Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xv, 152 p.; also includes graphics (some col.). Includes bibliographical references (p. 138-152). Available online via OhioLINK's ETD Center

El Síndrome de Down (SD) se determina por la trisomía del autosoma 21 humano, y sus alteraciones se asocian a sobreexpresión o bien desregulación de los productos génicos derivados de este cromosoma. La trisomía 16 (Ts16) murina es un modelo animal de este síndrome, debido a la gran homología entre los cromosomas humano y murino correspondiente. Desafortunadamente, la condición en el modelo murino no es viable. Frente a esto se han generado líneas celulares inmortalizadas derivadas de corteza de ratón normal y trisómico 16, denominadas CNh y CTb respectivamente. Estas líneas celulares sobreexpresan genes relacionados con el SD y muestran función colinérgica alterada (menor incorporación de colina de alta afinidad (IC), expresión de la acetilcolintransferasa (ChAT) y liberación fraccional de acetilcolina (LF) frente a una estimulación), similares a las previamente encontradas en cultivos primarios de corteza Ts16. También se han encontrado elevados niveles de Mioinositol en pacientes con SD, lo cual estar relacionado con la sobreexpresión del Transportador Na+ /Mioinositol SMIT1, cuyo gen se encuentra ubicado en cromosoma 16 murino. Con el objeto de dilucidar la participación de SMIT1 en la disfunción colinérgica descrita anteriormente, decidimos reducir la expresión de SMIT1 transfectando las células con secuencias de antisentido específicos para mRNA en nuestras líneas celulares derivadas de corteza cerebral de Ts16 (CTb) comparadas con la línea derivada de un animal normal (CNh). Posterior a la transfección, estudios de Western Blot (WB) mostraron una reducción en la expresión de SMIT1 en 10%, 40%, y 42% a 24, 48 y 10 72 horas respectivamente, comparadas con niveles de SMIT1 en cultivos de la líneas celulares trisómicas CTb sin transfectar (n=6 p<0,01). Una vez normalizada la expresión de SMIT1, estudiamos la IC en CTb y CNh, encontrando que la línea CTb exhibe una reducción en la IC de un 80%, 85% y a 2 y 5 minutos de incubación con 1µCi de [3 H]- colina respectivamente, comparada con CNh. A 72 horas post-transfección los niveles en la IC fueron esencialmente similares en ambos tipos celulares. Finalmente, la LF, la cual está reducida en CTb comparada con CNh después de una despolarización generada por glutamato, nicotina , muscarina y KCl, , muestra un progresivo incremento después del Knockdown del SMIT al ser estimulada con nicotina y muscarina, mostrando valores similares a CNh después de 48 horas post-transfección. Sin embargo, al inducir despolarización con KCl y estimular con Glutamato extracelular, las líneas CTB tranfectadas no exhibieron diferencias con respecto a la línea celular trisómica CTb sin transfectar. Los resultados sugieren que la normalización en la expresión de SMIT1 en la línea CTb mediante la transfección con oligonucleótidos antisentido revierten: 1) la reducción de la incorporación de 3H colina, y 2) el déficit en la liberación de acetilcolina frente a agonistas Colinérgicos (Nicotina y Muscarina). Los resultados pueden constituir un punto de partida para el diseño de una terapia farmacológica que apunte a recuperar las funciones cognitivas, en particular las relacionados con sistemas colinérgicos, en pacientes con SD.

