Thesis (Ph.D.)--University of Wyoming, 2008. / Title from PDF title page (viewed on August 3, 2009). Includes bibliographical references (p. 125-129).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Doctor en Bioquímica / La macroautofagia, comúnmente referida como “autofagia” es la principal vía de degradación de proteínas, organelos y material citoplasmático, permitiendo de este modo el reciclaje del material intracelular. Este proceso consiste en el englobamiento de fracciones citosólicas por una estructura multimembranar llamada “autofagosoma”, el cual posteriormente se fusiona con el lisosoma para formar el “autofagolisosoma”. Luego el material comprendido en el autofagolisosoma es degradado por enzimas hidrolíticas. Un estudio mostró que la inhibición de la enzima inositolmonofosfatasa (IMPasa) usando litio y L690.330, inducía una disminución de los niveles basales del IP3 y en consecuencia la generación de autofagia. Nuestros resultados confirmaron estos datos previos, demostrando que el pre tratamiento con mio-inositol revierte la autofagia inducida por litio y L-690.330. Además se demuestra que el pre tratamiento con mio-inositol también revertía la autofagia inducida por privación de nutrientes. IP3 es ligando de su receptor de IP3 (IP3R), el cual es el principal canal de Ca2+ a nivel del retículo endoplásmico. El principal objetivo de esta tesis es evaluar el rol del IP3R en la regulación de la autofagia. Los resultados mostraron que la disminución de los niveles proteicos del IP3R usando siRNA específicos, así como el tratamiento con antagonistas químicos del IP3R, tales como xestosponginas B y C, estimulaban significativamente el aumento en los niveles de autofagia. Además, xestospongina B, así como también la privación de nutrientes, indujo una pérdida en la interacción entre Bcl-2 y Beclin-1, los cuales interactúan en condiciones basales. El tratamiento con xestospongina B no perturbó los niveles de Ca2+, tanto en retículo endoplásmico como en el citosol, concluyendo que la autofagia inducida por xestospongina B es independiente de una fluctuación del Ca2+. Los experimentos de inmunoprecipitación mostraron que Beclin-1 (regulador clave en la inducción de la autofagia) interactúa tanto con IP3R así como con Bcl-2 en condiciones basales, y la interacción de este complejo es atenuado bajo condiciones de privación de nutrientes o por tratamiento con ABT737, el cual es un mimetizador de dominios BH3. Este resultado sugiere la presencia de un complejo proteico en la regulación de la autofagia. El papel del retículo endoplásmico en el desarrollo de la autofagia toma gran significancia debido al reclutamiento de proteínas clave (IP3R, Beclin-1 and Bcl-2). La relación entre autofagia y estrés de retículo no es clara y por lo tanto se evaluó el efecto de agentes inductores de estrés de retículo en la inducción de la autofagia. Los resultados mostraron que tunicamicina, tapsigargina y brefeldina-A (agentes inductores de estrés de retículo) activaron el UPR (respuesta a proteínas mal plegadas) e indujeron autofagia. La disminución de los niveles de proteínas claves en el desarrollo de la autofagia (Atg5, Atg10, Atg12, Vps34 y Beclin-1) usando específicos RNAs interferentes atenuaron la autofagia inducida por agentes inductores de estrés de retículo y xestospongina B. Además, la sobreexpresión de Bcl-2 y Bcl-XL con destinación a retículo endoplásmico atenuó la autofagia inducida por xestospongina B e inhibidores de la IMPasa. Esta tesis muestra novedosos resultados, los cuales dan cuenta de un complejo proteico IP3R/Beclin-1/Bcl-2 en la regulación de la autofagia. / Macroautophagy (herein referred to as “autophagy”) is the major catabolic pathway for entire organelles, long-lived/ aberrant proteins and superfluous portions of the cytosol. It consists of the stepwise engulfment of substrate elements into distinctive multimembraned “autophagosomes”, which after fusion with lysosomes form singlemembraned autophagolysosomes. Into the autophagolysosome, the engulfed material is degradated by lisosomal hidrolytic enzymes, leading the recyclage of intracellular material. A study has suggested that myo-inositol-1,4,5-trisphosphate (IP3) could regulate autophagy because inhibition of inositol monophosphatase (IMPasa) by lithium or L-690.330 stimulates autophagy through the depletion of IP3. Our results have confirmed that the reduction of intracellular IP3 levels by IMPasa inhibitors (lithium and L.690.330) stimulates autophagy, whereas the enhancement of IP3 levels by pre treatment whit mio-inositol inhibits the lithium and L.690.330 effect. Moreover we have demostred that autophagy induced by nutrient privation was also inhibited by treatment with mio-inositol, but the effect of nutrient privation in the intracellular IP3 basal levels was not evaluated. IP3 acts on the IP3 receptor (IP3R), an IP3‑activated Ca2+ channel of the endoplasmic reticulum membrane and consequently we wanted to evaluate de roll of IP3R in the regulation of autophagy. The results obtained in this thesis show that knockdown of the IP3 receptor (IP3R) with specifics small interfering RNAs and pharmacological IP3R antagonist (xestospongin B and C) are a strong stimulus for the induction of autophagy, in addition, xestospongin B (like nutrient starvation) induced loss in the interaction between Beclin-1 and Bcl-2. Moreover, the autophagy promoted by xestospongin B not produced alterations in the steady-state Ca2+ levels in the ER or in the cytosol, therefore the autophagy induced by xestospongin B was Ca2+-independent. Immunoprecipitation assays shown that Beclin- 1 (key protein in the regulation of autophagy) interacts with IP3R and Bcl-2 in basal conditions, and this interaction may be attenuated both by nutrient starvation or ABT737 treatment, which is a mimetic compound of BH3. These results suggest the presence of a protein complex in the regulation of autophagy. The treatment whit ER stressors such as tunicamycin, thapsigargin and brepheldine A induced Unfolded Protein Responses (UPR) and autophagy. The autophagy induced by these agents showed to be IRE1α dependent, but the inhibition of autophagy showed an increase in the cell death, indicating a pro survival function of the autophagy upon endoplasmic reticumum stress conditions. The autophagy induced by treatment with xestospongin B and ER stressors was inhibited by knockdown of Atg5, Atg10, Atg12, Vps34 and Beclin-1, which are keys proteins in the autophagic process. We have also evaluated the roll of Bcl-2 and Bcl-XL in the inhibition of autophgy, and the results showed that Autophagy triggered by IMPasa inhibitors and xestospongin B was inhibited by Bcl-2 and Bcl-XL over expression specifically targeted to ER but not Bcl-2 or Bcl-XL proteins targeted to mitocondria. Altogether, these results suggest that IP3R form a regulator complex with Bcl-2 and Beclin-1, which exerts a major role in the physiological control of autophagy

