Dissertations / Theses on the topic 'OYE Enzymes'

Dynamic structural fluctuations occurring over a broad range of timescales are now known to facilitate the catalytic function of enzymes, but there is less comprehensive experimental evidence linking fast-timescale, high frequency motions to the reaction coordinate. Interest in the role of such motions has recently surged and been the subject of intensive experimental efforts, in part due to the identification of enzymatic hydride tunnelling reactions. This mechanism involves transiently degenerate product and reactant states, which enable H-transfer to occur instantaneously without the need to surmount the activation barrier associated with traditional transition-state based models of enzyme catalysis. The primary gauge of tunnelling in enzyme-catalysed reactions is the identification of temperature dependent kinetic isotope effects (KIEs), i.e. the relative rates of a reaction where the transferred atom is substituted for an alternate isotope. The identification of temperature-, and also pressure-, dependent KIEs has resulted in the emergence of new models of describing enzymatic H-transfer. These invoke a role for fast-timescale protein motions that 'promote' transfer via tunnelling. A popular model system for studying enzymatic H-tunnelling reactions is Pentaerythritol tetranitrate reductase, which belongs to the Old Yellow Enzyme (OYE) family of ene-reductases. These nicotinamide coenzyme dependent oxidoreductases catalyse the stereospecific reduction of alpha/β-unsaturated alkene containing substrates. Here, the importance of donor-acceptor distances in determining the observed rate of PETNR reduction with NAD(P)H is probed via a detailed structural and kinetic analysis of site-directed variants. In addition, an investigation of distance-dependent Nuclear Overhauser effects via Nuclear Magnetic Resonance (NMR) spectroscopy is undertaken to assess active site organisation and measure donor-acceptor distances in PETNR-substrate complexes. A variable pressure NMR study reveals how NOE build- up is perturbed in high-energy conformers favoured as a result of the application of increased hydrostatic pressures. Recently there has been interest in exploiting the stereoselective properties of reactions catalysed by ene-reductase enzymes for use in biocatalytic reactions to produce industrially valuable compounds from renewable sources. The reactions of PETNR and additional OYE enzymes, Thermophilic old yellow enzyme and Xenobiotic reductase A, with both natural coenzymes and a set of synthetic Nicotinamide Coenzyme Biomimetics (NCBs) are also characterised. The NCBs represent affordable and fast-reacting alternatives to the physiological coenzymes. Reactions with NCBS are also shown to proceed via a tunnelling mechanism and furthermore, that enhanced donor-acceptor sampling correlates with the faster reactivity seen with these compounds.

The food-borne pathogene Listeria monocytogenes has been considered a significant threat to human health worldwide. It mainly infects individuals suffering insuffecint immunity such as pregnant women. During pregnancy, L. monocytogenes is capable of causing a serious damage to the mother and the fetus. It can spread to different organs including the placenta via adaptation to interacellular lifestyle. To maintain pregnancy, the levels of the hormones progesterone and β-estradiol increase and reduction in hormone levels was proposed to be associated with fetal death and abortion. The objectives of this project therefore were to investigate the role of pregnancy hormones on the growth and virulence of L. monocytogenes, and to identify bacterial genes with possible roles in binding to pregnancy hormones. It was obsereved that the growth of L. monocytogenes in the presence of progesterone under anaerobic condition was affected by the action of the hormone and the effect was dose/time-dependent of exposure as increasing concentrations showed greater effect on the bacterial growth. Interestingly, bacterial growth was restored within 24 h of exposure to the hormone. In parallel, a Tn917-LTV3 insertion library was constructed and a number of mutants isolated that had reduced growth in the presence of β-estradiol were identified. However, reduction in growth was not microbiologically significant. Furthermore, bioinformatics analysis was performed to identify listerial genes with possible role in hormones degradation. It was observed that L. monocytogenes encodes for a protein that is possibly involved in steroid degradation; therefore, gene expression and a clear-deletion mutant were performed to test this hypothesis. This revealed no significant role of this protein in the growth restoration observed in the presence of progesterone. Also, the deleted gene was investigated of its ability to reduce NADPH in the presence of a possible substrate (progesterone, β-estradiol). This showed that this gene could possess an enzymatic activity toward pregnancy hormones. An attempt to purify this protein for further investigation was performed and protein expression in a soluble form was unsuccessful. The findings presented in this thesis represent an important view when considering the relation between pregnancy hormones and L. monocytogenes; however, further investigations of hormone-degrading proteins from L. monocytogenes are needed. This knowledge may form the basis of a therapy to protect pregnant individuals.