ALVES, Karla Shangela da Silva. Estudo dos níveis salivares de mioinositol e quiroinositol em crianças saudáveis e portadoras de diabetes infanto-juvenil. 2012. 143 f. Dissertação (Mestrado em Odontologia) - Universidade Federal do Ceará. Faculdade de Farmácia, Odontologia e Enfermagem, Fortaleza, 2012. / Submitted by denise santos (denise.santos@ufc.br) on 2013-02-15T13:24:18Z No. of bitstreams: 1 2012_dis_kssalves.pdf: 1174860 bytes, checksum: d26be50d6f11fb6fddbf8330c2830139 (MD5) / Approved for entry into archive by Erika Fernandes(erikaleitefernandes@gmail.com) on 2013-02-18T15:47:18Z (GMT) No. of bitstreams: 1 2012_dis_kssalves.pdf: 1174860 bytes, checksum: d26be50d6f11fb6fddbf8330c2830139 (MD5) / Made available in DSpace on 2013-02-18T15:47:18Z (GMT). No. of bitstreams: 1 2012_dis_kssalves.pdf: 1174860 bytes, checksum: d26be50d6f11fb6fddbf8330c2830139 (MD5) Previous issue date: 2012 / 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. / A Diabetes mellitus é uma doença de causa múltipla, ocorrendo quando há 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 ação da insulina, alguns trabalhos sugerem que pode ser possivelmente mediado através do fosfoglicano inositol (IPGs), cujas algumas formas foram identificadas como: mioinositol e D-quiroinositol. Há estudos que relacionam a redução da glicemia em indivíduos diabéticos com o aparecimento desses inositóis nas secreções corpóreas, embora ainda não haja registro de identificação dessas moléculas na composição salivar. O objetivo deste estudo foi determinar a relação salivar do mioinositol e quiroinositol em crianças com diabetes tipo 1 e comparar a presença e concentração dessas substâncias com um grupo de crianças sadias (não diabéticas). Um total de 45 (quarenta e cinco) voluntários, 25 com diabetes tipo 1 descompensados e 20 sadios (não diabéticos), 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 líquida de alta eficiência (HPLC) para a identificação do mioinositol e quiroinositol. A partir dessa análise, foi observado uma menor concentração de quiroinositol (p=0,001, Kruskal- Wallis ANOVA seguido por método de Dunn’s) e uma maior da concentração de mioinositol (p=0,001, Kruskal- Wallis ANOVA seguido por método de Dunn’s) nas crianças afetadas em comparação com as crianças saudáveis. Os pacientes com diabetes tiveram a razão mio/quiroinositol maior que do grupo controle (p=0,001, Kruskal- Wallis ANOVA seguido por método de Dunn’s) e apresentaram uma correlação entre sua proporção o DM1(p= 0,001). O resultado desse estudo sugere que o mioinositol e o quiroinositol encontrado na saliva de crianças com DM1 podem influenciar no controle metabólico e desempenhar um papel de marcadores da DM1.

Les dérivés lipidiques et solubles des inositol phosphates sont largement impliqués dans la signalisation des cellules normales et des cellules cancéreuses. L’étude de ceux-ci est un domaine en pleine expansion et le représentant le plus connu de ces inositol phosphates est l’inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), responsable de la mobilisation calcique mais également point de départ du cycle des inositol phosphates hautement phosphorylés. Au cours de cette thèse, nous avons étudié le rôle des quatre enzymes capables de phosphoryler l’Ins(1,4,5)P3 :les 3 isoenzymes de l’inositol 1,4,5-trisphosphate 3-kinase (Itpk) l’Itpka, l’Itpkb et l’Itpkc clonées dans notre laboratoire, ainsi que l’inositol polyphosphate multikinase (IPMK), dans deux modèles cellulaires :les cellules souches embryonnaires murines (ESCm) ainsi que dans un modèle d’extension des neurites, les cellules PC12 stimulées au NGF.Dans le projet visant à étudier la fonction de ces quatre kinases dans les ESCm, nous avons fait ressortir deux points importants. Premièrement, grâce à l’étude des ESCm IPMK-/-, nous avons mis en évidence le rôle de l’IPMK dans la différenciation des ESCm d’une part en progéniteurs neuronaux et d’autre part en corps embryoïdes. Ces deux types de différenciation montrant une neurogenèse affectée dans les ESCm IPMK-/-. Deuxièmement, nous avons observé une augmentation d’expression de l’Itpkb dans les ESCm différenciées en corps embryoïdes comparé aux ESCm non différenciées.Dans le projet comparant la fonction des quatre kinases au cours de la différenciation des cellules PC12 en neurones sympathiques, nous avons montré que la surexpression de l’Itpka et de l’Itpkb inhibait l’élongation des neurites observées après stimulation des cellules au NGF. Cet effet inhibiteur sur l’élongation des neurites semble être expliqué par une modification de la réorganisation du cytosquelette d’actine induite par le NGF après surexpression de l’Itpka et de l’Itpkb. En conclusion, nous avons démontré que, bien qu’ayant le même substrat et permettant toutes la production d’inositol hautement phosphorylés, ces quatre enzymes pouvaient être différenciées par des fonctions spécifiques qu’elles exercent au sein de la cellule. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

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.

