. modesticaldum, an anaerobic photosynthetic bacterium, was also isolated and characterized at the biochemical level. These biochemical characterizations directly influenced recent studies on the high-resolution structure determination of intact CF1FO using electron crystallography on two-dimensional crystals. The availability of the functionally intact CF1FO purified at a large scale will lead to studies that investigate the possible crystallization conditions to ultimately determine its three-dimensional structure at atomic resolution. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2015
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Flow injection analysis with bioluminescence detectionNawawi, Mustaffa bin January 1987 (has links)
The detection of bacterial contamination of water, pharmaceutical products etc. is of great importance, and is most conveniently performed by the detection of bacterial ATP (Adenosine TriPhosphate) using the luciferin-luciferase bioluminescence system. This system uses unstable and expensive reagents, and emits transient light signals. In this study an FIA (Flow Injection Analysis) system was set up to monitor the light signal produced by the reaction. Using a luminometric detector (a liquid scintillation counter) with the FIA system, the reaction length, sample volume, flow rates, pH etc. were investigated.
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Investigating the Interaction Mechanism and Effect of ATP on Alpha-Synuclein Aggregation by NMR SpectroscopyKamski-Hennekam, Evelyn January 2022 (has links)
Recent studies suggest that Adenosine Triphosphate (ATP) can either enhance or inhibit the aggregation of amyloid proteins, depending on the interaction mechanism as well as specific protein properties. The connection between ATP and protein solubility is particularly important in Parkinson’s Disease (PD), where the aggregation of alpha-synuclein (αS) is closely linked to pathology. Since the greatest risk factor for PD is aging, and ATP levels decline dramatically with age and are greatly reduced in the brains of patients with early PD, it is possible that the modulating effect of ATP on protein solubility is a factor in PD onset. However, the driving mechanism behind the interaction of ATP and αS is currently unclear, as is the effect of physiologically-relevant ATP concentrations on early- and late-stage αS aggregation. Here, we determine using NMR spectroscopy that the triphosphate moeity of ATP drives its electrostatic interaction primarily with the N-terminal pseudo-apolipoprotein repeats of αS monomers. These interactions are modulated by magnesium and disrupt long-range N- to C-terminal contacts in αS monomers, causing a concentration-dependent enhancement of initial αS aggregation. We also show by Thioflavin T fluorescence as well as electron microscopy that ATP inhibits late-stage αS β-sheet fibril formation in a phosphate-dependent manner. Our NMR data reveals that ATP inhibits αS monomer-fibril interactions, suggesting that ATP attenuates αS secondary nucleation. Lastly, we show that the effects of ATP are different in the presence of PD-related αS mutations E46K and A53T. Overall, our study contributes a thorough characterization of the biologically- and pathologically-relevant interactions between ATP and αS, while also proposing a role for ATP in the age-related development of PD pathology. / Thesis / Master of Science (MSc) / Alpha-synuclein (αS) is a protein whose abnormal aggregation is characteristic of Parkinson’s Disease (PD). Adenosine Triphosphate (ATP) is a molecule that has recently been shown to reduce the aggregation of select disease-causing proteins. Therefore, the aim of this study is to characterize the interaction mechanism between ATP and αS, to explore how this interaction influences αS structural dynamics and to determine the effect of ATP on early- and late-stage αS aggregation. Another overall aim of this study is to characterize how the ATP-αS interaction is influenced by PD-related mutations in αS. To accomplish these aims, we will rely primarily on NMR spectroscopy as well as fluorescence and microscopy techniques. Our goal is to determine the role of ATP in αS aggregation as well as potentially connect the age-related decrease in ATP levels with PD, an age-related disease.
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The Effects of Neuropathy-Inducing Organophasphate Esters om Chick Dorsal Root Gangli Cell CulturesMassicotte, Christiane 09 December 2001 (has links)
Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN.
Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN. / Ph. D.
