Understanding how plants respond to stress is of importance, considering the increasing need to feed a growing population and supply its energy. Plants have complex systems for detecting, and responding to stresses. One stress-responsive system involves myo-inositol (Ins). Ins is a precursor for cell wall components, inositol trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol phosphate signaling molecules, and an alternate ascorbic acid (AsA) synthesis pathway. The enzyme, myo-inositol oxygenase (MIOX) is encoded by four genes in Arabidopsis and catalyzes the first step of Ins catabolism producing D-glucuronic acid (DGlcA). This research focuses on MIOX metabolism of Ins during plant growth and stress responses. I have examined miox mutants for alterations in metabolism and signaling. MIOX2 and MIOX4 expression patterns correlate with miox mutant root growth in varying nutrient conditions, and changes in flowering time. In miox2 mutants, I found an increase in Ins in most tissues, which was accompanied by cold- and abscisic (ABA)- sensitivity; however, miox4 mutants are ABA- insensitive, and have a small increase of Ins in flowers. MIOX2:GFP fusion protein accumulates in the cytoplasm and MIOX4:GFP accumulates in the cytoplasm and nucleus. Overexpresser MIOX4+ plants provide a model system to examine how directing carbon from Ins into DGlcA impacts Ins levels and Ins signaling. I have examined MIOX4+ plants for alterations in MIOX4 RNA and protein, and measured Ins by gas chromatography (GC). My results indicate that MIOX4+ tissues are impacted differently by the MIOX4 transgene, with decreases in Ins after seed imbibition, and increased Ins levels later in development. Ins depletion in seedlings was correlated with a decrease in Ins(1,4,5)P3. To determine the impact of reducing Ins and Ins(1,4,5)P3 in MIOX4+ seedlings, I examined processes known to involve Ins(1,4,5)P3 signaling. MIOX4+ seed have increased seed dormancy, NaCl-sensitivity, and ABA-insensitivity. These results suggest MIOX affects Ins signaling in response to ABA. Together, these data indicate that transcriptional control of MIOX2 and MIOX4 results in distinct roles in plant growth, and that MIOX2 and MIOX4 function in metabolic and signaling processes critical for growth, nutrient sensing, and stress responses. / Ph. D.

The complete role of inositol signaling in plants and humans is still elusive. The plant Arabidopsis thaliana contains fifteen predicted inositol polyphosphate 5- phosphatases (5PTases, E.C. 3.1.3.36) that have the potential to remove a 5-phosphate from various inositol second messenger substrates. To examine the substrate specificity of one of these Arabidopsis thaliana 5PTases (At5PTases), recombinant At5PTase1 was obtained from a Drosophila melanogaster expression system and analyzed biochemically. This analysis revealed that At5PTase1 has the ability to catalyze the hydrolysis of four potential inositol second messenger substrates. To determine whether At5PTase1 can be used to alter the signal transduction pathway of the major drought-sensing hormone abscisic acid (ABA), plants ectopically expressing At5PTase1 under the control of a constitutive promoter were characterized. This characterization revealed that plants ectopically expressing At5PTase1 had an altered response to ABA. These plants have stomata that are insensitive to ABA, and have lower basal and ABA-induced inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] levels. In addition, At5PTase1 mRNA and protein levels are transiently regulated by ABA. These data strongly suggest that At5PTase1 can act as a signal terminator of ABA signal transduction. Like the Arabidopsis At5PTase1, a human 5PTase, Ocrl, has the ability to catalyze the hydrolysis of a 5-phosphate from several inositol-containing substrates. The loss of functional Ocrl protein results in a rare genetic disorder known as Lowe oculocerebrorenal syndrome. To gather information concerning the specificity determinants of the Ocrl protein, a structure-function analysis of Ocrl was conducted using a vibrational technique, difference Fourier transform infrared (FT-IR) spectroscopy. Upon the introduction of Ins(1,4,5)P₃ substrate, structural changes in carboxylic acid and histidine residues were observed. The net result of changes in these residues indicates that upon Ins(1,4,5)P₃ introduction, a carboxylic acid-containing residue is protonated, and a histidine residue is deprotonated. This interpretation supports the idea that the deprotonation of the histidine residue is concomitant with the coordination of a divalent cation upon Ins(1,4,5)P₃ introduction. This work allows for the proposal of a new model for the role of the active site histidine of OCRL. / Ph. D.