A number of experimental studies have implied a link between diabetes-induced lipid accumulation in the myocardium and the development of cardiomyopathy. Since diabetics excrete large amounts of myo-inositol which is a lipotropic agent, this study was undertaken to investigate the effects of myo-inositol on the elevated myocardial lipid levels and the depressed cardiac performance of diabetic rats. Diabetes was induced in female Wistar rats (190-215 g) with streptozotocin (STZ) (55 mg/kg, i.v.). Three days after diabetes induction, myo-inositol was administered in the drinking water (2.5 g/kg/day) for a 8 week period. Untreated diabetics exhibited a loss of body weight, hyperglycemia, hypoinsulinemia and hypothyroidism. These effects were not altered after myo-inositol treatment. STZ-diabetes also produced a significant elevation of plasma and myocardial triacylglycerol, cholesterol and phospholipid. Myo-inositol treatment decreased these lipid levels. In addition, hearts from diabetic animals had a decreased ability to develop left ventricular developed pressure (LVDP) and both the rate of pressure rise (+dP/dt) and the rate of pressure decline (-dP/dt) were also reduced. Hearts from myo-inositol-treated diabetic animals showed a partial but definite improvement of cardiac function. As diabetes-induced hypothyroidism was not altered after myo-inositol supplementation, a combination treatment of both myo-inositol (2.5 g/kg/day, p.o. daily) and T₃ (30 ug/kg/day, s.c. daily) was then undertaken to determine whether heart function of diabetic rats could be further improved. STZ-diabetic rats were characterized by a loss of body weight, hyperglycemia and hypoinsulinemia; none of which were altered by either T₃ or myo-inositol plusT₃ treatment. T₃ treatment normalized the thyroid state of diabetic animals as shown by Tahiliani and McNeill (1984). However, plasma and myocardial triacylglycerol, cholesterol and phospholipid levels of diabetic rats either remained elevated or were further increased with T₃ or myo-inositol plus T₃ treatment. In addition, T₃ treatment alone did not prevent cardiac dysfunction in diabetic rats. There was, however, some improvement in heart function in the groups treated with both myo-inositol and T₃, but the improvement was not as pronounced as with myo-inositol treatment alone. / Pharmaceutical Sciences, Faculty of / Graduate

Die Inositol-Pyrophosphate (PP-InsPs) sind eine ubiquitäre Gruppe hochphosphorylierter eukaryotischer Signalmoleküle. Sie werden mit einer Vielzahl zentraler zellulärer Prozesse in Verbindung gebracht, doch fehlt oft ein detailliertes Verständnis der einzelnen Signalereignisse, was zum Teil auf einen Mangel an chemischen Werkzeugen zurückzuführen ist. Diese Arbeit beschreibt die chemische Synthese, Validierung und Anwendung von PP-InsP-Affinitätsreagenzien zur Identifizierung von Proteinbindungspartnern von Inositolhexakisphosphat (InsP6) und 5-Diphosphoinositol-Pentakisphosphat (5PP-InsP5), zwei wichtigen eukaryotischen Metaboliten. Die Affinitätsreagenzien wurden entwickelt, um InsP6 und ein metabolisch stabiles 5PP-InsP5-Analogon auf drei verschiedene Arten darzustellen. Die Anwendung dieser triplexierten Reagenzien auf Säugetier-Lysate lieferte einen ersten umfassenden Datensatz in HCT116- und HEK293T-Zellen. Die Interaktome wurden mittels quantitativer Proteomik annotiert und enthüllten Hunderte von potenziellen Proteinbindungspartnern. Die quantitative Analyse der InsP6- und 5PP-InsP5-bindenden Proteine zeigte Beispiele für hochspezifische Protein-Ligand-Interaktionen auf. Biochemische Untersuchungen ergaben, dass Inositol-5-Phosphatasen, PRPS1 und spezifische Phosphatidyl-Inositolphosphat-Kinasen potenziell unentdeckte Zielproteine von PP-InsPs sind. Darüber hinaus wurde durch die Entwicklung einer neuen Strategie der Myo-Inositol-Desymmetrisierung erstmals die Synthese eines Affinitätsreagens auf der Basis von 1,5-Bisdiphosphoinositol-Tetrakisphosphat (1,5(PP)2-InsP4) beschrieben. Die Affinitätsreagenzien und die proteomischen Datensätze stellen für die Gemeinschaft leistungsstarke Ressourcen dar, um künftige Untersuchungen zu den vielfältigen Signalmodalitäten von Inositolpyrophosphaten einzuleiten. / Inositol pyrophosphates (PP-InsPs) are a ubiquitous group of highly phosphorylated eukaryotic messengers. They have been linked to a panoply of central cellular processes, but a detailed understanding of the discrete signaling events is often missing, which can partially be attributed to a lack of chemical tools. This thesis describes the chemical synthesis, validation and application of PP-InsP affinity reagents to identify protein binding partners of inositol hexakisphosphate (InsP6) and 5-diphosphoinositol pentakisphosphate (5PP-InsP5), two important eukaryotic metabolites. The affinity reagents were developed to display InsP6 and a metabolically stable 5PP-InsP5 analog in three different ways. Application of these triplexed reagents to mammalian lysates provided a first comprehensive data set in HCT116 and HEK293T cells. The interactomes were annotated using quantitative proteomics and uncovered hundreds of potential protein binding partners. Quantitative analysis of InsP6 versus 5PP-InsP5 binding proteins highlighted examples of highly specific protein-ligand interactions. Biochemical studies primed inositol 5-phosphatases, PRPS1 and specific phosphatidyl inositol phosphate kinases as potentially undiscovered targets of PP-InsPs. Moreover, by developing a novel strategy of myo-inositol desymmetrization, the synthesis of an affinity reagent based on 1,5-bisdiphosphoinositol tetrakisphosphate (1,5(PP)2-InsP4) was described for the first time. The affinity reagents and the proteomic data sets constitute powerful resources for the community, to help launching future investigations into the multiple signaling modalities of inositol pyrophosphates.

