This research focused on the study of the amino sugar recycling and sialic acid degradation pathway as a possible entry point for N-acyl glucosamines for the production of sialic acid analogs. Meeting this objective would allow the development of a bacterial strain capable of producing non-natural nonulosonic acids that could be used in the development of medicines, vaccines or useful compounds for the study of interactions between pathogenic organisms and their host. The first step was to understand how N-acetyl-D-glucosamine-6-phosphate deacetylase reacts to different types of substrates in order to determine its tolerance to the size of acyl groups in acyl amino sugars. This was achieved by studying the enzymatic activity in an in vitro system. We determine that the enzyme has a preference for small and slightly bulky acyl groups. Then, an in silico docking modeling and an in vivo system experiment were carried out. These experiments allowed to confirm the previous results. The second project was carried out due to the uncertainty of whether the kinase involved in the catabolic pathway would be able to phosphorylate the substrates. By quantifying residual ATP, the high specificity of N-acetyl-D-glucosamine kinase could be verified. This result led us to think about the design of an organic synthesis strategy that would allow the phosphorylation of glucosamine in carbon 6. A simple synthetic route was designed based on the protection of the two most reactive moieties of the amino sugars and the reactivity of the hydroxy group on carbon 6. However, we had problems with the purification step of the final product due to its high polarity. The next stage of this investigation was to confirm the transformation of GlcNAc into ManNAc. For this, an NMR analysis was designed that would detect the presence of both sugars in the reaction system. The epimerization of ManNAc to GlcNAc was detected successfully. Notwithstanding, the reverse reaction could not be detected. Based on the results obtained in the previous stage, we realized that an error was made in the epimerization reaction since we placed the wrong kinase because we did not take into account its substrate specificity. Finally, we tried to produce sialic acid analogs in a fermentative system using different genetic variants of Escherichia coli. Two of the expected analogs, Neu5Pr and Neu5nBu, were obtained. In addition, NagA activity towards substrates with small acyl groups was confirmed.

Chitin ist ein Polysaccharid, welches aus N-Acetylglucosamin (GlcNAc) aufgebaut ist. Das Biopolymer kommt in der Natur in Invertebraten, Algen, Pilzen und Einzellern vor. Chitinasen hydrolysieren Chitin. Diese Enzyme sind essentiell für die Regulierung und Entwicklung von Arthropoden und Mikroorganismen. Sie fungieren in Pflanzen als Phytoalexine. <br> Das verstärkt das Interesse am Design neuer natürlicher und synthetischer Chitinase-inhibitoren, die als potentielle Insektizide, Fungizide, Antimalaria bzw. Antiasthmatika eingesetzt werden können. <br><br> Ziel dieser Arbeit war die Synthese von neuen Thioglycosidanaloga von N-Acetyl-chitooligosacchariden und deren Untersuchung als Enzyminhibitoren. <br><br> Die geschützten 4-O-Tf-galactopyranosylthioglycoside wurden aus den korrespondierenden p-Methoxyphenylglycosiden von GlcNAc nach einer neuen Methode in einer Stufe hergestellt. Die Reaktion der galacto-Triflate mit 2-Acetamido-3,4,6-tri-O-acetyl-2-desoxy-1-thio-ß-D-glucopyranose in Gegenwart von Natriumhydrid und 15-Krone-5 in THF lieferte die Pseudo-Trisaccharide.<br><br> Die Herstellung der Glycosyl-Thiazoline aus den entsprechenden p-Methoxyphenyl-glycosiden mit Lawesson's Reagenz ist nach einem neuen Reaktionsweg beschrieben wie auch die Synthese der Thioacetamide von GlcNAc-Oligomeren.<br><br> Die Pseudo-Oligosaccharide wurden als Inhibitoren der Chitinasen von Serratia marcescens, Chironomus tentans, Acanthocheilonema viteae und Hevaea brasiliensis sowie der N-Acetyl-glucosaminidase von Chironomus tentans getestet. In einigen Fällen ergab sich eine Hemmung (IC50) im µM-Bereich. / Chitin is a polysaccharide composed of N-acetylglucosamine (GlcNAc). The biopolymer is distributed in Invertebrates, algae, fungi and Protozoa. Chitinases hydrolyse chitin. These enzymes are essential for the regulation and development of Arthropoda and micro organisms. In bacteria chitinase degrade chitin as source of carbon and act in plants as phytoalexins. Currently, there is a great interest in the design of novel natural and synthetic chitinase inhibitors, which may act as potential insecticides, fungicides, antimalaria or antiasthmatics.<br><br> Thio-oligosaccharides and related thio-glycosides are rarely described in the literature. Thio-analogues of oligosaccharides derived from N-acetylglucosamine are of interest as potential enzyme inhibitors. The synthesis and enzymatic investigations of thio-glycoside analogues of N-acetyl-chitooligosaccharides are described in this thesis.<br><br> The protected 4-O-Tf-galactopyranosyl thioglycosides were prepared by a new method in one step from the corresponding p-methoxyphenyl glycosides of N-acetylglucosamine. Coupling of galacto-triflates with 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-ß-D-glucopyranose in the presence of sodium hydride and 15-crown-5 in THF, followed by deprotection, afforded the pseudo-trisaccharides. <br><br> The preparation of glycosyl thiazolines by a new manner from the analogues p-methoxyphenyl glycosides with Lawesson's reagent is also described however the synthesis of thioacetamides of N-acetylglucosamine.<br><br> The pseudo-oligosaccharides were tested for inhibition of chitinases from Serratia marcescens, Chironomus tentans, Acanthocheilonema viteae, and Hevaea brasiliensis, and also with N-acetyl-glucosaminidase from Chironomus tentans. Inhibition with IC50 values in the µM range was found in a few cases.