myo-Inositol synthesis and catabolism are crucial in many multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. myo-inositol monophosphatase (IMP) is a major enzyme required for the synthesis of myo-inositol and breakdown of inositol (1,4,5)-trisphosphate (InsP3), a potent second messenger involved in many biological activities. Arabidopsis contains a single canonical IMP gene, which was previously shown in our lab to encode a bifuntional enzyme with both IMP and L-galactose 1-phosphatase activity. Analysis of metabolite levels in imp mutants showed only slight modifications with less myo-inositol and ascorbate accumulation in these mutants. This result suggests the presence of other functional IMP enzymes in plants. Two other genes in Arabidopsis encode chloroplast proteins, which we have classified as IMP-like (IMPL), because of their greater homology to the prokaryotic IMPs such as the SuhB, and CysQ proteins. Prokaryotic IMP enzymes are known to dephosphorylate D-Inositol 1-P (D-Ins 1-P) and other substrates in vitro, however their in vivo substrates are not characterized. A recent study revealed the ability of IMPL2 to complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, which suggested an important role for IMPL2 in amino acid synthesis. The research presented here focuses on the characterization of IMPL functional roles in plant growth and development. To accomplish this I performed kinetic comparisons of the Arabidopsis recombinant IMPL1 and IMPL2 enzymes with various inositol phosphate substrates and with L-histidinol 1-phosphate, respectively. The data supports that IMPL2 gene encodes an active histidinol 1-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme which has the ability to hydrolyze D-Ins 1-P substrate and may be involved in the recycling of inositol from the second messenger, InsP3. Analysis of metabolite levels in impl2 mutant plants reveals that impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway. Together these data solidify the catalytic role of IMPL2 in histidine synthesis in plants and highlight its importance in plant growth and development. / Ph. D.

Myo-inositol is ubiquitous in nature and is found at the structural core of a diverse range of biologically important derivatives, including phosphatidylinositols, inositol phosphates and mycothiol. The synthesis of myo-inositol derivatives is notoriously difficult due to the need to control both regio- and enantioselectivity. As a result, synthetic routes to derivatives of this type are often lengthy and low yielding. The first biosynthetic step in the production of all myo-inositol metabolites is the isomerisation of D-glucose 6- phosphate to L-myo-inositol 1-phosphate as mediated by L-myo-inositol 1-phosphate synthase (INO1). For the protozoan parasite Trypanosoma brucei, INO1 is essential for survival and its version of the enzyme (TbINO1) has a high turnover. This makes TbINO1 an attractive candidate for the biocatalytic production of L-myo-inositol 1- phosphate, and a potential starting point for drastically shortened syntheses of important myo-inositol derivatives. The production of L-myo-inositol 1-phosphate by TbINO1 has been optimised to achieve complete conversion in reaction conditions that facilitate product isolation. Due to problems with an in-batch process, the TbINO1 enzyme was immobilised and the process was transferred to a flow system. This has allowed for production of significant quantities of L-myo-inositol 1-phosphate with a high level of purity. L-myo-inositol 1- phosphate obtained from the flow system has been used to prepare mycothiol glycosylation acceptor, 1,2,4,5,6-penta-O-acetyl-D-myo-inositol, in a concise synthesis with a greatly improved yield over the literature.

Les inositols phosphoglycans (IPGs) sont des médiateurs de l'action de l'insuline. Le DCI-IPG contenant du D-chiro-inositol (DCI) potentialise l'action de l'insuline en diminuant la glycémie. Il a déjà été observé qu'il y aurait une diminution de l'utilisation ou de la disponibilité tissulaire du DCI chez les femmes SOPK qui sont caractérisées par une résistance à l'insuline (RI). Notre hypothèse est qu'une augmentation de la clairance urinaire de 24h du DCI, (uCl[indice inférieur DCI]) ou de son précurseur le myo-inositol (MYO), pourrait résulter en une disponibilité moindre du DCI pour les tissus et une libération réduite du DCI-IPG par l'insuline, et ainsi contribuer à la RI globale chez l'humain, relié ou non au SOPK. Nous proposons que ce défaut serait corrigé par un agoniste des PPAR[gamma]. Nous avons déjà démontré