Methionine is essential for proper functioning of cellular processes such as protein synthesis, transmethylation and polyamine synthesis. Efficient recycling of methionine is important because of its limited bioavailability and metabolically expensive de novo synthesis. Further, cellular accretion of the nucleoside metabolites of the methionine salvage pathway compromises polyamine biosynthesis, transmethylation reactions and quorum sensing pathways, all critical reactions in cellular metabolism. 5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a key component of the methionine salvage pathway of plants and many bacterial species, including Escherichia coli, Enterococcus faecalis, Salmonella typhimerium, Haemophilus influenza and Streptococcus pneumoniae. In bacteria, this enzyme displays dual-substrate specificity for two methionine metabolites, 5’-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), and catalyzes the irreversible cleavage of the glycosidic bond to form adenine and the corresponding thioribose products, methylthioribose (MTR) and S-ribosylhomocysteine (SRH), respectively. In plants, MTAN is highly specific towards MTA and shows 0-16 % activity towards SAH. Plants rely on SAH hydrolase to metabolize SAH. Mammals do not have the nucleosidase enzyme and MTA is metabolized by MTA phosphorylase (MTAP). Like plants, mammals utilize SAH hydrolase to degrade SAH. Because MTAN is required for viability in multiple bacterial species and is not found in humans, it has been identified as a target for novel antibiotic development. This thesis describes the structural and functional characterization of bacterial and plant MTANs, with the aim of better understanding the molecular determinants of substrate specificity and the catalytic mechanism of this enzyme. The catalytic activities of representative plant MTANs from Arabidopsis thaliana, AtMTAN1 and AtMTAN2, were kinetically characterized. While AtMTAN2 shows 14 % activity towards SAH relative to MTA, AtMTAN1 is completely inactive towards SAH. As such, AtMTAN1 was selected for further examination and comparison with the bacterial MTAN from Escherichia coli (EcMTAN). The structures, dynamics and thermodynamic properties of these enzymes were analyzed by X-ray crystallography, hydrogen-exchange coupled mass spectrometry and isothermal titration calorimetry, respectively. Our studies reveal that structural differences alone do not sufficiently explain the divergence in substrate specificity, and that conformational flexibility also plays an important role in substrate selection in MTANs. MTANs from the pathogenic bacterial species, Staphylococcus aureus and Streptococcus pneumoniae, were examined kinetically and structurally. Comparison of the structures and catalytic activities of these enzymes with EcMTAN shows that the discrepancies in kinetic activities arefully explained by structural differences, as the overall structure and active sites of these bacterial MTANs are nearly identical. These experiments are in agreement with our proposal that dynamics play a significant role in catalytic activity of MTAN, and suggest that both structure and dynamics must be considered in future antibiotic design. To further our understanding on the catalytic mechanism of MTAN, the putative catalytic residues of AtMTAN1 were identified by structural comparison to EcMTAN and mutated by site-directed mutagenesis. The AtMTAN1 mutants were analyzed by circular dichroism and kinetic studies. Our results suggest that the catalytic mechanism is largely conserved between bacterial and plant MTANs, although the role of the putative catalytic acid remains to be confirmed.

<i>Claviceps purpurea</i>, a fungal pathogen, of ergot diseases in agriculturally important cereal crops, produces high levels of glycerides containing ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. A fatty acid hydroxylase (CpFAH) involved in the biosynthesis of ricinoleic acid was recently identified from <i>C. purpurea</i>. This research describes the biochemical characterization of a type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of this fatty acid into triglycerides from <i>C. purpurea</i>. Expression of CpDGAT2 in a quadruple mutant <i>Saccharomyses cerevisiae</i> H1246, in which all four triacylglycerol (TG) biosynthesis genes (DGA1, LOR1, ACAT1 and ACAT2) were disrupted, restored the ability of the mutant to synthesize TGs <i>in vivo</i>. <i>In vitro</i> enzymatic assays of microsomal preparations of the transformants indicated that CpDGAT2 preferentially use ricinoleic acid over linoleic acid, oleic acid and linolenic acids as acyl donor, and 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as acyl acceptor. CpDGAT2 did not show any activities for the formation of wax esters and estolides when 1-hexadecanol and triricinolein were used as acyl acceptors. Co-expression of CpFAH and CpDGAT2 in yeast resulted in increased accumulation of ricinoleic acids compared to expression of CpFAH along with the yeast native DGAT2 (ScDGA1) or expression of CpFAH alone. Northern blot analysis indicated that CpFAH is solely expressed in sclerotium cells and no transcripts of this gene were detected in mycelium and conidium cells. CpDGAT2 is more widely expressed in cell types examined except for conidiospores where the expression is low. The highest expression of CpDGAT2 was detected in 20 day-old sclerotium cells where the highest levels of ricinoleate glycerides are accumulated. Collectively, these data indicate CpDGAT2 and CpFAH are two key enzymes coordinating the biosynthesis and bioassembly of ricinoleic acid in <i>C. purpurea</i>.

