Inositol is important for normal growth and development in plants. The regulation of the inositol biosynthetic enzymes, <I>myo</I>-inositol phosphate synthase (MIPS) and <I>myo</I>-inositol monophosphatase (IMP) was investigated. The specific aims of this research were (1) to develop a tool to study MIPS protein accumulation in a model plant system, <I>Arabidopsis thaliana (At)</I> and potentially other plant species and (2) to determine the spatial expression patterns of <I>Lycopersicon esculentum</I> IMP-2 (<I>Le</I>IMP-2) at the cellular level. <I>Myo-inositol phosphate synthase (mips)</I> genes have been identified in plants, animals, fungi and bacteria. Alignment of the predicted amino acid sequences of <I>At</I>MIPS-1, -2 and <I>Glycine max</I> MIPS (<I>Gm</I>MIPS) indicated that <I>At</I>MIPS-1 and <I>Gm</I>MIPS are 87% identical, and <I>At</I>MIPS-2 and <I>Gm</I>MIPS are 89% identical. Based on these data, a <I>Gmmips</I> cDNA was fused at the N-terminus to a 6X histidine tag (5' GAC GAC GAC GAC GAC GAC 3'), cloned into an overexpression vector and overexpressed in <I>E. coli</I>. The fusion protein, HISMIPS, was extracted using denaturing conditions and purified using Ni²⁺ affinity chromatography. Anti-<I>Gm</I>MIPS antiserum from rabbit detected the recombinant HISMIPS protein (76 kD), and <I>Gm</I>MIPS (64 kD). Affinity purification by subtractive chromatography yielded anti-<I>Gm</I>MIPS antibody that detected <At</I>MIPS (66 kD) and a protein (34 kD) of unknown function. <I>At</I>MIPS accumulated to high levels in unopened flowers, opened flowers, and immature siliques (6 mm in length or less), but was not detectable in bolts, cauline or rosette leaves. The tomato <I>inositol monophosphatase (Leimp)</I> genes are a developmentally regulated multigene family. From analysis of sequences, <I>Leimp</I>-2 is intron-less and has the putative start site of translation located at +108 bp downstream from the putative start site of transcription. Investigation of the 5' UTR revealed the 3' end of a partial open reading frame (338 bp) highly homologous to the gene for calmodulin. Three light responsive elements and a cold responsive element were also identified in the 5' UTR. Transgenic <I>Leimp</I>-2::<I>uid</I>A plants were produced using the existing construct of the <I>Leimp</I>-2 promoter fused to the <I>uid</I>A gene (J. Keddie, University of California at Berkeley). Seedlings were preserved and sectioned. Using histological techniques, the analysis of the <I>Leimp</I>-2 promoter::<I>uid</I>A transgenic seedlings revealed that the <I>Leimp</I>-2 promoter causes expression at the base of the shoot apex and within leaflets of the first set of fully expanded leaves. Further, <I>Leimp</I>-2 promoter expression was localized to epidermal and cortex cells on the abaxial side of the 1st and 2nd fully expanded compound leaves. These studies of MIPS and IMP expression lay a foundation for a better understanding of the regulation of inositol biosynthesis in Arabidopsis, tomato, and other plant species. / Master of Science

Obesity is a pervasive condition associated with increased risk of dementia, cognitive impairment, and cerebral atrophy in later life. Given that the pathophysiology underlying obesity’s impact on the central nervous system is poorly understood, the current study examined the association between body mass index (BMI) and five cerebral metabolites of neurobiological significance: N-acetyl-aspartate (NAA), a marker of neuronal viability; choline-containing compounds, free choline, phosphocholine and glycerophosphocholine (Cho), markers of membrane breakdown and turn over; creatine (Cr), a marker of energy metabolism; myo-inositol (mI), an organic osmolyte and substrate for the synthesis of the secondary messenger, inositol triphosphate; and glutamate (Glu), a marker of excitatory neurotransmission and synaptic integrity. Fifty-five participants, aged 40-60 years, underwent neuropsychological testing, health screen and proton magnetic resonance spectroscopy (1H MRS) of the occipitoparietal grey matter. Concentrations of NAA, Cho, mI, and Glu were calculated as a ratio over Cr and examined in relation to BMI using multivariate multiple regression. Higher BMI was associated with elevations in mI/Cr (F(5,47)=3.583, p=0.008, ß=0.387, p=0.006), independent of age, sex, fasting glucose levels, and systolic blood pressure. The current study found that higher BMI is related to increased concentrations of the organic osmoltye and glial marker myo-inositol, potentially implicating plasma hypertonicity and neuroinflammation as mechanisms underlying obesity-related brain dysfunction. With validation and absolute quantification, studies of neurometabolites may improve identification of the pathological mechanisms underlying obesity’s consequences on cognition. / text

