Choline is necessary for the synthesis of phosphatidylcholine (PC), the predominant phospholipid species and an important lipid intermediate. Macrophages, critical mediators of innate immunity, have been implicated in lipid dysregulation associated with metabolic disease. Despite the importance of choline in lipid metabolism, few studies have investigated the relationship between choline metabolism and inflammation. My research revealed that macrophage polarization increased choline metabolism and the expression of the choline transporter CTL1. In addition, choline deficient macrophages showed altered cytokine secretion, suggesting choline metabolism may play an important role in regulating the immune response. This study also describes the generation of a novel CTL1-/- mouse, which showed decreased choline uptake and incorporation into lipids. As an in vivo model for choline deficiency, CTL1-/- mice represent an important model for the future study of choline metabolism. Altogether, these findings suggest an important relationship exists between choline metabolism and inflammation.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. AD is characterized clinically by cognitive, behavioral, and learning and memory impairments and neuropathologically by amyloid-beta (Aβ) plaques, neurofibrillary tangles (NFTs), neuroinflammation, and synaptic and neuronal loss. Although AD was first described over a century ago, there is no cure and the limited treatment options do little to slow the progression of the devastating disease. Because of these reasons, there has been a significant interest in preventative strategies for AD. Previous studies have shown that perinatal choline supplementation can improve learning and memory, dampen neuroinflammation, diminish cholinergic deficits, reduce brain amyloidosis, and increase neurogenesis in AD model mice. In these studies, we investigated the roles of perinatal choline supplementation on anxiety and exploratory behavior, spatial and fearful learning and memory, brain Aβ42 deposition, and gene expression in the AppNL-G-F AD mouse model. We found that there were slight anxiety-related behavior changes in AppNL-G-F mice compared to wildtype mice. AppNL-G-F mice had impaired spatial learning that could be prevented by perinatal choline supplementation at early ages (3- and 6-months-old) but not a later age (12-months-old). Fearful learning and memory deficits were found in AppNL-G-F mice at 9- and 12-months of age and could be abolished by perinatal choline supplementation. Perinatal choline supplementation significantly reduced Aβ42 deposition across the brain in AppNL-G-F mice. The AppNL-G-F transcriptome was characterized by changes in genes related to inflammatory, neuronal and synaptic, energy, and metabolic pathways and led to a distinct transcriptional signature from wildtype mice. Perinatal choline supplementation was able to significantly reduce the transcriptional deficits found in AppNL-G-F mice and led to the identification of choline responsive AD-pathology-associated genes (CRADPAGs). Lastly, some of the CRADPAGs identified using AppNL-G-F AD model mice correlated significantly with AD diagnosis, clinical dementia rating, and AD pathologies in a human data set. Together, the results from these studies and the previously published data suggest that adequate choline intake during development may serve as a strategy to prevent or reduce AD dementia and pathologies. / 2025-01-24T00:00:00Z

Le développement de sondes moléculaires fluorescentes pour le suivi ex vivo de phénomènes biologiques (communication nerveuse, par exemple) est en plein essor. Dans cette optique, des dérivés fluorescents de cyclotrivératrylènes (CTV), cavitands propices à la complexation de petites molécules organiques, ont été synthétisés. La stratégie utilisée consiste à introduire des groupements électro-donneurs et électro-attracteurs conjugués sur chaque unité aromatique du CTV afin d’avoir des systèmes de type « push-pull ». L’extension de la conjugaison entre les groupements électro-attracteurs et électro-donneurs a été envisagée afin d’améliorer les propriétés spectroscopiques des sondes. Des réactions de couplage organométallique de Sonogashira, ainsi que des cycloadditions alcynes-azotures catalysées par le cuivre (CuAAc), ont été conduites sur le squelette CTV dans ce but. La caractérisation spectroscopique des différentes sondes fluorescentes développées a été réalisée en milieu organique et en conditions physiologiques, pour celles dont la solubilité le permettait. L’étude des propriétés de reconnaissance de ces dernières, pour des ammoniums d’intérêt biologique (tels que les neurotransmetteurs, acétylcholine, dopamine et dérivés) en conditions physiologiques, a également été menée par spectrofluorimétrie, ainsi que par d’autres techniques (RMN, calorimétrie). La formation d’assemblages moléculaires en milieu aqueux a également été