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Biochemical Characterization of Arabidopsis Enzymes Involved in Inositol Pyrophosphate BiosynthesisAdepoju, Olusegun Adeboye 05 September 2019 (has links)
To compensate for the sessile nature of plants, thousands of years of evolution have led to the development of many sophisticated signaling pathways that help plants sense and respond appropriately to different environmental cues. One such signaling pathway is called inositol phosphate signaling. This research dissertation focuses on the inositol phosphate signaling pathway in plants, with emphasis on elucidating how a new class of signaling molecules collectively referred to inositol pyrophosphates are synthesized. Inositol pyrophosphates are an emerging class of "high-energy" intracellular signaling molecules containing one or two diphosphate groups attached to an inositol ring, with suggested roles in bioenergetic homeostasis and inorganic phosphate sensing. Information regarding the biosynthesis of this unique class of signaling molecules in plants is scarce, however the enzymes responsible for their biosynthesis in other eukaryotes have been well described. This work aims to characterize the biochemical activity of the kinase domain (KD) of the Arabidopsis plant diphosphoinositol pentakisphosphate kinase enzymes (AtVIP1 and AtVIP2), and elucidate the biosynthesis pathway of inositol pyrophosphates in plants. Our data indicate that AtVIP1-KD and AtVIP2-KD function primarily as diphosphoinositol pentakisphosphate 5 kinases that phosphorylate this substrate at the 1-position. We also discovered a previously unreported inositol hexakisphosphate kinase activity for the Arabidopsis inositol(1,3,4) triphosphate 5/6kinase enzymes, that can convert InsP6 to InsP7. Together, these enzymes can function in plants to produce inositol pyrophosphates, which have been implicated in signal transduction and phosphate sensing pathways. The significance and potential application of these findings in terms of reduced phytate content and phosphate pollution, improved plant fitness, and improved nutrient use efficiency are discussed. The future outlook of inositol phosphate signaling research is also discussed. / Doctor of Philosophy / Inositol Pyrophosphate Biosynthesis and Subcellular Distribution of Enzymes. Notably, InsP6 which represents the major precursor of PP-InsPs in plants is synthesized in the cytosol, however, it can also be transported into the vacuole by the ABC transporter MRP5. Subcellular localization of enzymes involved in PP-InsP biosynthesis including AtITPK and the kinase domains of AtVIP suggests that these molecules are present in the cytosol and nucleus, and to a smaller extent in the ER. Not shown are the full length and phosphatase domain of AtVIP, which are absent from the nucleus.
See document for accompanied illustration.
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Inositol phosphate generation in the heart : mechanisms and functional relevanceMatkovich, Scot J. Unknown Date (has links) (PDF)
The studies described in this thesis have used principally the rat neonatal cardiomyocyte (NCM) model to investigate previously unresolved questions regarding inositol phosphate signalling in the heart. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is known to be an arrhythmogenic molecule in the setting of cardiacischaemia and subsequent reperfusion, but the mechanisms responsible for its enhanced generation in pathological circumstances, as well as those suppressing its generation during phospholipase C (PLC)-coupled receptor stimulation under physiological conditions, have not been characterised. [3H]Inositol-labelling in combination with anion-exchange high performance liquid chromatography (HPLC)was used to gain an accurate picture of the changes in various [3H]InsP isomers induced by PLC stimulation.