Methionine is essential for proper functioning of cellular processes such as protein synthesis, transmethylation and polyamine synthesis. Efficient recycling of methionine is important because of its limited bioavailability and metabolically expensive de novo synthesis. Further, cellular accretion of the nucleoside metabolites of the methionine salvage pathway compromises polyamine biosynthesis, transmethylation reactions and quorum sensing pathways, all critical reactions in cellular metabolism. 5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a key component of the methionine salvage pathway of plants and many bacterial species, including Escherichia coli, Enterococcus faecalis, Salmonella typhimerium, Haemophilus influenza and Streptococcus pneumoniae. In bacteria, this enzyme displays dual-substrate specificity for two methionine metabolites, 5’-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), and catalyzes the irreversible cleavage of the glycosidic bond to form adenine and the corresponding thioribose products, methylthioribose (MTR) and S-ribosylhomocysteine (SRH), respectively. In plants, MTAN is highly specific towards MTA and shows 0-16 % activity towards SAH. Plants rely on SAH hydrolase to metabolize SAH. Mammals do not have the nucleosidase enzyme and MTA is metabolized by MTA phosphorylase (MTAP). Like plants, mammals utilize SAH hydrolase to degrade SAH. Because MTAN is required for viability in multiple bacterial species and is not found in humans, it has been identified as a target for novel antibiotic development. This thesis describes the structural and functional characterization of bacterial and plant MTANs, with the aim of better understanding the molecular determinants of substrate specificity and the catalytic mechanism of this enzyme. The catalytic activities of representative plant MTANs from Arabidopsis thaliana, AtMTAN1 and AtMTAN2, were kinetically characterized. While AtMTAN2 shows 14 % activity towards SAH relative to MTA, AtMTAN1 is completely inactive towards SAH. As such, AtMTAN1 was selected for further examination and comparison with the bacterial MTAN from Escherichia coli (EcMTAN). The structures, dynamics and thermodynamic properties of these enzymes were analyzed by X-ray crystallography, hydrogen-exchange coupled mass spectrometry and isothermal titration calorimetry, respectively. Our studies reveal that structural differences alone do not sufficiently explain the divergence in substrate specificity, and that conformational flexibility also plays an important role in substrate selection in MTANs. MTANs from the pathogenic bacterial species, Staphylococcus aureus and Streptococcus pneumoniae, were examined kinetically and structurally. Comparison of the structures and catalytic activities of these enzymes with EcMTAN shows that the discrepancies in kinetic activities arefully explained by structural differences, as the overall structure and active sites of these bacterial MTANs are nearly identical. These experiments are in agreement with our proposal that dynamics play a significant role in catalytic activity of MTAN, and suggest that both structure and dynamics must be considered in future antibiotic design. To further our understanding on the catalytic mechanism of MTAN, the putative catalytic residues of AtMTAN1 were identified by structural comparison to EcMTAN and mutated by site-directed mutagenesis. The AtMTAN1 mutants were analyzed by circular dichroism and kinetic studies. Our results suggest that the catalytic mechanism is largely conserved between bacterial and plant MTANs, although the role of the putative catalytic acid remains to be confirmed.

Addressing issues with sensitivity and specificity in TBI assessment this study compared the performance on neuropsychological tests and results from qEEG assessment between a heterogeneous TBI (N=20) group and a matched normal control group (N=20). The TBI group was performed worse on all measures. Significant differences in performance were found in the domains of information processing speed and executive function. Effect sizes of these differences were large. This was also true for the amplitude of the qEEG parameter P3NoGo along with P3Go latency and theta power in the temporal and frontal lobes. Binary logistic regression revealed higher sensitivity and specificity when combining neuropsychological tests and qEEG parameters, suggesting qEEG parameters in  combination with neuropsychological tests to be good assets in TBI assessment.