Hydrolytic enzymes such as chitinases and glucanases are implicated in plant defense responses against fungal pathogens. These enzymes are responsible for the breakdown of chitin and glucan, two major components of the fungal cell walls. Genes encoding these enzymes have been used to genetically engineer plants to enhance their protection against fungal pathogens. Western Australia has over 4000 endemic plant species and a largely unknown fungal biota. Given that fungi possessing chitinases and glucanases with novel activities have been isolated in other parts of the world, we propose that fungi from Western Australian soils may possess novel biochemical/enzymatic activities. The aims of this research project were to isolate chitinolytic and glucanolytic fungi from soil and to clone the genes encoding for chitinase and glucanase enzymes. To achieve these aims, fungi with activity against chitin and glucan were isolated, the activity quantified by colorimetric and inhibition assays and gene fragments with homology to known chitinase and glucanase genes were isolated and their sequences determined. Soil fungi were isolated from five locations in and around the Perth Metropolitan area of Western Australia with the use of a medium containing Rose Bengal that eliminates all actinomycetes and most bacteria and reduces the growth of fast growing mold colonies. Forty-one isolates were obtained by this method. Twenty four chitinolytic and glucanolytic fungal isolates were identified by growing them on chitin-containing media to select for those species that utilised chitin/glucan as a carbon source. These were assayed for production of exo- and endochitinolytic and glucanolytic enzymes. Enzyme activity was compared between crude and dialysed supernatants. Exochitinase activity was determined in the supernatants of 4-day old fungal cultures by the release of p-nitrophenol from p-nitrophenyl-N-acetyl-â-D glucosaminide. The supernatants were measured for endochitinase activity determined by the reduction of turbidity of suspensions of colloidal chitin. Glucanase activity was determined by release of reducing sugar (glucose) from laminarin. Supernatants from eleven of the twenty four isolates showed significant levels of enzyme activity. Eleven isolates were assayed for activity against purified cell walls of phytopathogenic fungi. Activity was determined by measuring reducing sugars in the fungal supernatants against cell wall preparations of six economically important plant pathogens. Chitinolytic activity was detected in seven isolates against cell wall preparations of Botrytis cinerea and Rhizoctonia solani, in four isolates against Fusarium solani and Sclerotinia sclerotium; in five isolates against Ascochyta faba and in six isolates against Leptosphaeria maculans. Similarly glucanolytic activity was detected in eight isolates against B. cinerea, in seven against R. solani, in two against F. solani, in three against S. sclerotium and A. faba and in one against L. maculans. The supernatants derived from the isolates were used in a bioassay to determine growth inhibition against live B. cinerea spores by measuring turbidity reduction. Growth inhibition was measured against a control (B. cinerea, grown in medium with no added supernatant). Boiled supernatant did not inhibit the growth of B. cinerea spores but there was 100% inhibition by the crude supernatant from ten of the twenty four isolates. Similarly, supernatants were used to assess growth inhibition against live mycelia cultures of F. solani and S. sclerotium. Growth inhibition of F solani ranged from 9- 59%, boiled and crude supernatants respectively whilst growth inhibition of S. sclerotium ranged from 46-75%, boiled and crude supernatants respectively. Two partial chitinase genes from the soil filamentous ungus Trichoderma asperellum,(ChiA and ChiB) and a novel glucanase gene from the filamentous fungus Aspergillus (Glu1) were cloned. ChiA, was 639 bp long, encoding 191 amino acids with identity to other chitinase genes. Two highly conserved regions, characteristic of glycosyl hydrolases from family 18, were present. ChiB, was 887 bp long and encoded a 293 amino acid sequence that was closely related to an endochitinase gene from the filamentous fungus Trichoderma asperellum. The two highly conserved regions corresponding to the substrate binding and active sites that characterise the glycosyl hydrolases from family 18, also found in ChiA, were found in this gene. Glu1 was 2844 bp long and encoded a 948 amino acid sequence that shared high identity with a â-1, 3-glucanase from the filamentous fungus Aspergillus oryzae. The sequence contained conserved regions found in glycosyl hydrolases from family 17 that encode for substrate binding, N-terminal sequences and putative asparagine linked glycosylation sites. The partial putative sequence ChiA is probably a pseudogene because it has two inframe stop codons. However, once the entire sequence of ChiB is known, both ChiB and the novel glucanase gene Glu1 could be useful contenders for engineering resistance in crop plants.