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Caractérisation de suppresseurs de la mort cellulaire programmée chez Arabidopsis thaliana / Characterization of suppressors of programmed cell death in Arabidopsis thalianaBruggeman, Quentin 14 November 2014 (has links)
La Mort Cellulaire Programmée (MCP) est un processus essentiel pour plusieurs aspects de la vie des plantes, incluant le développement et les réponses aux stress. Des analyses génétiques ont permis d’identifier plusieurs acteurs clés de la MCP chez Arabidopsis thaliana,, dont l’enzyme MIPS1, qui catalyse une étape limitante de la biosynthèse du myo-inositol (MI), composé cellulaire majeur à l’origine de nombreux dérivés. Une des caractéristiques les plus importantes du mutant mips1, désactivé pour cette protéine, est l’apparition de lésions sur les feuilles de rosette, dépendante des conditions lumineuses et due à de la MCP impliquant la voie de l’acide salicylique. Ces données avaient permis de révéler un rôle du MI, ou de ses dérivés, dans le contrôle de la MCP. Mon travail de thèse a consisté à rechercher et à caractériser des suppresseurs du mutant mips1 par deux approches complémentaires : une approche gène candidat par comparaison de transcriptome et une stratégie de génétique directe suite au crible de mutations secondaires extra-géniques abolissant le phénotype de mort cellulaire de mips1. Les analyses effectuées sur différents suppresseurs ont mis en évidence l’implication de plusieurs facteurs dans la MCP, tels que le facteur de polyadénylation CPSF30, d’une héxokinase ou encore de la protéine PCB2 intervenant dans la biosynthèse de la chlorophylle. La caractérisation de ces suppresseurs a permis de démontrer l’importance de différentes voies comme la maturation des ARNm, le métabolisme carboné primaire ou l’activité chloroplastique dans le contrôle de la MCP dépendante de l’accumulation de MI. Ce travail apporte de nombreuses perspectives, visant à mieux appréhender les différentes voies de régulation de la MCP indispensables pour un développement correct et pour faire face à des stress biotiques et abiotiques chez les plantes. / Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. Mutational analyses have identified several key PCD components in Arabidopsis thaliana, as the enzyme MIPS1 catalysing the limiting step of myo-inositol (MI) synthesis, crucial cellular compound at the root of many derivatives. One of the most striking features of mips1, disrupted for this protein, is the light-dependent formation of lesions on leaves due to Salicylic Acid (SA)-dependent PCD, revealing roles for MI or inositol derivatives in the regulation of PCD. My thesis work was to find and characterize suppressor of mips1 mutant using two complementary approaches: a gene candidate approach by transcriptomic comparisons and a strategy of direct genetic by screening for extra genic secondary mutations that abolish mips1 cell death phenotype. Analysis of different suppressors revealed the involvement of several factors in MCP, such as the polyadenylation factor CPSF30, a hexokinase or the protein PCB2 operating in chlorophyll biosynthesis. Characterization of these suppressors allowed us to demonstrate crucial role of functions as mRNA maturation, primary carbohydrate metabolism or chloroplastic activity in the regulation of MCP depending on MI accumulation. This work brings many opportunities, to better understand the different regulatory pathways of PCD essential for proper development and to cope with biotic and abiotic stress in plants.
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Galanin receptor ligandsRunesson, Johan January 2009 (has links)
In the nervous system galanin primarily displays a modulatory role. The galaninergic system consists of a number of bioactive peptides with a highly plastic expression pattern and three different receptors. The lack of receptor subtype selective ligands and antibodies have severely hampered the charac-terization of this system. Therefore, most of the knowledge has been drawn from experiments with transgenic animals, which has given some major conclusions, despite the compensatory effects seen in several animal studies. Therefore, the production of subtype selective ligands is of great importance to delineate the galanin system and slowly experimental data from receptor subtype selective ligand trials is emerging. This thesis aims at studying galanin receptor-ligand interactions and to increase and improve the utilized tools in the galanin research field, espe-cially the development of novel galanin receptor subtype selective ligands. Paper I demonstrates the potential to N-terminally extend galanin ana-logues and the successful development of a galanin receptor 2 (GalR2) selec-tive ligand. In addition, a cell line stably expressing galanin receptor 3 (GalR3) was developed, to improve and simplify future evaluations of sub-type selective galanin ligands. Paper II measures the affinities of M617 and M871 to GalR3 and demon-strates that M871 preferentially binds GalR2. Furthermore, the relatively high affinity of M617 was evaluated by assessing the contribution in recep-tor interaction of individual amino acid residues in the C-terminal part of the M617. In conclusion, this thesis has provided a novel design strategy for galanin receptor ligands and increased the understanding of ligand interactions with the GalR3. Furthermore, M1145 has together with new analogues proven to be highly GalR2 specific, holding promises to future delineation of the galaninergic system as a therapeutic target.