This work focuses on the determination of inositol in drugs and food supplements using high-performance liquid chromatography in HILIC mode. The work is divided into several sections, it discusses the characteristics of analyzed myo-inositol including methods for saccharides determination. A chapter is detached for high-performance liquid chromatography. One of the work goals was also chromatography columns comparison. The experimental part includes measurement conditions, methods of sample preparation evaluated data and results including the discussion. Two real samples were analyzed, in both of them the content of myo-inositol was declared by the producer.

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.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / A Diabetes mellitus à uma doenÃa de causa mÃltipla, ocorrendo quando hà falta de insulina ou quando a mesma nÃo atua de forma eficaz, causando um aumento da taxa de glicose no sangue (hiperglicemia). Ainda nÃo se sabe precisamente o mecanismo de aÃÃo da insulina, alguns trabalhos sugerem que pode ser possivelmente mediado atravÃs do fosfoglicano inositol (IPGs), cujas algumas formas foram identificadas como: mioinositol e D-quiroinositol. Hà estudos que relacionam a reduÃÃo da glicemia em indivÃduos diabÃticos com o aparecimento desses inositÃis nas secreÃÃes corpÃreas, embora ainda nÃo haja registro de identificaÃÃo dessas molÃculas na composiÃÃo salivar. O objetivo deste estudo foi determinar a relaÃÃo salivar do mioinositol e quiroinositol em crianÃas com diabetes tipo 1 e comparar a presenÃa e concentraÃÃo dessas substÃncias com um grupo de crianÃas sadias (nÃo diabÃticas). Um total de 45 (quarenta e cinco) voluntÃrios, 25 com diabetes tipo 1 descompensados e 20 sadios (nÃo diabÃticos), de ambos os sexos, com idades de 3 a 12 anos, foram selecionados e convidados a participar do estudo. Amostras de saliva foram coletadas e centrifugadas. Os sobrenadantes foram separados, liofilizados e purificados. Logo em seguida, foram analisados por cromatografia lÃquida de alta eficiÃncia (HPLC) para a identificaÃÃo do mioinositol e quiroinositol. A partir dessa anÃlise, foi observado uma menor concentraÃÃo de quiroinositol (p=0,001, Kruskal- Wallis ANOVA seguido por mÃtodo de Dunnâs) e uma maior da concentraÃÃo de mioinositol (p=0,001, Kruskal- Wallis ANOVA seguido por mÃtodo de Dunnâs) nas crianÃas afetadas em comparaÃÃo com as crianÃas saudÃveis. Os pacientes com diabetes tiveram a razÃo mio/quiroinositol maior que do grupo controle (p=0,001, Kruskal- Wallis ANOVA seguido por mÃtodo de Dunnâs) e apresentaram uma correlaÃÃo entre sua proporÃÃo o DM1(p= 0,001). O resultado desse estudo sugere que o mioinositol e o quiroinositol encontrado na saliva de crianÃas com DM1 podem influenciar no controle metabÃlico e desempenhar um papel de marcadores da DM1. / Diabetes mellitus is a disease of multiples causes that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces, causing a rise in blood glucose levels (hyperglycemia). It is not clear the action mechanism of insulin but it has been suggested that inositol phosphoglicans, such as myoinositol and D-chiro-inositol, can be important secondary messengers in insulin signal transduction. Although there are some studies linking a reduction in blood glucose levels in diabetic patients with the presence of these inositols in body secretions, there are not reports about the presence of these molecules in salivary composition. Thus, this study aimed to determinate the myoinositol and D-chiro-inositol salivary relation in children with type 1 diabetes and to compare the presence and concentration of these molecules with healthy children (non-diabetic). It has been selected and invited 45 volunteers of both sexes aged 3-12 years, 25 with decompensate type 1 diabetes and 20 healthy children. Saliva samples were collected and centrifuged. The supernatants were separated, purified and lyophilized. The identification of myoinositol and D-chiro-inositol were carried out by means of high-performance liquid chromatography (HPLC). The results showed that children with type 1 diabetes have a lower concentration of D-chiro-inositol and a higher concentration of myoinositol than healthy children. Consequently, the myo/chiro-inositol rate was higher in type 1 diabetes children and there is a correlation between the rate and type 1 diabetes incidence. In conclusion, our data suggests that myoinositol and chiroinositol found in the saliva of children with type 1 diabetes may influence in metabolic control and plays an important role as markers of type 1 diabetes.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20943号 / 理博第4395号 / 新制||理||1631(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 三木 邦夫, 教授 杉山 弘, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Phytic acid, also known as myo-inositol hexakisphosphate or Ins(1,2,3,4,5,6)P6, is the major storage form of phosphorus in plant seeds. Phytic acid is poorly digested by non-ruminant animals such as swine and poultry, and it chelates mineral cations including calcium, iron, zinc, and potassium, classifying it as an anti-nutrient. The excretion of unutilized phytic acid in manure translates to an excess amount of phosphorus runoff that can lead to eutrophication of lakes and ponds. Understanding the phytic acid biosynthetic pathway will allow for the development of low phytic acid (lpa) soybeans by the down-regulation of specific genes. The goal of this research was to elucidate the pathway(s) for phytic acid biosynthesis in soybean (Glycine max). We have isolated several myo-inositol phosphate kinase genes in soybean as possible candidates for steps in the biosynthetic pathway. We have characterized the genes for four myo-inositol(1,3,4)P3 5/6-kinases (GmItpk1-4), one myo-inositol(1,4,5)P3 6/3/5-kinase (GmIpk2), and one myo-inositol(1,3,4,5,6)P5 2-kinase (GmIpk1). We have examined expression in developing seeds and other tissues by Northern blot analysis and quantitative RT-PCR. We have expressed all six genes as tagged fusion proteins in E. coli, and verified enzyme activity on the proposed substrates. For each enzyme, we have conducted biochemical characterization to determine enzyme kinetics and substrate specificities. We have verified in vivo activity of GmIpk2 and GmIpk1 by complementing yeast mutants in the respective genes. Our studies indicate the likelihood that three of the genes may be involved in phytic acid biosynthesis: GmItpk3, GmIpk2 and GmIpk1. For future work, to more fully understand the contribution of each kinase gene to phytic acid biosynthesis, an RNA interference approach will be employed. The gene sequences identified in this study will be used to construct silencing vectors for use in future transformation of soybean embryogenic cultures to determine the effects of down-regulation on myo-inositol phosphate profiles. / Ph. D.

