Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: LaPlaca, Michelle; Committee Member: Backus, Deborah; Committee Member: Bellamkonda, Ravi; Committee Member: Lee, Robert; Committee Member: Prausnitz, Mark.

Thesis--University of California, Berkeley, 1959. / "Chemistry General" -t.p. Includes bibliographical references (p. 119-122).

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 112 pages. Includes Vita. Includes bibliographical references.

Orientador: Margarida Maria Barros / Banca: Luiz Edivaldo Pezzato / Banca: Carlo Rossi Del Carratone / Resumo: Essa pesquisa teve por objetivo avaliar o desempenho produtivo da tilápia do Nilo (Oreochromis niloticus) arraçoada com níveis crescentes de colina nas dietas. O período experimental foi de 109 dias. Foram utilizados 192 alevinos com peso médio inicial de 4,0 gramas, distribuídos em 32 tanques-rede de 200 L, numa densidade de seis peixes por tanque rede, sendo os tanques-rede alojados em aquários de 1000 L. O conjunto de aquários era dotado de sistema de filtragem de água por meio de biofiltro e sistema de aquecimento, sendo a temperatura mantida a 25,3 ± 0,2ºC. As rações foram formuladas de modo a apresentar 28,0% de proteína digestível e 3100,0 kcal ED/kg de dieta com mesma concentração de aminoácidos. O delineamento experimental foi inteiramente casualizado com oito tratamentos e quatro repetições. As rações foram suplementadas com colina (cloreto de colina 60,0%) de modo a apresentarem 100,0; 200,0; 400,0; 600,0; 800,0; 1000,0 e 1200,0 mg de colina por quilograma de ração, além de uma ração isenta de suplementação. Não foram observadas diferenças estatísticas para ganho de peso, taxa de sobrevivência e conversão alimentar aparente, porcentagem de extrato etéreo do filé e concentração de lipídeos no plasma. No entantohouve diferença estatística para o índice hepatossomático e porcentagem de extrato etéreo do fígado, sendo que 800,0 mg de colina/kg de dieta determinou maior ação lipotrófica. O oposto foi observado nos peixes do tratamento isento de suplementação. Concluiu-se que os diferentes níveis de colina não melhoram o desempenho produtivo dos peixes, pois a dieta basal supostamente supriu a exigência do peixe para colina, entretanto, a suplementação favoreceu a melhora no estado do fígado. / Abstract: The aim of this study was to evaluate the performance of Nile tilapia (Oreochromis niloticus) fed diets supplemented with increasing levels of choline. The experimental period was 109 days. One hundred and ninety two (initial weight 4.0 g) fingerlings were distributed in 32 net cages (200L), with a density of six fish per cage. These cages were allocated in 1000L aquariums connected to a bio-filter system and heater controlled temperature through thermostat (25.3 ± 0.2ºC). Feeds were formulated to contain 28% of digestible protein and 3100 kcal DE/kg with the same concentration of amino acids per treatment organized in totally random experimental design with eight treatments and four replicates. The feeds were supplemented with choline chloride (60%) presenting 100, 200, 400, 600, 800, 1000, 1200 mg of chlorine per kg of feed and one treatment with no supplementation. It was not observed any significant differences in performance, survival and apparent feed conversion, fillet ether extract and plasma lipids concentration among the treatments. However, there was a significant difference in the hepatosomatic index and liver ether extract percentage, showing that 800 mg of choline/kg determined a better lipotrofic action and the opposite was observed in fish fed diet with no choline supplementation. It was concluded that different levels of choline did not improve performance supposedly due to the amount of choline... / Mestre

Cocaine- and amphetamine-regulated transcript (CART) peptide, a family of neuropeptides, is shown to inhibit food intake upon intracerebroventricular injection to the rat. CART peptide-immunoreactivity (irCART) was detected in neurons of the dorsal motor nucleus of the vagus (DMNV) of postnatal day one (P1) rats, the earliest day examined. The number of labeled DMNV neurons reached the peak between P5 and P8 rats and gradually declined thereafter. Few irCART neurons were noted in the DMNV between P22 and P90 rats. Double-labeling the medullary sections from P5 and P8 rats with CART-antiserum and choline acetyltransferase (ChAT)-antiserum revealed that irCART neurons in the DMNV were ChAT-immunoreactive (irChAT), but not all irChAT neurons were irCART. Intraperitoneal injection of the retrograde tracer Fluorogold to P3 and P5 rats labeled DMNV neurons, the majority of which were also irCART. The number of irCART neurons in other regions of the brain and spinal cord generally showed an increase in adult rats as compared to that of the same regions in immature rats. Our result suggests that expression of irCART in DMNV neurons undergoes developmental changes such that few neurons appear to contain irCART in mature rats. As a corollary, CART may be a signaling molecule to the gastrointestinal tract during the critical period of early development.