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Elucidating the function of inositol pyrophosphate signaling pathways in Arabidopsis thalianaCridland, Caitlin A. 12 April 2022 (has links)
Phosphate (Pi) is an essential nutrient for plants, required for plant growth and seed viability. When Pi is limited, plants undergo dynamic morphological and metabolic changes to leverage available Pi, known as the Phosphate Starvation Response (PSR). The inositol phosphate (InsP) signaling pathway is a crucial element of the plant's ability to regulate the PSR and respond to changing energy conditions. InsPs are synthesized from the cyclic 6-carbon polyol scaffold, myo-inositol. Inositol hexakisphosphate (InsP6) is the most abundant InsP signaling molecule and can be phosphorylated by the multifunctional inositol tetrakisphosphate 1-kinase 1 (ITPK1) and diphosphoinositol pentakisphosphate (VIP) kinases, resulting in inositol pyrophosphates (PP-InsPs). PP-InsPs have high energy bonds and have been linked to Pi maintenance and energy homeostasis in yeast, plants, and mammals. However, the precise mechanism(s) by which PP-InsPs act within plant signaling pathways remains to be determined. Two approaches to understand the role of PP-InsPs in plants are described within this dissertation. The first approach analyzes genetic loss-of-function vip1/vip2 double mutants, and their responses to low Pi conditions. Specifically, vip1/vip2 double mutant gene expression and lipid remodeling patterns in response to low Pi were characterized. We found that vip1-2/vip2-2 had an impacted lipid remodeling response under low Pi conditions, whereas ipk1 had altered lipid composition under Pi-replete conditions. In a complementary approach, a gain-of-function in either the ITPK1 or the kinase domain of VIP (VIP2KD) were constructed in transgenic Arabidopsis thaliana plants. Both ITPK1 and VIP2KD transgenic plants contain elevated levels of the specific inositol pyrophosphate, InsP8. Elevated InsP8 in both types of plants results in changes in growth and senescence phenotypes, delayed time to flowering, Pi accumulation, and altered PSR gene expression. The data from both approaches suggest new roles for PP-InsPs in the regulation of the PSR and other signaling pathways in plants. To enhance my teaching and leadership skills, I participated in the Graduate Teaching Scholars (GTS) program. As a GTS, I worked with the Virginia Tech Research and Extension Experiential Learning (VT-REEL) program where I developed a structured mentorship program for undergraduate and graduate students and created a professional development workshop series. During the COVID-19 pandemic, I developed an online version of the VT-REEL program. Using inclusive pedagogy practices and surveys from the participants, we compiled the best practices for moving a summer undergraduate research program online. These practices come from surveyed participants in the 2020 and provides strategies that can be tailored to various online research experiences and be implemented in both online and in-person formats. / Doctor of Philosophy / Phosphate (Pi) is crucial for plant development and crop yield, but is often limited in soils. Pi-containing fertilizers are often added to supplement soils. Overuse of Pi-containing fertilizers can lead to Pi runoff and can devastate aquatic ecosystems. In addition, Pi is a limited, nonrenewable resource, with U.S. stores projected to be depleted in as little as 30 years. It is now crucial to develop crops that can feed a growing population with less Pi input. Here, we describe how changing levels of plant messenger molecules known as inositol pyrophosphates (PP-InsPs) impact the ability of plants to sense and respond to Pi. This knowledge advances understanding f how mineral nutrient physiology affects many plants traits, and can be harnessed to develop novel strategies to reduce Pi-application and overuse.
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Isolation and Characterization of D-Myo-Inositol-3-Phosphate Synthase Gene Family Members in SoybeanGood, Laura Lee 13 August 2001 (has links)
The objective of this research was to isolate genes encoding isoforms of the enzyme D-myo-inositol 3-phosphate synthase (MIPS, E.C. 5.5.1.4) from soybean and to characterize their expression, especially with respect to their involvement in phytic acid biosynthesis. A MIPS-homologous cDNA, designated GmMIPS1, was isolated via PCR using total RNA from developing seeds. Southern blot analysis and examination of MIPS-homologous soybean EST sequences suggested that GmMIPS1 is part of a multigene family of at least four similar members. The sequences of promoter and genomic regions of GmMIPS1 and GmMIPS2 revealed a high degree of sequence conservation. Northern and western blot analyses showed that MIPS transcript and protein are abundantly expressed early in seed development. Immunolocalization of MIPS protein in developing seeds confirmed expression of MIPS early in seed development and correlated MIPS protein accumulation in soybean seed tissue with tissues in which phytic acid is known to accumulate. The promoter region of GmMIPS1 was isolated and analyzed for possible seed-specificity using promoter:GUS fusions. Two GmMIPS1 promoter fragments were capable of conferring GUS expression when bombarded directly into developing soybean seeds. However, preliminary bombardment experiments into soybean cell suspension culture indicated that both promoter fragments drove expression of GUS in undifferentiated tissue, indicating a potential lack of seed-specificity. / Master of Science
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Régulation des récepteurs glutamatergiques dans différents modèles de vulnérabilité neuronaleValastro, Barbara January 2003 (has links)
Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.