mise en évidence (expériences de diffusion dynamique de la lumière, microscopie électronique en transmission), pour certaines sondes lors de ce travail. / The development of fluorescent probes for the ex vivo detection of biological phenomena (neuronal communication, for example) presents a growing interest. In this context, fluorescent cyclotriveratrylene (CTV) derivatives have been developed, as these cavitands are known to complex small organic molecules. The strategy used to get interesting spectroscopic properties, was to introduce conjugated electron-donating and electron-withdrawing groups on each aromatic unit of the CTV (leading to “push-pull” systems). To improve the spectroscopic properties of the probes, the conjugation between the electron-donating and the electron-withdrawing groups was extended. Hence, the Sonogashira organometalic coupling reaction and the copper catalyzed cycloaddition directly on CTV skeleton were used. The spectroscopic characterization of the new fluorescent probes synthesized was done, in organic solvent but also in physiological conditions when the solubility permits it. The recognition studies for biological ammoniums, such as neurotransmitters (acetylcholine, dopamine, and derivatives) were performed in physiological conditions by spectrofluorimetry but also other methods (like MNR or micro-calorimetry). The formation of molecular assemblies was also observed (by dynamic light scattering and transmission electron microscopy) during this work with some of the probes.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Nonalcoholic steatohepatitis (NASH) has been identified as a source of significant inter individual variation in drug metabolism. A previous ex vivo study demonstrated significant changes in hepatic Cytochrome P450 (CYP) activity in human NASH. This study evaluated the in vivo activities of multiple CYP isoforms simultaneously in prominent diabetic NASH mouse models. The pharmacokinetics of CYP selective substrates: caffeine, losartan, and omeprazole changed significantly in a diabetic NASH mouse model, indicating attenuation of the activity of Cyp1a2 and Cyp2c29, respectively. Decreased mRNA expression of Cyp1a2 and Cyp2c29, as well as an overall decrease in CYP protein expression, was found in the diabetic NASH mice. Overall, these data suggest that the diabetic NASH model only partially recapitulates the human ex vivo CYP alteration pattern. Therefore, in vivo determination of the effects of NASH on CYP activity should be conducted in human, and more appropriate models are required for future drug metabolism studies in NASH.

A Doença Hepática Gordurosa Não Alcoólica (DHGNA) é uma condição clínicopatológica comum, caracterizada por depósito de lipídeos no hepatócito do parênquima hepático. A esteatose hepática (EH) é um dos componentes da DHGNA e caracteriza-se pela presença de vacúolos de lipídeos, principalmente triacilgliceróis (triglicerídeos), dentro dos hepatócitos. Alterações na oxidação das gorduras no fígado ou redução na exportação de lipoproteínas de muito baixa densidade (VLDL) a partir do órgão são os principais mecanismos etiopatogênicos envolvidos com a EH. A patogênese da DHGNA é multifatorial e diversos fatores ou condições têm sido relacionados à predisposição para o seu desenvolvimento. Atualmente, diferentes tratamentos farmacológicos para DHGNA estão sendo propostos, mas ainda não há nenhum estudo comprobatório da sua eficácia. Objetiva-se avaliar os efeitos da suplementação da colina e do frutooligossacarídeo (FOS) na dieta de ratos Wistar, no modelo de esteatose hepática, induzido por dieta hiperglicídica. Foram utilizados 46 ratos machos, da raça Wistar adultos com peso variando entre 250 - 320 g, vindos do Biotério Central do Campus da USP Ribeirão Preto. Do lote inicial de animais foram distribuídos de forma aleatória nos diferentes grupos de estudo de I a IV, dependendo da indução ou não da esteatose. Considerou-se fase I o período correspondente a indução de esteatose e fase II quando se submeteu os animais a suplementação com nutrientes (Grupos III e IV), ou quando os animais receberam dieta padrão pós fase I (Grupo II) . Os animais do Grupo I (controle) receberam ração padrão do biotério que foi igual para todos os animais, sendo separado um lote da mesma ração para todo o experimento. Foi analisado as seguintes variáveis: Ingestão alimentar semanal, evolução do peso dos animais, nitrogênio urinário, amônia urinária, colesterol total e triacilgliceróis séricos, peso úmido de fígado e coração, nitrogênio e gordura tecidual, dosagem de vitamina E, malondialdeído (MDA) e glutationa no tecido hepático e análise histopatológica. Observamos que nenhum dos nutrientes empregados (colina e FOS) foi eficaz na redução da quantidade de gordura do fígado pela análise histológica. Nenhum dos nutrientes adicionados foi capaz de proteger o fígado da ação dos radicais livres, já que o MDA, um