O objetivo desta pesquisa foi o desenvolvimento, avaliação da estabilidade e da eficácia de formulações cosméticas contendo acetil hexapeptídeo-3 (AHP) ou acetil tetrapeptídeo-5 (ATP) ou mistura de di e tripeptídeos (DTP). Para tal, foram desenvolvidas 6 formulações à base hidroxietilcelulose (HEC), metilfenil polissiloxano (MFP) e álcool batílico e lecitina de soja (ABL) (F1), ou de HEC, MFP, ésteres de polietilenoglicol com cadeia alquílica com 12 a 20 carbonos (EP) (F2), ou de polímero de acrilatos (PA), MFP e ABL (F3) ou de PA, MFP e EP (F4), ou de HEC e MFP (F5) ou de PA e MFP (F6), contendo ou não (veículo) 10% de AHP, ou 10% de ATP ou 4% de DTP, as quais foram submetidas a um estudo de estabilidade física por determinação da reologia e a uma avaliação sensorial. Para o estudo de eficácia dos peptídeos objeto de estudo as formulações foram aplicadas nos antebraços, na face, na região dos olhos e da boca de 60 voluntárias, sendo realizadas medidas do conteúdo aquoso do estrato córneo, da perda de água transepidérmica (TEWL), do micro-relevo cutâneo e das propriedades mecânicas da pele (anisotropia), antes (basal), após 2 horas da aplicação única (efeitos imediatos) e após 15 e 30 dias de uso contínuo (efeitos a longo prazo) das formulações. Os dados foram analisados estatisticamente pelo teste paramétrico análise de variância. Nos testes de estabilidade física, as formulações de nos 1, 2 e 5 foram consideradas as mais estáveis e as formulações de nº 5 e 6 foram as que apresentaram melhor sensorial, sendo, portanto, selecionada a formulação de nº 5 como veículo para os estudos de eficácia. Na avaliação dos efeitos imediatos na pele, observou-se que as formulações estudadas proporcionaram um aumento significativo no conteúdo aquoso do estrato córneo dos antebraços, enquanto que, somente as formulações acrescidas dos peptídeos provocaram um aumento significativo neste conteúdo na face. Em relação ao micro-relevo cutâneo, as formulações contendo ou não os peptídeos provocaram uma redução do parâmetro Ser (aspereza da pele) e Rt (rugosidade) nos antebraços e a formulação contendo DTP provocou um efeito imediato significativo no parâmetro Sesm (textura da pele). Na avaliação dos efeitos a longo prazo, as formulações ocasionaram uma diminuição significativa na TEWL e as acrescidas dos peptídeos objetos de estudo proporcionaram um aumento significativo no conteúdo aquoso do estrato córneo na região dos olhos, enquanto que nos antebraços e na região da boca todas as formulações acrescidas ou não dos peptídeos ocasionaram um aumento nesse parâmetro. Em relação ao micro-relevo cutâneo, a aplicação das formulações contendo os peptídeos, na região da boca, provocou um aumento significativo nos valores de Sesm. A aplicação das formulações contendo os peptídeos objeto de estudo, nos antebraços ocasionou uma diminuição significativa da anisotropia da pele. A formulação nº 5 contendo os peptídeos objeto de estudo, apresentou efeitos imediatos na hidratação, aspereza, rugosidade e textura da pele e, efeitos a longo prazo, na hidratação, textura e anisotropia da pele, mostrando a importância do emprego destes peptídeos em produtos cosméticos com finalidades hidratantes e antienvelhecimento. / The aim of this study was the development, evaluation of the stability and the efficacy of cosmetic formulations containing acetyl hexapeptide-3 (AHP), or acetyl tetrapeptide-5 (ATP) or mixture of di- and peptides (DTP). For this purpose the 6 following formulations (F) were developed: F1- containing hydroxyethyl cellulose (HEC), methylphenyl polysiloxane (MPPS), batyl alcohol, and lecithin (BAL); F2- HEC, MPPS and C12-C20 PEG-8 alkyl ester (AE); F3- acrylate copolymer (AC), MPPS and BAL; F4- AC, MPPS and AE; F5- HEC and MPPS and F6- AC and MPPS, which were supplemented or not (vehicle) with 10% of AHP or 10% of ATP or 4% of DTP and submitted to the evaluation of physical stability by the determination of the rheologic behavior and also to the sensorial analysis. For clinical efficacy studies, formulations were applied to the volar forearm and to the face skin (eye corner and cheek region), of 60 female volunteers. Skin conditions in terms of skin moisture, transepidermal water loss (TEWL), skin mechanical properties (anisotropy) and skin microrelief, before and after 2 hours (immediate effects) and after 15 and 30-day period of daily application (long term effects). Results obtained were statistically analyzed using the ANOVA parametric test. In the rheological study, the formulations number 1, 2, and 5 were considered more stable than the others. Moreover, the formulations number 5 and 6 showed the best sensorial attributes, and for this reason the formulation number 5 was selected for the efficacy study. The evaluation of immediate effects of formulations showed that the application of the formulations studied in the volar forearm increased the water content of the stratum corneum, while only the formulations containing the peptides provoked an increased in this content, when face skin was analyzed. When the volar forearm skin microrelief was analyzed, the application of all the formulations studied provoked a significant decrease in skin roughness (Ser) and depth of roughness (Rt), furthermore only the formulation supplemented with the DTP showed a significant increase in the skin smoothness (Sesm). In the evaluation of long term effects, all formulations provoked a significant reduction in TEWL values and the formulations containing the peptides studied provoked a significant increase in the water content of the stratum corneum of eye corner; when the volar forearms and the cheek region were analyzed, all the formulations containing or not the peptides studied, provoked an increase in this parameter. When the skin microrelief was analyzed, only the formulation supplemented with AHP or ATP or DTP, provoked a significant increase in the skin smoothness (Sesm), when applied in the cheek region. The application of formulations containing the peptides studied in the volar forearms provoked a significant decrease in the skin anisotropy. The formulation nº 5 (F5) containing the peptides studied, showed immediate effects on skin hydration, roughness and smoothness, and also long-term effects on skin hydration, smoothness and mechanical properties, which demonstrated the importance of the use of these peptides in cosmetic formulations with hydration and anti-aging purposes.