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Efeito da estimulação purinérgica sobre a produção de melatonina em macrófagos da linhagem RAW 264.7 / Purinergic stimulation effect on melatonin production in RAW 264.7 macrophages lineageGarcia, Letícia D\'Argenio 22 February 2016 (has links)
A melatonina é um hormônio produzido de forma rítmica e no período de escuro pela glândula pineal bem como de forma não rítmica por diversos tecidos e células imunocompetentes. É sintetizada pela acetilação e metilação da serotonina pela ação das enzimas arilalquilamina N-acetiltransferase (AA-NAT) e acetilserotonina -O-metiltransferase (ASMT) que levam à formação de N-acetilserotonina (NAS) e melatonina (MEL), respectivamente. Nos últimos anos temos demonstrado que síntese de melatonina pela pineal pode ser negativamente modulada por mediadores inflamatórios e pelo ATP que atua como co-transmissor juntamente com a noradrenalina liberada no terminal nervoso simpático que a inerva. Perifericamente, contudo, estes mediadores inflamatórios apresentam um efeito contrário induzindo a produção de melatonina em células imunocompetentes. Estas observações levaram à criação da hipótese de um eixo imune-pineal. Esse trabalho teve como objetivo verificar o efeito do ATP sobre produção de melatonina em macrófagos da linhagem RAW 264.7 Os dados desse trabalho mostram que o ATP é capaz de induzir de maneira dose dependente a produção de melatonina em macrófagos através da modulação das enzimas AA-NAT e ASMT. Foi demostrado também que esse efeito é mediado pelo receptor P2X7 e que a melatonina produzida age autocrina e paracrinamente aumentando a fagocitose de particulas de zimosan. Com isso, podemos concluir que o ATP é um ativador endógeno do eixo imune-pineal / During the dark phase, melatonin is produced rhythmically by the pineal gland. Besides, a non rhythmical production is observed in several tissues and immunocompetent cells. In both scenarios, melatonin biosynthetic pathway is dependent on serotonina methylation by the action of arylalkylamine N-acetyltransferase (AA-NAT), leading to the formation of N-acetylserotonin (NAS), which will be a further target for acetylserotonin O-methyltransferase (ASMT), the last step in melatonin synthesis. In the last years, we have demonstrated that melatonin synthesis by the pineal gland may be negatively modulated by inflammatory mediators such as adenosine triphosphate (ATP), which also acts as co-transmitter released along with noradrenalin by the sympathetic nerve terminals. However, these inflammatory stimuli have the opposite effect inducing melatonin production in immunocompetent cells. These observations led us to the hypothesis of an immune-pineal axis. Therefore, this study aimed to investigate if the ATP was able to induce melatonin production in RAW 264.7 macrophages. ELISA assays demonstrate that high doses of ATP induce melatonin synthes. Accordingly, ATP is able to induce an increase in the expression of AA-NAT as well as the ASMT. Herein, we also demonstrated that this effect is mediated by P2X7 receptor, and melatonin-ATP induced acted as an autocrine and paracrine message that increases the phagocytosis of zymosan particles. Therefore, we conclude that ATP is an endogenous activator of the RAW 264.7
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Caractérisation de lATP synthétase mitochondriale (complexe V) de lalgue verte Chlamydomonas reinhardtii. Spécialisation et évolution de lenzyme chez les Chlorophyceae.Lapaille, Marie 28 April 2010 (has links)
Résumé
Le complexe V mitochondrial (F1FO-ATP synthétase) catalyse la phosphorylation de lADP par le phosphate inorganique en utilisant la force proton-motrice générée par la chaîne de transport délectrons. Ce complexe protéique possède deux domaines : un secteur associé à la membrane, FO, impliqué dans la translocation des protons, et un domaine extrinsèque, F1, qui catalyse la synthèse dATP. Les deux secteurs sont connectés par deux bras : un bras central qui couple la translocation des protons à la région catalytique, et un bras latéral qui est considéré comme faisant partie du stabilisateur (stator) de lenzyme.
Au cours de ce travail, nous nous sommes intéressés à lenzyme de deux algues appartenant à la classe des Chlorophyceae, Chlamydomonas reinhardtii et Polytomella sp.. L'enzyme des deux algues présente une composition sous-unitaire atypique, les sous-unités classiquement retrouvées chez les eucaryotes et impliquées dans larchitecture du bras périphérique ou dans la dimérisation du complexe en étant absentes. En contrepartie, 9 sous-unités dorigine évolutive inconnue sont associées à lenzyme. Elles ont été appelées Asa1 à 9 pour ATP Synthase Associated protein.
Chez C. reinhardtii et Polytomella sp., lATP synthétase présente une stabilité accrue de sa forme dimérique in vitro, et, in vivo, les cellules de C. reinhardtii sont insensibles à loligomycine, un puissant inhibiteur de la translocation de protons au travers de FO.
Nous avons dans un premier temps tenté détablir la composition sous-unitaire du complexe V chez des espèces appartenant aux différentes classes de Chlorophytes (Chlorophyceae, Trebouxiophyceae, Prasinophyceae et Ulvophyceae) en combinant analyses génomiques et protéomiques. Plusieurs sous-unités Asa ont ainsi pu être détectées chez des algues appartenant à divers ordres de Chlorophyceae. Au contraire, les analyses de séquences disponibles chez les autres classes de Chlorophytes (Trebouxiophyceae, Prasinophyceae et Ulvophyceae) indiquent une composition canonique de lenzyme.