Amine transaminases are enzymes that catalyze the mild and selective formation of primary amines, which are useful building blocks for biologically active compounds and natural products. In order to make the production of these kinds of compounds more efficient from both a practical and an environmental point of view, amine transaminases were incorporated into multi-step one-pot reactions. With this kind of methodology there is no need for isolation of intermediates, and thus unnecessary work-up steps can be omitted and formation of waste is prevented. Amine transaminases were successfully combined with other enzymes for multi-step synthesis of valuable products: With ketoreductases all four diastereomers of a 1,3-amino alcohol could be obtained, and the use of a lipase allowed for the synthesis of natural products in the form of capsaicinoids. Amine transaminases were also successfully combined with metal catalysts based on palladium or copper. This methodology allowed for the amination of alcohols and the synthesis of chiral amines such as the pharmaceutical compound Rivastigmine. These examples show that the use of amine transaminases in multi-step one-pot reactions is possible, and hopefully this concept can be further developed and applied to make industrial processes more sustainable and efficient in the future.

QC 20170113

Abstract Collagen prolyl 4-hydroxylases catalyze the hydroxylation of proline residues in collagens. The vertebrate enzymes are α2β2 tetramers in which the β subunit is identical to protein disulphide isomerase (PDI). Two isoforms of the catalytic α subunit have been identified in vertebrates, forming type I [α(I)]2β2 and type II [α(II)]2β2 collagen prolyl 4-hydroxylase tetramers. This thesis reports on the cloning and characterization of a third vertebrate α subunit isoform, α(III). The recombinant human α(III) isoform associates with PDI to form an active type III collagen prolyl 4-hydroxylase tetramer, and its Km values for the cosubstrates are very similar to those of the type I and II enzymes, those for a peptide substrate and an inhibitor being found to lie between the two. The α(III) mRNA is expressed in all tissues studied but at much lower levels than the α(I) mRNA. A novel mixed tetramer PHY-1/PHY-2/(PDI-2)2 was found to be the main collagen prolyl 4-hydroxylase form produced in the nematode Caenorhabditis elegans in vivo and in vitro. However, mutant nematodes can compensate for the lack of the mixed tetramer by increasing the assembly of PHY-1/PDI-2 and PHY-2/PDI-2 dimers, these forms also being unique. The catalytic properties of the recombinant mixed tetramer were characterized, and it was shown by the analysis of mutant worms that PHY-1 and PHY-2 represent the only catalytic subunits needed for the hydroxylation of cuticular collagens. The roles of the two catalytic sites in a collagen prolyl 4-hydroxylase tetramer were studied by using the C. elegans mixed tetramer and a hybrid C. elegans PHY-1/human PDI dimer. An increase in the chain length of the peptide substrate led to an identical decrease in the Km values in both enzyme forms. It is thus clear that two catalytic sites are not required for efficient hydroxylation of long peptides, and their low Km values most probably result from more effective binding to the peptide-substrate-binding domain. Inactivation of one catalytic site in the mixed tetramer reduced the activity by more than 50%, indicating that the remaining wild-type subunit cannot function fully independently.

Cross-linked films of ε-caprolactone (CL) and 1,5-dioxepan-2-one (DXO) having various mole fractions of monomers and different cross-link densities were prepared using 2,2’-bis-(-caprolactone-4-yl) propane (BCP) as cross-linking agent and Sn(Oct)2 as catalyst. Reaction parameters were examined to optimize the film-forming conditions. Networks obtained were elastomeric materials, easy to cast and remove from the mould. Effect of CL content and cross-link density on the final properties of the polymer network was evaluated. Thermal, mechanical and surface properties of the films were controlled by monomer feed composition and cross-link density. The films have potential to be used for tissue engineering applications as shown by preliminary cell growth studies. To avoid organometallic catalysts in the synthesis of poly(1,5-dioxepan-2-one) (PDXO), the enzyme-catalyzed ring-opening polymerization (ROP) of DXO was performed with lipase-CA (derived from Candida antarctica) as a biocatalyst. A linear relationship between number-average molecular weight (Mn) and monomer conversion was observed, which suggested that the product molecular weight can be controlled by the stoichiometry of the reactants. The monomer consumption followed a first-order rate law with respect to monomer and no chain termination occurred. Effect of reaction water content, enzyme concentration and polymerization temperature on monomer conversion and polymer properties was studied. An initial activation by heating the enzyme was sufficient to start the polymerization as monomer conversion occurred at room temperature afterwards. Terminal-functionalized polyesters and tri-block polyesters were synthesized by lipase-CA catalyzed ROP of DXO and CL in the presence of an appropriate alcohol as initiator. Alcohol bearing unsaturation introduced a double bond at the chain end of the polyester, which is a useful pathway to synthesize comb polymers. Dihydroxyl compounds were used as macro-initiators to form tri-block polyesters. The enzyme-catalyzed polymerization of lactones has been shown to be a useful method to synthesize metal-free polyesters.