The objective of this research was to isolate and characterize soybean genes involved in phytic acid metabolism for use in genetic engineering strategies to improve phosphorus utilization. A soybean phytase from germinated cotyledons was purified 28,000-fold to apparent homogeneity and was determined to be a glycosylated homodimer with 70 kD subunits. Soybean phytase preferred phytate as substrate (Km = 60 mM) and was capable of removing of all six phosphate groups from phytate. The pH and temperature optima for soybean phytase activity were 4.5 and 58*C, respectively. The N-terminus and four internal peptides from the purified soybean phytase were sequenced by Edman degradation. The amino acid sequence data were used to design degenerate oligonucleotide primers for PCR amplification of the soybean phytase coding sequence. A protein 547 amino acids in length was predicted from the 1641 bp coding sequence. The phytase protein showed significant similarity to plant purple acid phosphatases (PAPs) and contained the conserved metallo-phosphomonoesterase active site motif. The soybean phytase coding sequence was placed under the control of a constitutive 35S CaMV promoter in a soybean biolistic transformation vector and was introduced into "Williams 82" suspension culture cells by particle bombardment. Stably transformed cell suspension lines were recovered. DNA blot analysis demonstrated that the recombinant soybean phytase coding sequence had integrated into the genomes of two cell lines. Expression of the transgene was confirmed by RNA blot analysis. Phytase activity was three to four fold higher in these two lines compared to control non-transformed cultures. A soybean L-myo-insoitol-1-phosphate synthase (MIPS) cDNA was isolated from total RNA from developing seeds. The protein encoded by the soybean MIPS cDNA showed 87-91% homology to MIPS protein sequences from other plant species. RNA blot analysis of staged developing soybean seeds revealed that MIPS is transcribed early in the cotyledonary stage of development. Compared to other soybean tissues, MIPS expression levels were highest in developing seeds. DNA blot analysis demonstrated that multiple copies of the MIPS gene are present within the soybean genome. / Ph. D.

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.

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.

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.

Receptor-mediated phosphoinositide hydrolysis was studied in 3H-myo-inositol labelled rat cerebral cortical slices. Several CNS neurotransmitter receptor agonists stimulated the hydrolysis of inositol phospholipid(s). Maximal responses to receptor stimulation showed the order, muscarinic cholinergic > adrenergic > serotonergic > histaminergic. Potassium ion depolarisation and a Ca2+ ionophore also stimulated the accumulation of 3H-inositol phosphate(s). The metabolic sequence of muscarinic receptor-mediated phosphoinositide breakdown was examined in detail. Carbachol stimulated the sustained accumulation (> 45 min.) of 3H-Ins P1, 3H-Ins P1, 3H-Ins P2, 3H-Ins P3 and of a novel 3H-inositol phosphate identified as Ins-1,3,4,5-P4. Kinetic studies showed that muscarinic receptor activation results in the rapid (< 5 sec.) increased accumulation of 3H-Ins P2, 3H-Ins P3 and 3H-Ins P4 while the onset of 3H-Ins P1 accumulation is delayed. Using hplc, the Ins P3 fraction was resolved into two components with the retention times of Ins-1,3,4-P3 and Ins-1,4,5-P3. Stimulated accumulation of Ins-1,3,4-P3 was preceded by that of the other polyphosphates. The probable formation of Ins-1,3,4-P3 via Ins-1,3,4,5-P4 dephosphorylation is discussed. A phospholipid precursor for Ins-1,3,4,5-P4 could not be identified but production of this molecule via an ATP-dependent, Ins-1,4,5-P3 3-kinase was confirmed. Studies of the rates at which the separate 3H-inositol phosphates are hydrolysed in stimulated tissue suggest considerable flux through this kinase reaction and indicate that the majority of the 3H-Ins P1, and 3H-Ins P2 accumulating in response to agonist result from 3H-tris- and 3H-tetrakisphosphate metabolism. Pharmacological data support these conclusions for conditions of both high and low receptor occupancy. Lithium ions markedly affected muscarinic receptor-mediated 3H-inositol phosphate metabolism, dose-dependently potentiating stimulated 3H-Ins P3, and 3H-Ins P2 accumulations while concomitantly attenuating those of 3H-Ins P3 and particularly 3H-Ins P4. The latter effects were half-maximal at 1 mM Li+, exhibited a delayed onset, were not related to receptor desensitization but may be indirect consequences of Ins P1 phosphomonoesterase inhibition. The significance of these actions is discussed in the context of the potential second messenger roles of Ins-1,4,5-P3 and Ins-1,3,4,5-P4.

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