et publié que les frères de femmes SOPK sont de bons modèles de RI et qu'ils présentent aussi une altération dans le métabolisme des inositols. Un groupe de 11 frères et 21 hommes contrôles ont été étudiés et les résultats ont fait l'objet d'un article original que j'ai écrit et inclus dans ce mémoire. Dans cette étude, nous avons déterminé que la uCl[indice inférieur DCI] était diminuée de moitié et qu'au contraire les concentrations plasmatiques à jeun du DCI étaient plus que 3 fois augmentées pour le groupe des frères par rapport au groupe témoin. Par ailleurs, 16 hommes et 9 femmes témoins ainsi que 2 femmes SOPK ont été recrutés dans le cadre d'un autre projet pilote au cours duquel une hyperglycémie provoquée orale (HGPO) et un clamp euglycémique-hyperinsulinémique ont été effectués. Le rythme d'apparition des acides gras libres (Ra[indice inférieur AGL]), la production endogène du glucose, les métabolismes oxydatif et non-oxydatif des hydrates de carbone (HC), la uCl[indice inférieur DCI], les concentrations plasmatiques du DCI et du MYO ainsi que la bioactivité du DCI-IPG ont été déterminés.Les résultats obtenus au cours de cette étude pilote ont été analysés avec les résultats obtenus pour les frères de femmes SOPK et inclus dans la section 2 de ce mémoire. Nous avons mesuré une sensibilité à l'insuline plus basse ainsi que des valeurs d'aire sous la courbe (AUC) pour le glucose et l'insuline plus élevées pour les frères que pour les groupes témoins. En regard des inositols plasmatiques, les niveaux de DCI des femmes sont plus bas que ceux des hommes peu importe le groupe et une uCl[indice inférieur DCI] plus élevée a été observée chez les 2 groupes témoins par rapport au groupe des frères. Nos résultats démontrent donc que les frères sont de bons modèles de RI. Nos hypothèses de départ n'ont pu être vérifiées étant donné le peu de patients insulinorésistants inclus dans l'étude pilote. Cependant, pour les hommes, nous avons observé une corrélation positive entre la uCI[indice inférieur DCI] et les niveaux d'insuline. Ce projet est toujours en cours dans le but de recruter des sujets possédant un plus large éventail de RI afin de vérifier nos hypothèses.

Le myo-inositol (MI) est un soluté organique impliqué dans diverses fonctions physiologiques de la cellule dont la signalisation cellulaire. Il est également un osmolyte compatible reconnu. Trois co-transporteurs de type actif secondaire responsables de son absorption ont été identifiés. Deux d’entre eux sont couplés au transport du sodium (SMIT1 et SMIT2) et le troisième est couplé au transport de protons (HMIT). L’objectif de cette étude a été la caractérisation du transport du MI par SMIT2 dans des membranes en bordure en brosse (BBMv) issues du rein de lapin et de l’intestin de rat ainsi qu’après expression dans les ovocytes de Xenopus laevis. La quantification de l’ARNm de SMIT1 et de SMIT2 dans le rein nous a appris que SMIT1 est majoritairement présent dans la médullaire alors que SMIT2 est principalement localisé dans le cortex. Ces résultats ont été confirmés par immunobuvardage en utilisant un anticorps dirigé contre SMIT2. Grâce à l’inhibition sélective de SMIT1 par le L-Fucose et de SMIT2 par le D-chiro-inositol (DCI), nous avons démontré que SMIT2 semble le seul responsable du transport luminal de MI dans le tubule contourné proximal avec un Km de 57 ± 14 µM. Pour ce qui est de l’intestin, des études de transport de MI radioactif ont démontré une absence de transport de MI chez le lapin alors que l’intestin de rat présente un transport de MI très actif. Une quantification par qRT-PCR nous a permis de constater que l’intestin de lapin ne semble pas posséder les transporteurs de MI nécessaires. Comme pour le rein, SMIT2 semble le seul transporteur de MI présent au niveau du pôle apical des entérocytes intestinaux chez le rat. Il est chargé du prélèvement du MI de l'alimentation avec un Km de 150 ± 40 µM. Les analyses fonctionnelles exécutées sur SMIT2 de rat en électrophysiologie après expression dans les ovocytes de Xenopus laevis donnent sensiblement les mêmes résultats que pour les BBMv de rein de lapin et d’intestin de rat. Dans les ovocytes, SMIT2 présente une grande affinité pour le MI (270 ± 19 µM) et le DCI (310 ± 60 µM) et aucune affinité pour le L-fucose. Il est ii également très sensible à la phlorizine (16 ± 7 µM). Une seule exception persiste : la constante d’affinité pour le glucose dans les BBMv d’intestin de rat est 40 fois plus petite que celle observée sur les ovocytes de Xenopus laevis. Nous avons également testé la