L'activité de produits imidazolés vis-à-vis de l'inhibition de la farnésyl transférase a conduit au développement d'un programme de recherche industriel ayant pour base d'une part un azole et d'autre part un hétérocycle permettant une reconnaissance enzymatique spécifique. Dans le cadre de ce programme, nous nous sommes intéressés aux synthèses de divers hétérocycles de structures polyazanaphtalènes. Ainsi, des 6-acylisoquinol-1(2H)-ones, des 6- et 7-formyl-1,8-naphtyridin-2-ones et des 6- et 7-formyl-1,5-naphtyridin-2-ones, pouvant être substituées en position 3 par un groupe méthyle et en position 4 par un groupe phényle ont été préparées. La méthodologie employée pour la préparation de ces molécules repose essentiellement sur l'utilisation des réactions de métallation sur des dérivés para-substitués du N-allylbenzamide et des dérivés substitues des 2- et 3-aminopyridines. Les synthèses mises en oeuvre ont permis d'accéder en peu d'étapes à des systèmes hétérobicycliques différemment substitués avec de bons résultats.

The main objective of this research was to develop a protocol in which pyridoxal phosphate (PLP) would act as a tag to identify PLP-dependent enzymes from complex mixtures or cell lysates. Following the purification of a PLP-dependent enzyme (CysM), a method was developed to reduce the PLP-lysine Schiff base to form a chemically stable bond between the PLP and the protein. The reduced protein was enzymatically digested resulting in multiple peptide fragments with one or more containing PLP (bound to the active site lysine). These fragments were analyzed by monitoring the absorbance or fluorescence using High Performance Liquid Chromatography. Immobilized Metal Ion Affinity Chromatography (IMAC) was then used to enrich the PLP-peptide(s) from the peptide mixture. The PLP-bound peptide(s) was then analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS). More specifically, sodium borohydride (NaBH4) was used to reduce the Lysine-PLP bond in CysM. This reaction was monitored by either UV-vis spectroscopy or mass spectrometry. Trypsin was used to enzymatically digest the reduced CysM before it was enriched with IMAC and analyzed with LC-MS. Since the objective of this project was to develop a method which could be applied to a cell lysate, IMAC was used as an enrichment method to separate the PLP-peptide(s) from other peptides within the mixture. The PLP-peptide(s) was then located in the peptide mixture by monitoring the absorbance at 325 nm. The LC-MS results of the full reaction before IMAC treatment versus the final column, when monitoring the mass spectrum, showed that the treatment using the IMAC column separated the PLP-peptides from all other peptides within the sample. Using IMAC to enrich specifically the PLP-peptides, followed by analysis with LC-MS, may be a useful method for studying PLP-dependent enzymes within the proteome or the "PLP-ome."

