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.

Author of the Thesis: Bc. Andrea Matějková Supervisor of the Thesis: doc. Ing. Monika Šorfová, Ph.D. Title of the Thesis: Relation of function of the pelvic floor to respiration Year of the presentation of the thesis : 2016 Powered by TCPDF (www.tcpdf.org)

The fields of virtual reality and gesture-based input devices are growing and becoming more popular. In order for the two technologies to be implemented together, an understanding of gestures in relation to virtual objects and users' expectations of those gestures needs to be understood. Specifically, this thesis focuses on arm gestures for the rotation of virtual objects. Participants in the study were first asked to freely perform an arm gesture they felt should execute a task. Next, participants were asked to perform specific rotation tasks with pre- configured arm gestures on four objects. There were two types of objects: those that could only be rotated on one axis and those that could be rotated on two axes. Each object type was represented by a familiar small and large object: combination lock, water wheel, baseball and beach ball. Data on how quickly they could complete the rotation tasks was collected. After performing the tasks on each of the four objects, participants were asked to rate the intuitiveness of each gesture as well as their preferred gesture for the specific task. The captured data showed that when users were presented with virtual representations of familiar physical objects, most of them expected to rotate the objects with the same gestures they would use on the actual physical objects. Considering 1-axis objects, an arm-based twist gesture outperformed other arm-based gestures in both intuitiveness and efficiency. Also with 2-axis objects, an arm-based horizontal/vertical gesture outperformed others in both intuitiveness and efficiency. Interestingly, those gestures were most efficient for each object type regardless of the size of the object being rotated. This would indicate that users are able to mentally separate the physical and virtual experiences. Larger objects require different rotation gestures than smaller objects in the physical world, but that requirement is non-existent in a virtual world. However, while the mind can separate between the physical and virtual worlds, there is still an expected connection. This is based on the fact that the gestures most preferred for the rotation tasks are the same gestures used for similar physical tasks.

Universiẗat, Diss., 2006--Giessen.

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.