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1	Transforming Dihydroxyacetone Phosphate-Dependent Aldolases Mediated Aldol Reactions From Flask Reaction Into Cell-Based Synthesis & Studying The Mechanism Of Chemical Desialylation In The Life Processes Wei, Mohui 09 May 2016 (has links) Dihydroxyacetone phosphate (DHAP)-dependent aldolases have been intensively studied and widely used in the synthesis of carbohydrates and complex polyhydroxylated molecules. However, the strict specificity toward donor substrate DHAP greatly hampers their synthetic utility. We transformed DHAP dependent aldolases mediated in vitro reactions into bioengineered Escherichia coli (E. coli). Such flask-to-cell transformation addressed several key issues plaguing in vitro enzymatic synthesis: 1) it solves the problem of DHAP availability by in vivo hijacking DHAP from glycolysis pathway of bacterial system, 2) it circumvents purification of recombinant aldolases and phosphatase, and 3) it dephosphorylates resultant aldol adducts in vivo, thus eliminating the additional step for phosphate removal and achieving in vivo phosphate recycling. The engineered E. coli strains tolerate a wide variety of aldehydes as acceptor, and provide a set of biologically relevant polyhydroxylated molecules in gram scale. Sialic acids exist in abundance in glycan chains of glycoproteins and glycolipids on the surface of all eukaryotic cells and some prokaryotic cells. Their presence affects the molecular properties and structure of glycoconjugates, modifies their functions and interactions with other molecules. The sialylation status, referring to the expression levels and linkages of sialic acids on the cell surface, is determined by the dynamic balance between sialylation and desialylation (removal of sialic acids). Sialylation is mainly regulated through expression and activity of sialyltransferases. And the mainstream idea attributes desialylation to the sialidases. However, more and more emerging evidences support the existence of ROS/RNS mediated chemical desialylation process under some pathological conditions. We used electrochemical oxidation of sialic acid conjugates to mimic ROS mediated chemical desialylation. Such electrochemical desialylation mimicry reveals that 1) β-linked sialic acid is much more difficult to de desialylated than α-linked sialic acid, 2) electron withdrawing residue and bulky underlying residue can facilitate the desialylation, 3) α- 2,3-linked sialic acid is easier to be desialylated than α-2,6- and α-2,8-linked sialic acid. This information is highly valuable for identifying the ROS species participated in ROS mediated desialylation and unveiling corresponding mechanisms. The mechanism of ROS mediated desialylation was proposed to go through radical decarboxylation. DHAP-dependent aldolase Metabolic engineering E. coli synthetic machinery Electrochemical desialylation Desialylation mechanism
2	Study of the pathophysiological role of nitric oxide on the amyloid-induced toxicity attending to the biochemical modifications and cellular damages Guix Ràfols, Francesc Xavier 22 January 2009 (has links) Aquesta tesi demostra que el peroxinitrit produït com a conseqüència del pèptid beta-amiloide (A) contribueix l'augment de la relació A42/A40 que ocorre a la malaltia d'Alzheimer. L'A42 contribueix a l'aparició de la malaltia degut a la seva major toxicitat (quan es compara amb l'A40) que resulta d'una gran estabilitat i capacitat agregativa. A més el peroxinitrit incrementa la toxicitat d'aquest degut a què potencia la seva agregació en forma d'oligomers altament tòxics. De fet els oligomers formats de nitro-A42 presenten una major toxicitat que aquells formats de A42 . En conjunt aquest resultats senyalen l'important paper que l'A42 té en la malaltia d'Alzheimer. Per altra banda, des de la identificació dels agregats d'A i la subseqüent formació dels cabdells neurofibrilars (NFT) com a els dos trets distintius de la malaltia, un gran esforç s'ha dedicat a establir els mecanismes moleculars que uneixen ambdós processos. Aquesta tesi demostra que el peroxinitrit format a partir de l'agregació de d'Ai la conseqüent nitrotirosinació de proteïnes, potencia l'agregació de la proteïna tau en forma de fibres. D'aquesta forma, la nitrotirosinació de la proteïna triosafosfat isomerasa (TPI) podria ser el vincle entre la toxicitat derivada del agregats d'Ai la patologia derivada de la proteïna tau. Per tant, la nitrotirosinació de la TPI podria explicar la progressió temporal que ocorre als cervells de pacients amb la malaltia d'Alzheimer des de la toxicitat induïda per l'Ai l'aparició dels NFT. Els resultats presentats en aquesta tesi podrien obrir nous aspectes en la recerca de la malaltia d'Alzheimer així com en altres malalties que cursin amb estrès oxidatiu i plegament erroni de proteïnes. / This thesis demonstrates that amyloid ß-peptide (Aß)-induced peroxynitrite