<i>Claviceps purpurea</i>, a fungal pathogen, of ergot diseases in agriculturally important cereal crops, produces high levels of glycerides containing ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. A fatty acid hydroxylase (CpFAH) involved in the biosynthesis of ricinoleic acid was recently identified from <i>C. purpurea</i>. This research describes the biochemical characterization of a type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of this fatty acid into triglycerides from <i>C. purpurea</i>. Expression of CpDGAT2 in a quadruple mutant <i>Saccharomyses cerevisiae</i> H1246, in which all four triacylglycerol (TG) biosynthesis genes (DGA1, LOR1, ACAT1 and ACAT2) were disrupted, restored the ability of the mutant to synthesize TGs <i>in vivo</i>. <i>In vitro</i> enzymatic assays of microsomal preparations of the transformants indicated that CpDGAT2 preferentially use ricinoleic acid over linoleic acid, oleic acid and linolenic acids as acyl donor, and 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as acyl acceptor. CpDGAT2 did not show any activities for the formation of wax esters and estolides when 1-hexadecanol and triricinolein were used as acyl acceptors. Co-expression of CpFAH and CpDGAT2 in yeast resulted in increased accumulation of ricinoleic acids compared to expression of CpFAH along with the yeast native DGAT2 (ScDGA1) or expression of CpFAH alone. Northern blot analysis indicated that CpFAH is solely expressed in sclerotium cells and no transcripts of this gene were detected in mycelium and conidium cells. CpDGAT2 is more widely expressed in cell types examined except for conidiospores where the expression is low. The highest expression of CpDGAT2 was detected in 20 day-old sclerotium cells where the highest levels of ricinoleate glycerides are accumulated. Collectively, these data indicate CpDGAT2 and CpFAH are two key enzymes coordinating the biosynthesis and bioassembly of ricinoleic acid in <i>C. purpurea</i>.

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Taiwans whale watching industry has been prosper rapidly since 1997 while not any law administering it .Due to free market structure the resource of whale watching would be excessly exploited and the whale watching industry will not exist any more. There are three research topics on the institutional design of the industry in my article. Namely , the administering department and its related rules for whale watching industry to be established, the cost analysis for governance structure of institution, and the study of franchise for Taiwan¡¦s current whale watching industry. According to Williamson¡]1991¡^,I present three kinds of organization style¡]i.e. market ,hierarchy organization and their mixed form¡^ and their key differences to figure out the most efficient one. And finally I come to a conclusion that the franchise is the most efficient of the three kinds of organizational style and can be a reference structure for making law on Taiwan¡¦s whale watching industry.

Recently, Taiwan GDP has been increased continuously, outdoor sports are getting popular and we have 2 days off per a week. More and more people are encouraged to join the healthy outdoor sports. Golf is one of the quality, healthful outdoor sports. Also there are many new golf courses, driving ranges, golf shops. It helps golf to be very popular. At present, the top 4 golf manufacturing companies are listing theirs stocks in Taiwan markets such as Dynamic (OTC), Ota (OTC), Fusen (TSEC), Advance (preparing). Each company has its own strength and weakness. Also each company has a complete manufacturing strategy both in Taiwan and China. How does Dynamic use Benchmarking theory to upgrade their company strategy. The writer is interested in the research on the topic. In order to combine the theory and active operation, the writer designs the researching range as below: 1. The decision for the topic of Benchmarking. 2. Choose good company for Benchmarking 3. The trend of the external environment. 4. Use the resource of industrial specificity to upgrade the competitive strength.

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Programmable site-specific endonucleases are useful tools for genome editing and may lead to novel therapeutics to treat genetic diseases. TALENs can be designed to cleave chosen DNA sequences. To better understand TALEN specificity and engineer TALENs with improved specificity, we profiled 30 unique TALENs with varying target sites, array length, and domain sequences for their ability to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results collectively suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches, yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. We engineered a TALEN variant, Q3, that exhibits equal on-target cleavage activity but 10-fold lower average off-target activity in human cells. Our results demonstrate that identifying and mutating residues that contribute to non-specific DNA-binding can yield genome engineering agents with improved DNA specificities.