Lanalyse de la stabilité de la forme dimérique du complexe de différentes espèces d'algues vertes sur BN PAGE (Blue Native PolyAcrylamide Gel Electrophoresis) suggère également que la présence dun dimère stable est caractéristique aux Chlorophyceae. Par ailleurs, leur croissance, respiration, et niveaux d'ATP sont à peine affectés par la présence d'oligomycine à des concentrations inhibitrices chez les représentants des autres classes de Chlorophytes.
Les nombreuses particularités communes aux algues appartenant à cette classe suggèrent que la perte d'éléments canoniques du stator est apparue lors de la séparation des Chlorophyceae et a été accompagnée du recrutement de nouvelles sous-unités. Ce réarrangement drastique de la composition de stator et du module de dimérisation pourrait avoir conféré de nouvelles propriétés à lenzyme, notamment une meilleure stabilité et une plus grande résistance à loligomycine.
Nous avons également étudié la fonction de la sous-unité atypique Asa7 en inactivant son expression chez C. reinhardtii. Bien que la perte de la sous-unité Asa7 n'aie aucun impact sur la bioénergétique des cellules ou sur la structure mitochondriale, elle déstabilise lenzyme in vitro et rend la croissance, la respiration, et de le niveau d'ATP sensible à oligomycine.
L'impact de la perte de l'activité ATP synthétase mitochondriale chez un organisme photosynthétique a été étudié chez C. reinhardtii par linactivation de l'expression du gène ATP2, codant pour la sous-unité catalytique beta. Les résultats démontrent que, en l'absence de beta, l'ATP synthétase ne peut plus être assemblée et les cellules deviennent dépendantes de la photosynthèse. La respiration en présence ou en absence du découpleur CCCP suggère que le passage des protons à travers la membrane interne mitochondriale est bloqué chez la souche mutante. Enfin, la morphologie des mitochondries est affectée, et les chloroplastes montrent un réaménagement massif de l'appareil photosynthétique, suggérant des répercussions importantes sur la synthèse dATP par les chloroplastes. Ces résultats contribuent à la compréhension des interactions entre organites bioénergétiques chez les organismes photosynthétiques.
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Calcium Signaling Mechanisms Mediate Clock-Controlled ATP Gliotransmission among Immortalized Rat SCN2.2 Cell CulturesBurkeen, Jeffrey Franklin 2009 August 1900 (has links)
The hypothalamus is an integral part of the brain's regulation of mammalian physiology and behavior. Among many functions, this regulatory center activates the sympathetic nervous system, maintains appropriate body temperature, controls food intake, and controls release of hormones from the pituitary gland. Deep within the hypothalamus lie a paired cluster of cells, the suprachiasmatic nuclei (SCN), which function as the chief circadian pacemaker. The goal of the present thesis research was to study rhythmically controlled ATP gliotransmission. I used an immortalized SCN2.2 hypothalamic cell line to determine the mechanism by which ATP signaling is regulated in this context. Additionally, this research aimed to elucidate if clock-controlled ATP gliotransmission is fundamentally distinct from stimulus-evoked calcium-dependent mechanisms that regulate intercellular ATP signaling among astrocytes.
In this thesis, I show that there are multiple ATP signaling mechanisms present among SCN2.2 cells. cAMP-dependent signaling mediates clock-controlled ATP accumulation but not stimulus-evoked ATP signaling. In addition, pharmacological studies suggest that disparate purinergic receptor-mediated mechanisms are involved in the regulation of clock-controlled versus stimulus-evoked ATP signaling.
Rhythmic accumulation of ATP in SCN2.2 cultures is modulated by calcium-dependent processes. Peaks in ATP accumulation coincide with elevated mitochondrial calcium levels, while troughs in ATP accumulation coincide with periods of high cytosolic calcium levels, suggesting a possible mechanistic link between circadian shifts in intracellular calcium handling and ATP handling in SCN2.2 cells. Clock-controlled ATP accumulation in SCN2.2 cells is not a by-product of rhythmic cell cycle or rhythmic cell death.
Overall, my research suggests that the ATP accumulation rhythm in SCN2.2 cells is likely an output of the biological clock, mediated by astrocytic calcium signaling processes, and not an output of cell division or cell death. Estimation of ATP accumulation in SCN2.2 cultures at peak time points suggests that clock-controlled ATP release is critical to the function of astrocytes in the mammalian brain, perhaps in the regulation of brain metabolism, the regulation of sleep/wake physiology, or the integration of both.
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