<p>Gliomas are tumors of the central nervous system for which improvements in treatment are critically needed. Mutations in IDH1 and IDH2, which encode the cytosolic and mitochondrial NADP+-dependent isocitrate dehydrogenases, respectively, are frequent in gliomas. Here, we summarize recent literature concerning gliomas, the normal cellular functions of IDH1/2, the epidemiology of IDH1/2 mutations, and the understanding of the function of IDH1/2 mutations in cancer. We then show in vitro using liquid chromatography-mass spectrometry that a function of many IDH1/2 mutations is to produce 2-hydroxyglutarate. Next, we use a mass spectrometry based platform to characterize metabolic changes in a glioma cell line expressing IDH1/2 mutants and show that the IDH mutants are associated with lowered N-acetylated amino acids both in this cell line model and in primary tumor tissue. Finally, we develop and characterize a Drosophila melanogaster (fruit fly) model of IDH1/2-mutated cancer by expressing the mutated Drosophila homolog of IDH1 in fly tissues using the UAS-Gal4 binary expression system. These results delineate downstream molecular players that likely play a role in IDH1/2-mutated cancer and provide a model organism for interrogation of genetic networks that interact with IDH1/2 mutation. These findings refine our understanding of glioma pathogenesis and may inform the design of new glioma therapies.</p> / Dissertation