博士
輔仁大學
食品營養博士學位學程
103
Studies evidence suggests the important role of elevated one-carbon metabolic stress and genetic modifiers of metabolic one-carbon stress to human hepatocellular carcinoma（HCC）development. Disturbance of one-carbon metabolism by a methyl-deficient diet has been shown to promote tumour progression in animal liver. Literatures association folate status with risks of HCC is very few. HCC patients frequently suffered from marginal folate deficiency, which has been demonstrated as the risk factor of HCC progression, but the folate status of HCC patients in relation to tumour progression and survival have not been assessed. Genetic polymorphism of one-carbon metabolic enzyme and HCC development Currently evidence is limited and results inconsistent. Whether genetic polymorphism affect the folate status of HCC patients or the interaction with folate which modulate the risk of HCC survival were not assessed. Whether this polymorphism has impact on oxidative DNA damage associated with HCC development remains unexplored. The aims of this study was to investigate (1)Genetic polymorphisms of one-carbon enzymes interactively modify metabolic folate stress and risks of HCC development (2)The relationship between folate status and tumour progression in HCC patients, and (3)Associations of folate status, the MTHFR C677T genotypes, and oxidative DNA damage with risks of advanced HCC malignancy and survival in HCC patients. A hospital-based case-control study, detected serum folate and Hcy levels for metabolic one-carbon stress of subjects(199 HCC patients and 199 healthy subjects), genetic polymorphisms of 3 major one-carbon enzymes at 5 loci were characterized. Ninety eligible patients with HCC were recruited through the Department of Internal Medicine of Hospital participated in a cross-sectional study. Analysis serum folate status of HCC patients, and detect the index of liver damage, clinical complications and tumor progression. Finally, the collection and analysis of red blood cell folate status, MTHFR C677T gene polymorphism, and the pathologic data of oxidative p53 gene damage of HCC patients, conduct the study of progression and survival. T variant allele at the MTHFR 677 loci, in relation to the other genotyped variant alleles (MTHFR 1298C, MS 2756G, TSER 2R, and TS3’UTR 1494 +6bp insertion), was associated with a significant 40% reduction in the risk of HCC. After adjusting for age, gender and Hcy levels, subjects with normal in relative to deficient serum folate had 86% reduction in HCC risks, among those with normal folate level, the compound MTHFR CC-TSER 2R variant allele was associated with 2-fold increased HCC risks (OR=0.14 v.s. 0.30, 95%CI=0.10-0.80), whereas the compound MTHFR T-TSER 2R variant alleles were with 2-fold reduction of HCC risks (OR=0.14 vs. 0.07, 95%CI=0.02-0.20). Serum folate showed inverse correlations with tumour progression：tumour size(＞5cm), tumour multiplicity(numbers＞1) and metastasis. Patients with low blood folate status had increased risks for advanced tumour progression in large tumours (OR=7.10, 95%CI=2.27-21.90), tumour multiplicity (OR=3.20, 95%CI=1.07-3.51) and metastasis (OR=4.5, 95%CI=1.11-18.4) relative to those with normal folate status. By adjustment for oxidative risk factors of HCC, the compound CT and TT genotypes in relative to the CC wild-type were associated with 83% reduced lymphocytic p53 oxidative lesions of HCC patients with RBC folate lower than 688 ng/mL. Such genetic protective effects by the CT/TT genotypes were 2-fold enhanced among those with high RBC folate. For those with non-folate-deficient status, the compound CT and TT vs. CC genotypes were associated with 80% reduced risks of advanced HCC stages. Such protection was negated by adjustment of lymphocytic p53 oxidative lesions (OR=0.60, 95%CI=0.31-1.41). Multivariate Cox proportional hazards analysis showed that the CT/TT genotypes vs. CC wild-type were better survival outcome of HCC patients. The data suggest that the MTHFR 677T and TSER 2R variant alleles interact to appropriately reduce risks of HCC in a normal folate status. Low blood folate status could be a risk factor for tumour progression. Reduced MTHFR activities by the MTHFR T allele may interact with RBC folate as the risk modifiers of lymphocytic p53 oxidative lesions, lower risks of late-stage HCC and a favorable survival of HCC patients. In this study, providing an important care indicator of nutrition intervention and genotype for prevention and prognosis of HCC.

碩士
輔仁大學
營養科學系碩士班
102
Hedgehog (Hh) pathway increased tumor cells invasion and migration ability via regulates Epithelial-mesenchymal transition (EMT) and Hh signaling pathway is aberrantly activated in cancer. Folate status and genetic polymorphisms of one carbon enzyme: methylenetetrahydrofolate reductase (MTHFR) C677T and thymidylate synthase 5‘-untranslated region (TS5` UTR) polymorphism are associated with DNA promoter methylation. The aim of this study was to investigate the relationship among tumor folate status, genetic polymorphisms of one carbon enzyme, sonic hedgehog (Shh) signaling pathway molecules, invasion-associated biomarkers and colorectal cancer (CRC) progression. We collected 99 cancer tissues and pair-non cancer tissues of CRC in Tainan Chi Mei Medical Center. We used L. casei to analyze folate status, determining MTHFR C677T and TS5`UTR genotype by Q-PCR and the promoter methylation status of Shh by MS-PCR in tissues. In addition, using Q-RT-PCR analyze mRNA expression of Shh, Hedgehog-interacting protein (HIPP), glioma-associated oncogen homolog 1 (Gli1), β-catenin and E-cadherin. The folate status was significantly higher in T3 stage than T4 stage of the CRC tissue, but no relationship with genetic polymorphisms of MTHFR C677T and TS5`UTR. The genetic polymorphisms of MTHFR C677T and TS5`UTR were the same between CRC and non CRC tissue. Compared with MTHFR 677CC genotype, CT/TT genotype reduced the Shh overexpression in CRC tissue, but HHIP, E-cadherin and β-catenin mRNA expressions were no different in the CRC tissue and non CRC tissue.The mRNA expression of HHIP, E-cadherin and β-catenin were significantly lower in 3R/3R genotype than in 2R/2R+2R/3R genotype in the CRC tissue. The mRNA expression of Shh was overexpression and the mRNA expression of HHIP, E-cadherin and β-catenin were significantly decreased in early stage of metastasis in CRC tissue. In folate status and mRNA △△Ct expression of Shh, we found Shh expression and folate status were increased. Methylation of Shh promoter showed unrelated to folate status, genetic polymorphisms of one carbon enzyme and the mRNA expression of Shh. In conclusion, genetic polymorphisms of one carbon enzyme showed unrelated to folate status and the folate status was positive correlation with Shh mRNA expression. Shh pathway andEMT regulated early stage of CRC metastasis. The folate status and genetic polymorphisms of one carbon enzyme affected Shh pathway may not through hypermethylation of Shh promoter.