The intracellular second messenger InsP₃ is a vital molecule in the regulation of Ca²⁺ signalling. Ca²⁺ mediates a wide range of cellular activities from fertilisation and cell differentiation through to apoptoisis. Using X-ray crystal structure data and molecular modelling, a series of novel InsP₃ analogues were designed as selective InsP₃R-antagonists. Two novel synthetic routes have been developed for the synthesis of these analogues. The first route uses a Ferrier-II rearrangement to provide enantiopure inositol intermediates, whereas, the second route employs a diastereomeric resolution to obtain the enantiopure inositols. The successful synthesis of InsP₃ and a series of 5-position modified analogues are reported herein.

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.

Lactobacillus casei BL23 expresses two enzymes encoded by the genes iolG1 and iolG2. They have been putatively assigned as myo-inositol dehydrogenases by sequence comparison. The enzyme catalyzes the reversible conversion of myo-inositol to scyllo-inosose and the concurrent reduction of NAD+ to NADH. iolG1 was subsequently determined to be a myo-inositol dehydrogenase but iolG2 was determined to be a scyllo-inositol dehydrogenase. Sequence analysis and kinetics by themselves did not provide insight as to why the enzymes are functionally different. This manuscript provides a structural rationalization for the differences in stereoisomer selectivity by X- ray crystal structure analysis and comparison. High resolution apo, binary, and ternary crystal structures for iolG1 and iolG2 wild type enzymes were determined. For iolG1 the ternary structures were determined for myo-inositol and d-chiro-inositol and for iolG2 the scyllo-inositol bound structure was determined. The high resolution structure information revealed the composition of their respective active sites and showed that subtle differences in critical amino acids for each enzyme define the orientation of the inositol stereoisomer for inline transfer of a hydride to NAD+. Mutagenesis studies of a closely related myo-inositol dehydrogenase from Bacillus subtilis were carried out. The wild type structure for BsIDH had already been determined and characterized. A portion of the results in this manuscript briefly explore structures of dehydrogenase mutants which validate the structural role of residues involved in cofactor selectivity