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Cyanide and central nervous system : a study with focus on brain dopamineCassel, Gudrun January 1993 (has links)
The brain is a major target site in acute cyanide intoxication, as indicated by several symptoms and signs. Cyanide inhibits the enzyme cytochrome oxidase. This inhibition causes impaired oxygen utilization in all cells affected, severe metabolic acidosis and inhibited production of energy. In this thesis, some neurotoxic effects of cyanide, in particular, the effects on dopaminergic pathways were studied. In a previous study, decreased levels of striatal dopamine and HVA were found after severe cyanide intoxication (5-20 mg/kg i.p.). However, increased striatal dopamine were found in rats showing convulsions after infusion of low doses of cyanide (0.9 mg/kg i.v.), at the optimal dose rate (the dose rate that gives the treshold dose). Increased striatal dopamine synthesis was observed in rats after cyanide treatment and in vitro. Furthermore, in rat, as well as in pig striatal tissue, cyanide dose- dependently increased the oxidative deamination of 5-HT (MAO-A) and DA (MAO-A and -B) but not that of PEA (MAO-B). Thus cyanide affects both the synthesis and metabolism of dopamine. In rats, sodium cyanide (2.0 mg/kg, i.p.) decreased the striatal dopamine Dj- and D2-receptor binding 1 hour after injection. Increased extracellular levels of striatal dopamine and homovanillic acid were also shown after cyanide (2.0 mg/kg; i.p.). DOPAC and 5-HIAA were slightly decreased. This indicates an increased release or an extracellular leakage of dopamine due to neuronal damage caused by cyanide. Thus the effects of cyanide on dopamine Dj- and D2~receptors could in part be due to cyanide-induced release of dopamine. Because of reported changes in intracellular calcium in cyanide-treated animals, the effects of cyanide on inositol phospholipid breakdown was studied. Cyanide seemed not to affect the inositol phospholipid breakdown in vitro. The effects of cyanide on the synthesis and metabolism of brain GAB A were also examined. A decreased activity of both GAD and GAB A-T were found in the rat brain tissue. The reduced activity of GAB A-T, but not that of GAD returned to the control value after adding PLP in the incubation media. The cyanide-produced reduction of GABA levels will increase the susceptibility to convulsions, and could partly be due to GAD inhibition. In conclusion, cyanide affects the central nervous system in a complex manner. Some effects are probably direct. The main part, however, appears to be secondary, e.g. hypoxia, seizures, changes in calcium levels or transmitter release produced by cyanide. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1993, härtill 7 uppsatser</p> / digitalisering@umu
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Connections Between Inositol Phosphate Signaling and Energy Responses in PlantsWilliams, Sarah Phoebe 19 November 2015 (has links)
The ability for an organism to sense and respond appropriately to its environment is often critical for survival. One mechanism for this is the inositol phosphate (InsP) signaling pathway. This work focuses on the role of InsP signaling in maintaining energy homeostasis in the plant. InsP signaling is connected to energy sensing in plants via a protein complex containing both the inositol polyphosphate 5-phosphatases (5PTase13) and the Sucrose non-Fermenting Related Kinase 1 (SnRK1). SnRK1 is considered a fuel gauge for the plant cell that senses energy status and reprograms growth appropriately. While the SnRK1.1 gene has been well studied, the role other SnRK1 isoforms play in energy or stress signaling is less well understood. This work examined the role of 3 SnRK1 isoforms in energy signaling, finding that SnRK1.1 and SnRK1.2 are regulated and function differently in Arabidopsis. The second part of this work focuses on the inositol pyrophosphates, which are a novel group of InsP signaling molecules containing diphosphate or triphosphate chains (i.e. PPx) attached to the inositol ring. These PPx-InsPs are emerging as critical players in the integration of cellular metabolism and stress signaling in non-plant eukaryotes. Most eukaryotes synthesize the precursor molecule, myo-inositol (1,2,3,4,5,6)-hexakisphosphate (InsP6), which can serve as a signaling molecule or as storage compound of inositol, phosphorus, and minerals. Even though plants produce huge amounts of InsP6 in seeds, almost no attention has been paid to whether PPx-InsPs exist in plants, and if so, what roles these molecules play. This work details the presence of PPx-InsPs in plants and delineates two Arabidopsis gene products (AtVip1 and AtVip2) capable of PP-InsP5 synthesis. We further examined the subcellular location of enzymes connected to PPx-InsP synthesis as well as the developmental and tissue specific patterns of expression of the genes that encode these enzymes. We localized the enzymes involved in InsP6 and PPx-InsP production to the nucleus and endoplasmic reticulum (ER). The subcellular compartmentalization of PPx-InsP signaling may be unique to plants. An increased understanding in the pathways involved in energy sensing and metabolic response may reveal novel strategies to improve crops for yield and viability in the future. / Ph. D.