Inositol pyrophosphates (PP-InsPs) are a family of molecules recently discovered to be implicated in a number of plant pathways such as auxin regulation, phosphate (Pi) sensing, and jasmonate-(JA)-regulated plant defense. Transgenic plants that overexpress inositol tetrakisphosphate 1-kinase (ITPK1) and the kinase domain of the dual domain diphosphoinositol pentakisphosphate kinase 2 (VIP2KD) have been previously studied to display uniquely elevated PP-InsPs. Here it is reported that the JA defense pathway is constitutively upregulated in VIP2KD OX plants, resulting in a lower rate of herbivory on the transgenic plants. ITPK1 OX, although also having elevated PP-InsPs, was fed upon by insect larvae comparably to Wild-Type Arabidopsis (WT). The data implicate VIP2, InsP8, and possibly the PP-InsP biosynthesis as a whole. / Master of Science in Life Sciences / Plants and insects have been evolving defenses against each other since they first emerged together post-Cambrian explosion. They each have evolved targeted metabolic pathways to produce chemicals with which to repel, harm, or even trick one another. In Arabidopsis thaliana, one of the most widely studied defense mechanisms is the jasmonic acid defense pathway, which responds to the herbivory of insects like caterpillars by setting off an array of genetic switches. The plant enters a stressed state wherein it represses the genes focused on growth and development and encourages the expression of genes focused on protecting vital resources and thwarting the attacker. This work examines a connection between the phosphate-sensing pathways and the jasmonic acid defense pathways in plants, and the following data show that plants with elevated inositol pyrophosphates (a phosphate storage molecule) are resistant to the herbivory of common pest caterpillars.

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

The myo-inositol signaling pathway in plants allows them to sense external environmental stimuli and respond to them. This signaling pathway depends on the dynamic levels of the second messenger, inositol(1,4,5)trisphosphate, which in turn is regulated by inositol polyphosphate 5-phosphatases (5PTases). Previous studies have shown that 5PTase 13 binds an important energy sensor, Sucrose non-fermenting (Snf) 1-related kinase (SnRK1.1) and also a novel protein P80. Studies from the lab have also shown that P80 is a part of a deubiquitinating enzyme complex along with WDR20 and Ubiquitin-specific protease called UBP3. Our p80 mutants have a root deficient phenotype under low energy conditions which is normalized by addition of sucrose. p80 mutants show reduced growth and early senescence under specific environmental conditions. This is opposite in nature to SnRK1.1 overexpressors. In this study, I have examined the possible interaction of P80 with SnRK1. I have studied the effects of expression of the exogenous SnRK1.1:GFP transgene under the control of the 35S CaMV promoter in the p80 mutant. This will facilitate the delineation of mechanisms that plants use for the control of energy sensing. I also examined the effects of the overexpression of SnRK1.2:GFP in the p80 mutant. Further, I have explored the presence of a new class of molecules, inositol phosphate molecules (InsPs) containing pyrophosphate bonds (PPx) in p80 mutants. Recent evidence has shown that this class of molecules has roles in sensing and signaling. I have demonstrated that InsP7 is present in developing seeds and vegetative tissue of higher plants. I have also demonstrated that p80 mutants have an alteration in the levels of PPx-InsPs. In addition, I have established spatial expression patterns of two enzymes involved in the synthesis of PPx-InsPx, known as VIP kinases. These studies will help resolve how PPx-InsPs are regulated in plants and thus help in their functional characterization. / Master of Science