碩士
國立臺灣海洋大學
食品科學系
93
This study examined the restriction enzyme map and open reading frames（ORF) position of one expression sequence from Antrodia camphorata .The results were presented in graphic forms to provide basic data for the improvement of food genetic research . The expression sequence from Antrodia camphorata was used as the input information to the Jemboss and NEBcutter softwares, the results from both databases were identical. They helped to predict the restriction enzyme diagram slice and the position of ORF . The procedure Remap marked the position of enzyme incise on the sequence of DNA in the software of Jemboss. If one would like to predict the ORF between the start codon and the stop codon, one can make use of the getorf and plotorf functions.They could complement each other and help locate the longest ORF within frames , and predict translated protein. Basic Local Alignment Search Tool（ BLAST) was used to search the protein sequence database and found the sequence of similar block.

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
O cancro é uma das maiores causas de morte a nível mundial, prevendo-se que o número de novos casos supere os 24 milhões nas próximas duas décadas. O processo pelo qual as células adquirem propriedades cancerígenas é atualmente considerado como sendo dependente da aquisição de mutações, cujas consequências se traduzem em danos no genoma. O tipo de mutação mais comum é o que envolve a substituição de um nucleótido que, quando tem apresenta uma frequência de pelo menos 1% na população, é denominado polimorfismo de nucleótido único (SNP, do inglês single nucleotide polymorphism). Dependendo da região em que ocorre e da alteração funcional ao qual pode estar associado, o SNP pode conferir tanto um efeito protector como um factor de risco aumentado para o desenvolvimento de doença. A síndrome mielodisplásica (SMD) é um conjunto de doenças relacionadas com o sistema hematopoiético, caracterizada por uma hematopoiese ineficaz, displasias, e ainda uma susceptibilidade acrescida para o desenvolvimento de leucemia mieloide aguda (LMA). A transcrição de genes supressores tumorais, como é o caso do P15 e do P16, pode ser inactivada pela metilação aberrante da região promotora, nomeadamente das ilhas CpG, o que tem adquirido especial interesse em neoplasias hematológicas, em particular na SMD. Nutrientes provenientes da dieta, como é o caso do folato e da vitamina B12 podem influenciar a produção de grupos metil e, consequentemente, o processo de metilação. Para além disso, a SMD parece estar relacionada com SNPs em enzimas que regulam o ciclo do carbono, o mecanismo responsável tanto pela metilação do DNA como pela síntese de nucleótidos. Uma vez que a hipometilação global e a hipermetilação localizada do DNA são características de tumores humanos, incluindo a SMD, e o suprimento de folato está associado a estas características, foi colocada a hipótese de que polimorfismos em genes que intervêm neste ciclo, nomeadamente os genes MTR, SCL19A1, MTRR e CBS, podem afectar o estado de metilação do DNA e, assim, influenciar não só o desenvolvimento de SMD, como a progressão da doença para LMA. Neste sentido, o principal objectivo deste trabalho foi a avaliação da relação entre os polimorfismos genéticos associados aos genes MTR (rs1805087 e rs2229276), SLC19A1 (rs1051266), MTRR (rs162036) e CBS (844ins68), que regulam o ciclo do carbono, e o risco associado ao desenvolvimento de SMD. Analisamos ainda a influência das variantes x polimórficas nos níveis de folato e vitamina B12, bem como o seu papel no grau de metilação das sequências LINE-1 e dos genes P15 e P16. Para isto, foram genotipadas as amostras de 77 doentes e 80 controlos não neoplásicos, para cada um dos genes em estudo. Os genótipos associados aos genes MTR e MTRR foram obtidos através da técnica ARMS-PCR, tendo sido utilizada a técnica de RFLPPCR para a análise do gene SLC19A1 e o PCR convencional para o gene CBS. O grau de metilação da sequência LINE-1 foi avaliado por ensaios COBRA e a frequência de metilação dos genes P15 e P16 foi obtida pela realização da técnica MS-PCR. Posteriormente, foi avaliada a força de associação entre as variantes polimórficas e o risco de desenvolvimento de SMD, através do cálculo do risco relativo por análise de regressão logística. Avaliou-se ainda a relação entre os polimorfismos genéticos, a progressão da doença para LMA e a sobrevivência global dos doentes, com recurso a regressão logística e análise de Kaplan-Meier. De seguida, analisou-se a possibilidade de o risco para o desenvolvimento de SMD ser influenciado pelos perfis génicos ou haplotipos. Por fim, o teste exacto de Fisher foi usado ara avaliar relação entre as variantes polimórficas e o grau de metilação dos genes P15 e P16 e a sequência LINE-1. Da avaliação dos polimorfismos no risco de desenvolvimento da doença, observámos que o genótipo GG do variante rs2229276 associada ao gene MTR pode constituir um factor protector para o desenvolvimento de SMD (OR=0.29) e que a presença do alelo G parece estar relacionada com uma menor predisposição para a doença (OR=0.33). No que diz respeito à associação entre as variantes polimórficas e a presença ou ausência de alterações citogenéticas, o genótipo heterozigótico (WT-Ins) do gene CBS (844ins68) mostrou-se relevante em indivíduos com cariótipo normal para as alterações mais comuns na SMD (OR=0.22). Pela análise das curvas de Kaplan-Meier, observou-se que os indivíduos portadores do genótipo AG para o polimorfismo rs162036 do gene MTRR apresentam um aumento da progressão da doença para LMA, bem como uma tendência, embora sem significado estatístico, para uma diminuição da sobrevivência dos doentes. Para além disso, a avaliação dos níveis de vitamina B12 parece estar relacionada com o genótipo GG do gene MTR (rs2229276). Em relação ao polimorfismo rs1051266 relativo ao transportador SLC19A1, este parece influenciar o estado de metilação do gene P16 nos doentes com SMD (OR=3.80). xi Os resultados deste estudo permitem-nos concluir que a presença de variantes polimórficas desempenha um papel importante na SMD e realçam a importância do estudo de genes envolvidos no ciclo do carbono na patogénese desta doença