capacité de certains transporteurs de sucre présents à la surface des membranes apicales des entérocytes à prélever le MI. Vu que l'inhibition de ces transporteurs (SGLT1 et GLUT5) ne changeait rien au taux de MI radioactif transporté, nous en avons conclu qu'ils ne sont pas impliqués dans son transport. Finalement, l’efflux de MI à partir du pôle basolatéral des entérocytes n’est pas effectué par GLUT2 puisque ce dernier lorsqu'il est exprimé dans des ovocytes, est incapable de transporter le MI. / Myo-inositol (MI) is an organic solute involved in various aspects of cell physiology, including cell signaling. It is also known as a compatible osmolyte. Three secondary active MI cotransporters have been identified; two are Na+- coupled (SMIT1 and SMIT2) and one is H+-coupled (HMIT). The main aim of this study was to characterize MI uptake throught SMIT2 as expressed in epithelial cells and in Xenopus laevis oocytes. In order to achieve the characterization of this transport system, we used purified brush border membrane vesicles (BBMv) isolated from rabbit kidney and rat intestine. We first performed a quantification of mRNA levels in rabbit kidney using real time PCR for both SMIT1 and SMIT2. We found that SMIT1 is mainly expressed in the renal medulla while SMIT2 is mainly localized in the renal cortex. This result was confirmed on Western blots using an antibody raised against SMIT2. Through inhibition studies using selective substrates for SMIT1 (inhibited by L-fucose) and SMIT2 (inhibited by D-chiroinositol), we showed that SMIT2 seems to be responsible for all the apical transport of MI into the proximal convoluted tubule with a Km of 57 ± 14 µM. By transport studies we established that rabbit intestine seems to lack apical transport of MI while rat intestine has a very active uptake of this molecule. qRT-PCR quantification confirmed the absence of MI transporters in rabbit intestine. As for kidney, SMIT2 seems to be the only transporter responsible for apical MI uptake in enterocytes with a Km of 150 ± 40 µM. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from rat intestine and rabbit kidney. SMIT2 displays high affinities for MI (270 ± 19 µM), DCI (310 ± 60 µM) and no affinity for L-fucose. SMIT2 is very sensitive to phlorizin (Pz; 16 ± 7 µM). Although these functional characteristics essentially confirmed those found in rat intestine, a iv discrepancy exists between the two systems studied. Indeed, the affinity constant for glucose was approximately 40-fold lower in vesicles than in oocytes. We also tested the ability of SGLT1 and GLUT5, other sugar transport systems present in enterocytes apical membranes, to perform MI uptake. Because the inhibition of these transporters did not alter radiolabeled MI uptake, we concluded that they had no significant contribution to MI transport in rat intestine. Finally, the basolateral efflux of MI was not mediated by GLUT2 because when expressed in oocytes, this transporter was not able to transport MI.

It has been six decades since the discovery of lithium for the treatment of bipolar disorder. There is, as yet, no conclusive evidence as to how lithium produces this therapeutic effect, since it is known to interact with multiple cellular targets. One of the most credible targets is the enzyme, inositol monophosphatase (lMPase), which plays a crucial role in cell signalling. My aim was to find a novel IMPase inhibitor and evaluate it as a possible lithium-like mood stabiliser by using enzyme, cell and whole animal experiments. To achieve this, I created recombinant human and mouse IMPase enzymes and then used these for screening and crystallisation. I used two different approaches for the small-molecule screening: substrate-based virtual screening and drug repositioning using a library of compounds with clinically proven safety. I identified ebselen as a novel IMPase inhibitor suitable for drug repositioning. I determined thatebselen inhibited IMPase noncompetitively, likely through a covalent modification on a cysteine. In cell cultures, ebselen was found to inhibit not just IMPase but other steps that resulted in accumulation of higher inositol phosphates. When injected intraperitoneally into mice, ebselen crossed the blood- brain barrier and exhibited inhibition of IMPase ex vivo. Moreover, in mice, ebselen simulates some, but not all, of the behavioural effects of lithium. I have determined that ebselen inhibits IMPase and acts as a partial lithium mimetic. Given that ebselen is safe in man, it warrants clinical testing for the treatment of bipolar disorder.