The enzyme spermidine/spermine N1-acetyltransferase (SSAT) is a candidate biomarker for various cancers as its activity in cancerous tissues is significantly increased. An artificial molecule, amantadine, is exclusively acetylated by SSAT to acetylamantadine (AcAm), levels of which in urine can serve as a proxy biomarker for malignancy. Current method of AcAm detection is laborious, time-consuming, and lacks the possibility of transforming to a point-of-care device. In this thesis, two different approaches were applied to detect AcAm in deionized water and in human urine using optical methods. The first one was fluorescence-based indicator displacement assay using cucurbit[7]uril as the receptor molecule. The second was programmed gold nanoparticle disaggregation with cucurbit[7]uril as a molecular linker. / Graduate

No description available.

Flavonoids are natural compounds commonly ingested in herbs and vegetables. They are believed to have a positive impact on human organism, in particular by their antioxidant, hepatoprotective and anti-cancer effects. In these days, it is possible to consume high concentrations of these compounds in form of dietary supplements. However it is not clear, whether flavonoids in such unnaturally high concentrations are still beneficial or rather harmful. It has already been proven, that flavonoids can influence the activity of biotransformation enzymes and interfere e.g. with the process of carcinogenesis and drug metabolism. For that reason it is important to investigate the impact of an increased intake of flavonoids. The aim of this thesis was to investigate the influence of dihydromyricetin (a potential drug to cure alcohol use disorder) and its structurally similar flavonoid myricetin on the activity of enzymes, cytochrome P450 2E1 (CYP2E1) and N­acetyltransferases 1 and 2 (NAT1/2). The research included the determination of the impact of a premedication by these flavonoids on the expression and activity of CYP2E1 and NAT1/2. The inhibition capacity of myricetin and dihydromyricetin towards the activity of CYP2E1 and NAT1/2 was also investigated. After the flavonoid premedication of rats the...

Doctor of Philosophy / Biochemistry and Molecular Biophysics / Timothy P. Durrett / The membrane bound O-acyltransferase (MBOAT) family contains multi-pass membrane proteins that add fatty acids to different compounds. Despite their importance in economic activity and human health, little is known about the localization of the active site and regions important for determining substrate specificity of MBOATs. Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) is the only known MBOAT enzyme that exhibits a high preference for acetyl-CoA, the shortest possible acyl-CoA. EaDAcT catalyzes the transfer of the acetate group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol. Our goal was to investigate the structural features and the amino acid residues that define substrate specificity of EaDAcT to provide insights into the mechanism by which MBOAT family controls substrate selection. By mapping the membrane topology of EaDAcT we obtained the first experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains with both the N- and C- termini oriented toward the endoplasmic reticulum lumen. The MBOAT signature region including the putative active site His-257 of the protein is embedded in the third transmembrane domain close to the interface between the membrane and the cytoplasm. In order to identify amino acid residues important for acetyltransferase activity, we isolated and characterized orthologs of EaDAcT from other acetyl-TAG producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved residues of DAcTs revealed that Ser-253, His-257 and Asp-258 are essential for enzyme activity of EaDAcT, suggesting their involvement in the enzyme catalysis. Alteration of residues unique to acetyltransferases did not alter the acyl donor specificity of EaDAcT, implying that multiple amino acids are important for substrate recognition. Together, this work identifies the structural features of EaDAcT and offers an initial view of the amino acids important for activity of the enzyme.