Cancer is a leading cause of death worldwide, predicted to rise more than 24 million new cases globally in the next two decades. The process by which normal cells become progressively transformed into malignant derivatives is now known to require the acquisition of mutations, which arise as a consequence of damage to the genome. The most common mutational change is associated with the substitution of a single nucleotide, called single nucleotide polymorphism (SNP) when its frequency on the population is, at least, of 1%. Depending on the region where it occurs and the functional changes that it may cause, a SNP may confer a protective effect or modulate an increased susceptibility to disease development. Myelodysplastic Syndrome (MDS) is a family of clonal hematopoietic stem cells disorders that involves a multistep pathogenesis characterized by dysplasia, innefective hematopoiesis and susceptibility to transformation into Acute Myeloid Leukemia (AML). Transcriptional inactivation of tumor suppressor genes, such as P15 and P16, by promoter CpG island hypermethylation has been subject of interest in haematological malignancies, namely in MDS. Dietary factors such as folate and vitamin B12 could influence the supply of methyl groups and, therefore the biochemical pathways of methylation processes. Moreover, MDS is thought to be associated with SNPs in enzymes from one-carbon metabolism, the pathway that is responsible for DNA methylation and nucleotide synthesis. Since global hypermethylation and targeted hypomethylation are considered defining characteristics of human tumours, including MDS, and folate depletion in certain human cells lines can result in global hypomethylation and also targeted hypermethylation, we hypothesized that genetic variability in 1-carbon metabolism, namely in MTR, SLC19A1, MTRR and CBS genes, can affect the methylation status and contribute, not only to MDS development, but also to progression to AML. In this context, our major goal was to evaluate the interplay between genetic polymorphisms in enzymes involved in the folate mechanism, namely MTR rs1805087 and rs2229276, SLC19A1 rs1051266, MTRR rs162036 and CBS 844ins68, and the risk of developing MDS. Furthermore, we assessed the influence of these genetic polymorphisms in folate and vitamin B12 levels in MDS patients, as well as the role of gene variants in the degree of P15, P16 and LINE-1 methylation. To this end, we genotyped 77 MDS patients and 80 healthy xiv controls, assessing ARMS-PCR for MTR and MTRR genes, RFLP-PCR for determining the variants for SLC19A1, and conventional PCR for detecting CBS variants. P15 and P16 methylation status were obtained by MS-PCR, while LINE-1 level of methylation was performed by COBRA assay. Posteriorly, we evaluated the strength of association between polymorphic variants and the development of MDS by calculating the associated odds ratio, through logistic regression analysis. We also evaluated the relation between genetic profiles, evolution to AML and overall survival in MDS patients, by logistic regression and Kaplan-Meier curves analysis. Next, we investigated the influence of genetic profiles and haplotypes on the risk of MDS development. The influence of genetic polymorphisms on P15, P16 and LINE-1 methylation was performed using Fisher’s exact test. Concerning odds ratio for risk disease, our main results revealed that AG genotype from MTR rs2229276 is a protector factor for the development of MDS, presenting a 0.29- fold decreased associated risk. Adittionally, the G allele also proved to have a protector effect on MDS, with a 0.33-fold decreased risk on disease development. In relation to cytogenetic alterations, the CBS 844ins68 WT-Ins genotype was found to be significantly associated with the absence of the most common cytogenetic abnormalities in MDS (OR=0.22). Subsequently, the analysis of Kaplan-Meier curves showed that heterozygous (AG) individuals for MTRR rs162036 not only presented a significant increase to AML evolution (p=0.049), but also show a tendency to be associated with a worse overall survival. In relation to folate and vitamin B12 levels, only vitamin B12 proved to be influenced by polymorphisms, in this case, by GG genotype from MTR rs2229276 variant (p=0.039). The odds ratio relating P15 and P16 methylation and genetic variants showed that GG genotype from SLC19A1 rs1051266 negatively influences P16 methylation in MDS patients (OR=3.80). The results of this study allow us to accomplish that polymorphisms play important roles in MDS and highlight the importance of the study of one-carbon metabolism in the pathogenesis of the disease