marcador indireto da geração do estresse oxidativo, manteve-se com valores elevados mesmo na fase de tratamento. Ocorreu diminuição dos níveis de triacilgliceróis em todos os grupos submetidos à indução de esteatose, do início ao final do experimento. O frutooligossacarídeo foi capaz de reduzir os níveis de colesterol sérico, em relação aos seus níveis basais, quando suplementado após indução de esteatose. / Non-alcoholic fatty liver disease (NAFLD) is a common clinical pathological condition characterized by fat accumulation in the the hepatic parenchyma hepatocyte. Liver steatosis (HS) is one of the components of NAFLD and is characterized by the presence of lipids vacuoles, mainly triacylglycerol, within the hepatocites. Alterations in fat oxidation in the liver or very low density proteins lipoproteins tranport from the organ are the main etiopatogenic mechanisms involved in HS. NAFLD patogeny is multifactor, thus several factors have been associated the propensity to develop it. Presently, many drug treatments for NAFLD are being suggested, however, there have been no studies that prove their efficacy so far. The aim of this study was to assess the effects of choline and fructooligosaccharide (FOS) suplementation in Wistar rats with HS induced by high glicid diet. Forty six adult male Wistar rats weighing between 250g and 320g from the USP (Ribeirão Preto-USP) central vivarium were used. They were divided randomly into different study groups from I to IV depending on whether steatosis would be induced or not. Phase I of the study was the period corresponding to steatosis induction and phase II was when the animals received nutrient suplementation (Groups III and IV) or when they received standard diet (Group II). Group I animals (control) received the usual vivarium food, which was the same for all of them. A certain amount of that same food was kept aside for the duration of the experiment. The following variables were analyzed: weekly food intake, weight gain, urine ammonia, urine nitrogen, total cholesterol and serum triacylglycerol, liver and heart humid weight, nitrogen and tissue fat, vitamin E, malondialdehyde (MDA) and glutathione content in the liver tissue and hitopathological analysis. As observed, neither of the nutrients (choline and FOS) was efficient in reducing the amount of fat in the liver. Neither of the nutrients added was able to protec the liver from free radicals, once the MDA, a indirect marker for oxidative stress generation, showed high levels even during the treatment phase. There was a reduction in triacylglycerol levels in all steatosis induced groups, from the beginning to the end of the experiment. Fructooligosaccharide was able to reduce the levels of serum cholesterol, in relation to its basal levels, when suplemented after steatosis induction.

The ability of bacteria to detect their surroundings and enact an appropriate response is critical for survival. Translation of external signals into a coherent response requires specific control over the transcription of DNA into RNA. Much of the regulation at this step is accomplished by transcriptional regulators, proteins that bind to DNA and alter gene expression. A wide-spread variety of regulators in bacteria is the AraC-family. These regulators are divided into two conserved domains and respond to a variety of compounds owing to different N-terminal domains. A subfamily of these regulators, GATase1-like AraC-family transcriptional regulators (GATRs), is described. These proteins contain an N-terminal domain with structural characteristics similar to enzymes that synthesize amine-containing compounds. Members of this subfamily of transcriptional regulators are found in a wide range of bacteria, however, few are characterized. A relatively high number of GATRs are encoded in the Burkholderia thailandensis genome. Therefore, we utilized this bacterium as a model to explore the function and diversity of these regulators. GATRs in B. thailandensis divided into two groups based on bioinformatics analysis. The first group includes three members which we identified that contribute to the positive regulation of glycine betaine (GB) catabolism. GB can be utilized as a nutrient source or as a potent osmoprotectant. The regulation of this pathway in B. thailandensis differs from previously established models due to the interplay of these regulators. Homologs of two other GATRs in this group were identified that regulate carnitine and arginine catabolism. The second group of GATRs contains uncharacterized members with no known functions. A genetic strategy for engineering constitutive GATRs was developed and employed to investigate the transcriptional regulons of these GATRs. This approach yielded the identification of a novel GATR that represses expression of an operon producing a formaldehyde detoxification system, and is the first example of a GATR that functions as a repressor.