Bacterial regulatory small RNAs (sRNAs) act as crucial regulators in central carbon metabolism by modulating translation initiation and/or degradation of target mRNAs in metabolic pathways. This work demonstrates that a noncoding sRNA, SdhX, is produced by RNase E-dependent processing from the 3 ́ untranslated region (3 ́ UTR) of an operon encoding three enzymes of the tricarboxylic acid (TCA) cycle. In Escherichia coli, SdhX negatively regulates ackA (encoding an enzyme critical for degradation of the signaling molecule acetyl phosphate) without affecting the downstream pta gene that encodes the enzyme critical for acetyl phosphate synthesis. SdhX abundance is tightly coupled to the transcription signals of the TCA cycle genes but escapes all known post-transcriptional regulation. Therefore, SdhX expression directly correlates with transcriptional input to the TCA cycle, providing an effective mechanism for the cell to link the TCA cycle with acetate metabolism pathways. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

O desempenho animal pode ser melhorado pelo incremento na digestibilidade dos alimentos. Um dos elementos neste processo é a acurada caracterização da composição química. Objetivando avaliar quatro métodos para determinar o teor de lignina, foram estudadas cinco gramíneas: Brachiaria brizantha cv. Marandú, Brachiaria brizantha cv. Xaraés (MG-5), Panicum maximum cv. Mombaça, Pennisetum purpureum cv. Cameroon e Pennisetum purpureum cv. Napier. As frações fibrosas da parede celular (PC), fibra em detergente neutro (FDN) e fibra em detergente ácido (FDA) aumentaram conforme as plantas amadureceram, refletindo as mudanças na composição dos componentes da parede celular (celulose, hemicelulose e lignina). Os valores de PC foram superiores aos da FDN indicando solubilização da pectina e outros oligossacarídeos da parede celular na solução de detergente neutro. O método LDA apresentou os menores teores de lignina, evidenciando a solubilização de parte da lignina na solução de detergente ácido. Os resultados de LPer foram maiores que os de LDA, que pode ser devido à oxidação da celulose e pectina pelo permanganato de potássio. Os teores de LK foram maiores que os de LDA possivelmente por contaminação protéica, mas, menores que os de LPer. Os teores de LBA foram maiores que os outros três métodos. A digestibilidade acompanhou, de forma inversa, o estádio de maturidade das plantas. A digestibilidade in vitro apresentou forte correlação negativa com os teores de lignina para todos os métodos, menos para LPer. Encontrou-se um valor de relação de 2,23, entre os métodos LDA e LBA, que, ao ser aplicado, nos teores de LDA, resultou em reta similar ao da LBA. Chama a atenção como este valor de 2,23 é muito próximo ao 2,4 utilizado nas equações B2 e C das frações de carboidratos do \"Cornell Net Carbohydrate & Protein System\" e nas equações do National Research Counsil de 1996, para corrigir o teor de lignina. O método LBA é um método fácil e conveniente para determinar a concentração de lignina em forrageiras e uma boa opção para uso rotineiro nas análises de laboratório. / Animal performance can be improved by enhancing feed digestibility. One of the elements for this process is an accurate characterization of feedstuff chemical composition. With the objective of evaluating four methods used today for lignin determination, five grasses were used: Brachiaria brizantha cv. Marandú, Brachiaria brizantha cv. Xaraés (MG-5), Panicum maximum cv. Mombaça, Pennisetum purpureum cv. Cameroon e Pennisetum purpureum cv. Napier, All fibrous fractions, neutral detergent fiber (NDF), acid detergent fiber (ADF) and cell wall (CW), increased as the plants matured, reflecting the changes in the CW composition (cellulose, hemicellulose and lignin). The values obtained for CW were higher than those obtained for NDF, indicating solubilization of pectin and other cell wall oligosaccharides in the neutral detergent solution. The ADL method produced the lowest lignin values, reflecting lignin solubilization by the acid detergent solution. PerL results were higher than those of ADL, possibly due to hemicellulose and pectin oxidation by potassium permanganate. The values for KL were higher than those of ADL, possibly due to protein contamination, but were lower than PerL values. ABL values were the highest among all methods. Digestibility inversely followed plant maturity throughout the study. In vitro dry matter digestibility showed high negative correlation with lignin contents. A 2,23 ratio between ADL and ABL methods was found, which when applied to ADL values, resulted in a curve similar to ABL method curve. It is interesting to note that, this value of 2,23 is very close to the 2,4 used in carbohydrate fractions B2 and C of the \"Cornell Net Carbohydrate & Protein System\", for the correction of lignin content. The ABL method is easy and convenient for total lignin content determination in forages.