Thesis advisor: Mary F. Roberts / Myo-inositol and its phosphorylated derivatives are found across all domains of life, and these molecules play crucial roles in a wide variety of cellular processes. While the biosynthesis of inositol is an evolutionarily conserved pathway, there are a wide variety of enzymes that use inositol and its derivatives as substrates. This thesis explores two such enzymes; a phosphatidylinositol- specific phospholipase C (PI-PLC) produced by <i>Staphylococcus aureus</i>, and AF2372, a dual action inositol monophosphatase/ fructose bisphosphatase produced by the <i>Archaeoglobus fulgidus</i>. At the outset of this work, the structure of the <i>S. aureus</i> PI-PLC was unknown, but some interesting biochemical properties about the enzyme had been observed. The structure of AF2372 had been reported, but a structure had not yet been solved in the presence of osmolytes known to thermoprotect the enzyme. Both the <i>S. aureus</i> PI-PLC and AF2372 catalyze the cleavage of phosphorylated inositol compounds, but share no mechanistic, structural, or taxonomical similarities. Protein crystallography is a powerful tool, and with it I have been able to study these two enzymes at a molecular level, providing insight into complex biological questions about each enzyme. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Mary F. Roberts / Crystallization studies in presence of organic osmolytes were conducted to better understand the specific mechanism of compatible solute binding to the inositol monophosphatase of Archaeoglobus fulgidus. The synthesis of a-diglycerol phosphate, one of the natural osmolytes of A. fulgidus, was also completed for kinetic testing of its I-1-Pase thermoprotective properties and for crystallization trials. / Thesis (MS) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Grado de magister en fisiología / La plasticidad muscular es la capacidad que presentan las células musculares esqueléticas de adaptarse a diferentes estímulos externos, modificando su fenotipo. La frecuencia de estimulación eléctrica (EE) (in vitro como in vivo) induce la transición fenotípica de un músculo. De modo interesante, el EE de baja frecuencia induce marcadores de transición fenotípica rápido/lenta por una vía dependiente de la activación del IP3R. Además, se ha establecido al uniportador de Ca2+ mitocondrial como un regulador del trofismo muscular. Hipotetizamos que la regulación de los niveles de RNAm del uniportador de Ca2+ mitocondrial y de sus proteínas reguladoras en respuesta a EE de baja frecuencia es dependiente de la activación del IP3R. Fibras musculares adultas aisladas desde el músculo flexor digitorium brevis (fdb) de ratones C57BL/6J de 8 a 10 semanas de edad fueron expuestas a EE de baja frecuencia en presencia o ausencia de inhibidores de la vía propuesta. Se observó que el estímulo eléctrico de baja frecuencia resulta en una disminución de los niveles de RNAm de MCU, MICU1, MICU2 y EMRE, mientras que el EE de alta frecuencia no genera modificaciones. Las fibras musculares esqueléticas pre-incubadas con apirasa (enzima que degrada ATP) y xestospongina B (inhibidor de los receptores de IP3) previenen la disminución de los niveles de RNAm de MCU, MICU1, MICU2 y EMRE mediada por un EE de baja frecuencia. El ATP extracelular exógeno (agregado al medio de incubación) resulta en una disminución de los niveles de RNAm de MCU, MICU1, MICU2 y EMRE). Además, este efecto de EE no es prevenido por actinomicina D (inhibidor de la transcripción). Este trabajo contribuye a la comprensión de los mecanismos moleculares involucrados en la plasticidad muscular en respuesta a ejercicio físico y en particular, al rol de la mitocondria en estos mecanismos. / Muscular plasticity is the ability of the skeletal muscle cells of different external stimuli, modifying their phenotype. The frequency of electrical stimulation (ES) (in vitro as in vivo) induces the phenotypic transition of a muscle. Interestingly, low frequency ES induces rapid / slow phenotypic transition markers by a pathway dependent on the activation of IP3R. In addition, the mitochondrial Ca2 + uniporter has been established as a regulator of muscle trophism. We hypothesized that the regulation of mRNA levels of the mitochondrial Ca2 + uniporter and its regulatory proteins in response to low frequency EE is dependent on the activation of IP3R. Adult muscle fibers isolated from the flexor digitorium brevis muscle (fdb) of C57BL / 6J mice from 8 to 10 weeks of age were exposed to low frequency ES in the presence or absence of inhibitors of the proposed pathway. It was observed that the low frequency electrical stimulus results in a decrease in mRNA levels of MCU, MICU1, MICU2 and EMRE, while high frequency ES does not generate modifications. Skeletal muscle fibers pre-incubated with apirasa (enzyme that degrades ATP) and xestospongin B (inhibitor of IP3 receptors) prevent the decrease of mRNA levels of MCU, MICU1, MICU2 and EMRE mediated by a low frequency ES. Exogenous extracellular ATP (added to the incubation medium) results in a decrease in mRNA levels of MCU, MICU1, MICU2 and EMRE). In addition, this effect of ES is not prevented by actinomycin D (transcription inhibitor). This work contributes to the understanding of the molecular mechanisms involved in muscle plasticity in response to physical exercise and, in particular, to the role of mitochondria in these mechanisms. / 30/08/2019