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During conditions of osmotic stress, some plants accumulate compatible osmolytes such as glycine betaine or choline-0-sulphate. Choline is required as a precursor for synthesis of both osmolytes and choline is also required by all plants as a component of phospholipids. In the betaine accumulator spinach, choline synthesis requires three sequential N-methylations of phosphoethanolamine (PEA) to generate phosphocholine (PCho), with the first N-methylation being catalyzed by S-adenosyi-L-methionine: PEA Nmethyltransferase (PEAMeT). Choline synthesis and, more particularly the activity of PEAMeT, are up-regulated by salinity (Summers and Weretilnyk, 1993). This thesis reports on the partial purification and preliminary characterization of PEAMeT from spinach. A variety of column chromatography matrices including DEAE Sepharose, phenyl Sepharose, w-aminohexyl agarose, hydroxylapatite, phenyl Superose, Mono Q and adenosine agarose, have been used to purify PEAMeT. A 5403- fold purified preparation yielded a specific activity of 189 nmol· min-1 • mg-1 protein. SDS-PAGE analysis of this preparation revealed a number of polypeptide bands but only one which photoaffinity cross-linked to [3H]SAM. The estimated native molecular weight (MW) of PEAMeT was found to be 77 kDa by gel filtration chromatography and an estimated MW of 54 kDa was determined by SDS-PAGE. SDS-PAGE analysis of samples photoaffinity crosslinked to [3H]SAM gave a slightly higher estimated MW of 57 kDa. Effects of various factors on PEAMeT assay conditions were evaluated using partially purified PEAMeT preparations. PEAMeT activity as a function of pH gave a unimodal curve with an apparent pH optimum at 7.8 with 1 00 mM HEPES-KOH buffer. In vitro PEAMeT activity was inhibited by phosphate, PCho, S-adenosyi-L-homocysteine, ca+ 2, Mn+2 and co+2 but not by choline, betaine, ethanolamine, mono- and dimethylethanolamine or Mg+2 • Phosphobase N-methyltransferase activities present in preparations enriched for PEAMeT activity can catalyse the reaction sequence PEA- PMEA - PDEA - PCho. Under optimized assay conditions using PEA as the sole substrate, PMEA, PDEA and PCho were quantified and were detected in the order: PMEA (77%) > PDEA (17%) > PCho (6%). Thus a single enzyme, PEAMeT, is capable of converting PEA to PCho in leaves of spinach. The existence of a second enzyme which converts PMEA to PCho has also been reported for leaves and roots of spinach (Weretilnyk and Summers, 1992). The presence of two enzymes with overlapping activities raises questions regarding the roles of these two enzymes in choline metabolism. For example, do these enzymes also have overlapping functions in choline synthesis, particularly under conditions of osmotic stress? / Thesis / Master of Science (MSc)

Les analogues de choline constituent une nouvelle classe d'antipaludiques qui inhibent la biosynthèse de la phosphatidylcholine (PC) de Plasmodium, parasite responsable du paludisme. Les études conduites ont mis en relief des particularités uniques de ces composés. Nous avons élucidé le mécanisme d'action biochimique de l'albitiazolium, actuel candidat clinique, caractérisant chacune des 5 étapes conduisant à la biosynthèse de PC. L'albitiazolium affecte en premier lieu l'entrée de choline dans le parasite intraerythrocytaire, choline et albitiazolium utilisant le même transporteur et affecte de façon différentielle les autres étapes de synthèse. L'activité antipaludique est fortement antagonisée par la choline indiquant que le mécanisme d'action primaire est bien l'inhibition de la synthèse de PC. L'accumulation des analogues de choline dans le parasite intracellulaire leur permet de restreindre leur toxicité aux seuls érythrocytes infectés. Des études comparatives réalisées chez Plasmodium et Babesia montrent une double compartimentation de l'albitiazolium uniquement chez Plasmodium, l'une d'elles correspondant à la vacuole digestive. L'accumulation chez Plasmodium est glucose-dépendante et exige aussi le maintien des gradients ioniques dans la cellule. Bien que les analogues de choline exercent leur effet antiparasitaire dès les premières heures de contact, l'effet dit « cheval de Troie » exige des conditions particulières pour les mesures d'activités pharmacologiques, nous amenant à comparer différents tests d'activité. Seuls les tests isotopiques basés sur l'incorporation d'hypoxanthine ou d'éthanolamine après un cycle parasitaire entier et le test fluorescent au SYBR green appliqué après 72h obtiennent des résultats fiables quel que soit le mécanisme d'action des antipaludiques. Enfin, des études de pharmacocinétique / pharmacodynamie montrent une exposition plasmatique supérieure chez les souris infectées par Plasmodium, due au recyclage de l'albitiazolium après son accumulation dans l'érythrocyte infecté. / Choline analogues form a new class of antimalarial drugs that inhibit the biosynthesis of phosphatidylcholine (PC) in Plasmodium, the malaria-causing parasite. The studies presented here highlighted the unique features of these compounds. We elucidated the biochemical mechanism of action of albitiazolium, the current clinical candidate, characterizing each of the 5 steps leading to the biosynthesis of PC. Albitiazolium primarily affects the entry of choline into the intraerythrocytic parasite and choline and albitiazolium use the same carrier. The other steps of synthesis are differentially affected. Antimalarial activity is strongly antagonized by choline indicating that the primary mechanism of action is the inhibition of PC synthesis Accumulation of choline analogs in the intracellular parasite allows them to restrict their toxicity to infected erythrocytes. Comparative studies in Plasmodium and Babesia show a double compartmentalization of albitiazolium only in Plasmodium, one of them corresponding to the food vacuole. Accumulation in Plasmodium is glucose-dependent and requires maintaining ionic gradients in the cell.Although choline analogues exert their antiparasitic effect in the first hours of contact, the “Trojan horse effect” requires specific conditions for the determination of pharmacological activity, leading us to evaluate various tests of activity. Only the isotopic tests based on hypoxanthine or ethanolamine incorporation after one parasite cycle and the fluorescent SYBR green assay applied after 72 hours give reliable results regardless of the mode of action of the tested antimalarials. Finally, pharmacokinetics/pharmacodynamics studies in Plasmodium-infected mice revealed that albitiazolium is recycled after its accumulation in the infected erythrocyte leading to increased plasma levels.