Choline oxidase catalyzes the oxidation of choline to glycine betaine. The reaction includes betaine aldehyde as an intermediate. FAD is reduced by the alcohol substrate, betaine aldehyde intermediate and oxidized by molecular oxygen to give hydrogen peroxide. In this study, the Ser101Ala variant of choline oxidase was prepared to elucidate the contribution of the hydroxyl group of Ser101 in the proton and hydride transfer reactions for proper preorganization and reorganization of the active site towards quantum mechanical tunneling. The thermodynamic parameters associated with the enzyme-catalyzed OH and CH bond cleavages and the temperature dependence of the associated solvent and substrate kinetic isotope effects were investigated using a stopped-flow spectrophotometer. The proton and hydride transfer have been shown to be occurring via quantum tunneling in CHO-S101A enzyme.

Introduction [11C]choline is a very effective PET radiopharma-ceutical for the study of prostate cancer. To support the increasing demand for [11C]choline, several different synthetic approaches have been described in the literature, including different automated production methods using remote-controlled synthesis modules [1–4]. The most popular method uses a C18 Sep-Pak as solid support for methylation and, subsequently, a CM Sep-Pak for purification [2]. We report an optimized method for producing [11C]choline using only one CM Sep-Pak for both reaction and purification as was shown in the literature [4]. For synthesis of [11C]choline we used two modules Tracerlab FXC for preparation of methylation reagent [11C]CH3I and GPF-101 for [11C]choline synthesis. Material and Methods TracerlabFXC GE, GPF-101 Veenstra Instrument, 2-(dimethylamino)-ethanol (DMAE) ABX, Sep-Pak Light Accell Plus CM cation-exchange cartridges Waters used without conditioning, precursor 50 µL of DMAE dissolved in 25 µL of ethanol and loaded on a CM Sep-Pak. Schematic diagram of the automated system for the production of [11C]choline is given below. [11C]CH4 was produced in two standard Nitra target IBA irradiation of mixture 90 % N2/10 % H2 with 15 MeV protons using dual beam. Results and Conclusion [11C]CH4 was prepared in the targets and connected with Tracerlab FXC. [11C]CH3I was pre-pared in a loop in which allowed to react of elemental iodine at a temperature 720 oC. Con-version to [11C]CH3I usually is around 50% uncorrected activity. Activity is within the range 15–18 GBq of [11C]CH3I and time of production 10 min. Synthesis of [11C]choline is based on the reaction DMAE with [11C]CH3I on a Accell Plus CM cation-exchange column which serves both as a support for reaction and for separation of choline from DMAE by ethanol washing. The basic parameters are shown in TABLE 1. Beam current 2X 20 µA Irradiation time 30 min DMAE 50 µl Synthesis time from EOB 25 min Absolute yield without correction 6.6 GBq Radiochemical purity > 99 % Residual DMAE in product < 5 ppm Ethanol < 1000 mg/L pH 4.5–8.5 TABLE 1. Reaction parameters and result of production of [11C]choline syntheses Conclusion We have applied a simple synthesis method for [11C]choline preparation using automated commercial equipments with one column used both for reaction and separation purpose. The main advantage of using one column is lower contamination of the product [11C]choline with DMAE. When for synthesis of [11C]choline two columns C18 for synthesis and CM for separation is used, higher contamination of DMAE can be found in the product due to a release of DMAE from C18 column.

Thesis (Ph. D.)--Ohio University, August, 2002. / Title from PDF t.p. Includes bibliographical references (leaves 232-247).