contributes to the switch of the Aβ42/Aβ40 ratio that occurs in Alzheimer's disease (AD). Since Aβ42 is more toxic due to its higher aggregation and stability, it contributes to the trigger of the disease. In addition the aggregation of Aβ42 in form of the highly toxic oligomers is incremented by the presence of peroxynitrite. Moreover, these nitro-Aß42 oligomers are more toxic than those non-nitrated. All these results support the important role of peroxynitrite in AD etiology. Furthermore, since the identification of Aß accumulation and the subsequent formation of neurofibrillary tangles (NFT) as the two defining pathological hallmarks of AD, a fair amount of research on AD has been driven by the need to find the molecular mechanism linking Aß and NFT. This thesis shows the Aß-induced peroxynitrite, and the consequent nitrotyrosination of proteins, promotes tau fibrillization. Thus triosephosphate isomerase (TPI) nitrotyrosination could be the link between Aß-induced toxicity and tau pathology. Therefore, TPI nitrotyrosination may explain the temporal progression from Aß toxicity to NFT formation in AD brain. The work presented in this thesis could open a novel angle in the research of the pathophysiology of AD and could also have an impact to the research in other neurodegenerative diseases involving oxidative stress and protein misfolding. GAP free radicals fibrils familiar AD DHAP cerebral cortex BACE1 APH-1 antioxidants amyloid-beta peptide protein amyloid precursor alzheimer's disease advanced glycation end products acetylcholine plaques peroxinitrite peròxid d'hidrogen pèptid beta-amiloide Pen2 òxid nítric sintasa òxid nítric oligomers nitrotirosinasa nicastrina neurona metilglioxal malaltia d'alzheimer hipocamp glioxalase glicolisis GAP helicoidals aparellats filaments fibriles èstres oxidatiu DHAP cortex cerebral cabdells neurofibrilars BACE1 APH-1 antioxidants anió superòxid AD familiar AD esporàdic acetilcolina glycolysis glyoxalase hippocampus hydrogen peroxide methylglyoxal neurofibrillary tangles neuron nicastrin nitric oxide nitric oxide synthase nitrotyrosination oligomers oxidative stress pair helical filaments 616.8
3	Improved techniques for CE and MALDI-MS including microfluidic hyphenations foranalysis of biomolecules Jacksén, Johan January 2011 (has links) In this thesis, improved techniques for biomolecule analysis using capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and hyphenations between those have been presented.A pre-concentration method which is possible to apply in both techniques, has also been investigated. In this work the off-line MS mode has been used either in the form of fractionation (Paper I) or by incorporating the MALDI target in the CE separation system (Paper II).In Paper I, a protocol for CE-MALDI analysis of cyanogen bromide digested bacteriorhodopsin (BR) peptides as model integral membrane protein peptides were established. Also, an improved protocol for partially automated manufacturing of a concentration MALDI-target plate is presented. The design of the targets was suitable for the fractions from the CE. A novel technique for the integration of CE to MALDI-MS using a closed-open-closed system is presented in Paper II, where the open part is a micro canal functioning as a MALDI target window. A protein separation was obtained and detected with MALDI-MS analysis in the micro canal. A method has been developed for detection of monosaccharides originating from hydrolysis of a single wood fiber performed in a micro channel, with an incorporated electromigration pre-concentration step preceding CE analysis in Paper III. The pre-concentration showed to be highly complex due to the fact that several parameters are included that affecting each other. In Paper IV a protocol using enzymatic digestion, MALDI-TOF-MS and CE with laser induced fluorescence (LIF) detection for the investigation of the degree of substitution of fluorescein isothiocyanate (FITC) to bovine serum albumin (BSA), as a contact allergen model system for protein-hapten binding in the skin, is presented. The intention of a further CE-MALDI hyphenation has been considered during the work. In Paper V 2,6-dihydroxyacetophenone (DHAP) was investigated, showing promising MALDI-MS matrix properties for hydrophobic proteins and peptides. 2,5-dihydroxybenzoic acid (DHB) was undoubtedly the better matrix for the hydrophilic proteins, but its performance for the larger and hydrophobic peptides was not optimal. Consequently, DHAP can be used as a compliment matrix for improved analysis of hydrophobic analytes. / QC 20101214 Capillary electrophoresis Hydrophobic peptides/proteins Prestructured target Off-line interface Silicon micro canal Matrix Bovine serum albumin DHAP Hyphenations Hydrophobicity Single wood fiber analysis Pre-concentration Concentration platform Analytical chemistry Analytisk kemi

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