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Etude des effets de l'inactivation des isoformes B et C de l'enzyme Ins(1,4,5)P3 3-kinase chez la souris. Rôle de l'Ins(1,4,5)P3 3-kinase B dans le développement des lymphocytes T.Pouillon, Valérie 28 January 2004 (has links)
L’Ins(1,4,5)P3 joue un rôle évident dans la signalisation cellulaire : il permet la libération du Ca 2+ des stocks intracellulaires par son action au niveau de récepteurs spécifiques. Pour mettre fin à son action, l’Ins(1,4,5)P3 peut être dégradé par une Ins(1,4,5)P3 5-phosphatase en Ins(1,4)P2, un métabolite inactif. L’Ins(1,4,5)P3 peut aussi être transformé en Ins(1,3,4,5)P4 par une Ins(1,4,5)P3 3-kinase. L’Ins(1,3,4,5)P4 semble posséder des capacités de signalisation propres ou au contraire liées à celles de l’Ins(1,4,5)P3.
L’Ins(1,3,4,5)P4 est aussi le point de départ de toute une série d’inositol hautement phosphorylés, dont les rôles ne sont pas clairs. Trois isoformes de l’Ins(1,4,5)P3 3-kinase existent (A, B et C). Ces isoformes possèdent un domaine catalytique carboxy-terminal bien conservé. Par contre, les domaines amino-terminaux sont spécifiques et leur permettraient d’établir des interactions ou de subir des régulations propres. Pour tenter d’élucider le rôle fonctionnel de l’Ins(1,3,4,5)P4, nous avons généré et analysé des souris déficientes pour les isoformes B et C de cette enzyme.
Les souris déficientes pour l’Ins(1,4,5)P3 3-kinase C ne présentent pas de phénotype évident, ce qui suggère que son rôle n’est pas crucial ou que son absence peut être compensée par une autre enzyme.
Les souris déficientes pour l’Ins(1,4,5)P3 3-kinase B, par contre, présentent une immunodéficience caractérisée par une absence spécifique des lymphocytes T αβ périphériques. Cette absence fait suite à un blocage dans la différenciation du précurseur du lymphocyte, le thymocyte. Les caractéristiques de la signalisation induite par le récepteur de surface (TCR) permettent la sélection des thymocytes, de manière à constituer un pool de lymphocytes T restreints pour le MHC et tolérants pour le soi. Nous avons montré que ces phénomènes de sélection étaient défectueux dans les thymocytes mutants, du fait de leur hyporéactivité à la stimulation par le TCR. Le mécanisme responsable de cette hyporéactivité n’est pas encore élucidé. A première vue, la mobilisation de Ca 2+ ne semble pas altérée dans ces thymocytes mutants en réponse à des stimulations classiques. Cependant, d’autres types de stimulation, se rapprochant plus de celles réellement rencontrées par le thymocyte in vivo, doivent encore être investigués. L’intégrité d’autres voies de signalisation cruciales du lymphocyte T doit aussi être vérifiée.
En conclusion, l’isoforme B de l’Ins(1,4,5)P3 3-kinase et l’Ins(1,3,4,5)P4 qu’il produit jouent un rôle crucial dans la différenciation du thymocyte, par un mécanisme qui reste encore à déterminer.
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