碩士
國立屏東科技大學
農企業管理系所
101
The potential customer for One PowerBio Technology Company.On the magin products ofby theproduction of naturalfruits and vegetablesenzyme products. This study was to investigate the One Power Bio Technology Company. in product marketing, sales channels, and to explore the company's practices and promotional products to enhance product sales effectiveness. There are two margin part types of enzyme products with enhanced and maintenance, maintenance-type herbal ingredients belonging to the lowest, enhanced herbal ingredients belonging to a higher,mainly due to the effectivepotential consumersare willing to buyproductsto enhancethe attractiveness ofthe relationship. Aims to understandthe enterpriseforthe production ofdifferent products"ProductAttributes" and "product involvement" for different"demographics" and "lifestyle" of thedifference betweenthe objectof this studyis onlylockedthemedical cosmeticclinic andthe peopleofpossiblesources ofpotential consumersasa resultof itsmain object of study, such asPingtungand Kaohsiungareasforpotential consumersclinicsdistributed300questionnaires werecollectedthroughquestionnaires, the use of statistical analysis（ttest, chi-square test andsingle-factoranalysis of variance）verifyinfluence consumerattitudesand behavioralfactors

碩士
國立中興大學
食品暨應用生物科技學系所
97
Trehalose is a novel multi-functional disaccharide. It serves a variety of functions in organisms and protects organisms to survive harsh environments. In addition, it can be a sweetener, and be a useful compound to preserve biomaterials in the food, cosmetic, and pharmaceutical industries by its ability of stabilizing macromolecules. The most economical pathway for trehalose production is a reversible conversion of maltose into trehalose by trehalose synthase (TSase). In this study, we developed a new bifunctional gene combined with a recombination gene from a thermophilic Clostridium thermosulfurogenes β-amylase and a thermophilic TSase (PTTS#N503P) which was synthesized using overlap extension PCR and expressed in Escherichia coli system to express the active fusion enzymes. Our aims were focused on the directly converted of starch into trehalose in one-step by the recombinant fusion enzyme (PTTS#N503P–CTBA). Since the reaction temperature was increased to 60 °C, the novel PTTS#N503P–CTBA fusion enzymes represented 2-fold higher catalytic efficiency than that of PTTS#N503P enzymes. Also, the optimal reaction temperature of recombinant fusion enzyme was changed. The combination of TSase (PTTS#N503P) and thermophilic β-amylase resulted in a broad pH range of 5.0-7.0 with high enzyme activity. For enzyme thermodynamics result, the PTTS#N503P–CTBA represented slightly higher half-life time than that obtained by PTTS#N503P at 60 °C. For the effect of various metal ions, higher concentration (10 mM) of Cesiurn and Magnesiurn could help to stabilize the enzyme with higher catalytic activity. Overally, our novel fusion enzyme (PTTS#N503P–CTBA) is more thermostable and acid-resistant than the wild type. Since it can be used as a functional biocatalyst to directly convert maltose into high value-added of trehalose in one-step accompany with lower cost, we believe it will be another highly potential and useful choice for further industrial applications.