Helicobacter pylori infects and persistently colonizes the stomach, which results in gastritis and in some individuals peptic ulcer disease or gastric cancer. Adherence of H. pylori to the epithelium is an important factor for development of disease. Attachment is mediated by the adhesins BabA and SabA that binds the ABO/Leb blood group antigens and sialylated glycoconjugates respectively.  High-affinity attachment could be anticipated to be of disadvantage for H. pylori because epithelial cells have a fast turnover rate and the dislocated and shed epithelial cells would carry attached bacteria to the acidic gastric juice in the lumen. However, here we describe that H. pylori manage to adapt to this innate clearance mechanism by unique acid regulatory binding properties of its adhesins. We propose that pH regulated binding properties enable bacteria to detachment from host cells for chemotactic guided motility and successful return to the more neutral epithelium for a fresh restart of the infectious cycle. By comparison of BabA from different stomach loci we identified amino acid key position for acid regulated binding activity. Previous studies found lower prevalence of Leb-binding among H. pylori isolates from southern Europe compared to Sweden. Here we tested if the reduced prevalence of Leb-binding could be explained by a novel binding mode; in among Spanish strains, we identified S812 that demonstrates preference for multivalent binding to ABO antigens in glycolipids; we found that 812 BabA had drifted in its preferred binding epitope away from the consensus a1,2fucosylation and towards the blood group A and B derivatives. Such epitope drift might in particular optimize binding to ABO antigens in densely packed lipid rafts. In parallel, we studied the influence of BabA for disease progression by an inventory of gastric biopsies. BabA correlated both with the oncoprotein CagA, the VacAs1 toxin and, in addition, to severe disease progression. We further correlate BabA expression with positive secretor phenotype and stronger adhesion of H. pylori in vitro. For functional adherence studies in vitro, we constructed a recombinant Leb-expressing cell lineage that supports BabA mediated H. pylori attachment.

<p>Koi and goldfish have been released into rivers in South Africa since the 1800&rsquo / s for food and sport fish and have since spread extensively. These fish are present in most of the river systems in South Africa and pose an additional threat the indigenous cyprinids in the Western Cape. Monogenean parasites of the genus Gyrodactylus are of particular concern, as their unique biology renders them a possible threat. Gyrodactylus kherulensis and G. kobayashii were identified from koi and goldfish respectively imported from Asia, Europe and locally bred fish. Morphometrics and the use of statistical classifiers, which includes univariate (ANOVA and Kruskal-Wallis), bivariate (Pearson&rsquo / s correlation) and multivariate (Principal Component Analysis) placed the two species within their respective groups. There was some intraspecific variation among the different populations collected from the various locations, especially in the hamulus and ventral bar features, but the marginal hooklets, however, remained static for both helminth species.</p>

Protein kinases are enzymes that catalyse the phosphoryl transfer from the g-phosphate of ATP to acceptor amino acids in proteins. The specificity of selected model protein kinases was studied at three different levels using a) novel bi-substrate-analogue inhibitors, b) synthetic peptide substrates and c) mutated protein substrate analogues. A new class of protein kinase bi-substrate-analogue inhibitors was designed on the basis of adenosine-5’-carboxylic acid derivatives, where a short arginine containing peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. These compounds showed high inhibitory potential against two basophilic protein kinases, the protein kinase A (PKA) and protein kinase C (PKC), with IC50 values in the nanomolar range, but no inhibitory activity towards the acidophilic kinases CK1 and CK2. The inhibitors were efficiently applied for affinity purification of PKA using MgATP as well as L-arginine as eluting agents. Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings and its substrate specificity was studied using a set of synthetic peptides. These were derived from the phosphorylatable sequence RVLSRLHS(15)VRER of maize sucrose synthase 2 (SuSy2), and a consensus sequence motif A/LXRXXSXRZR (where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine) was defined from a study using arrays of systematically varied peptides attached to cellulose membrane (SPOTsTM membranes). The SuSy2 derived peptides were also found to be efficient substrates for mammalian PKC, but showed low reactivity in the case of PKA. On the basis of this peptide motif, a positionally oriented peptide library approach based on ESI-MS detection of phosphopeptides in initial velocity conditions was designed for quantitative kinetic characterization of protein kinase specificity profiles. On the basis of the obtained data an optimal peptide substrate for PKC, FRRRRSFRRR, was designed. The specificity of protein kinase A was studied using site-directed mutagenesis in the phosphorylation site of L-type pyruvate kinase (L-PK), and comparison of the obtained data with the data from previous studies on structurally altered peptide substrates revealed that amino acid alterations in short peptide substrates cause stronger effects on the phosphorylation rate than the corresponding alterations in the protein substrate L-PK.

It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease. The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (&lt;6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma.

Mast cells (MC) are potent inflammatory cells that are known primarily for their prominent role in IgE mediated allergies. However, they also provide beneficial functions to the host, e.g. in bacterial and parasitic defence. MCs react rapidly upon stimulation by releasing potent granule-stored mediators, and serine proteases of the chymase or tryptase families are such major granule constituents. As a first step towards a better understanding of the biological function of these proteases, we have determined the extended cleavage specificities of four mammalian mast cell chymases, by utilizing a substrate phage display approach. The specificities of these enzymes have then been used to compare their functional characteristics. The major mucosal MC chymase in mice, mMCP-1, was found to possess a strict preference in four amino acid positions of the peptide substrate. Using this sequence to search the mouse proteome for potential in vivo substrates led to the identification of several very interesting potential novel substrates. Some of them may explain the increased epithelial permeability provided by this enzyme. Human MCs, express only one single α-chymase, and the rodent α-chymases have secondarily gained elastase-like primary cleavage specificity. However, rodents express additional chymases, the β-chymases, and rodent β-chymases may have adopted the function of the α-chymases. The cleavage specificities of the human chymase and two rodent β-chymases were therefore determined (rat rMCP-1 and mouse mMCP-4). N-terminal of the cleaved bond the three chymases showed similar preferences, but C-terminal the human chymase and mMCP-4 shared a high preference for acidic amino acids in the P2´ position and therefore seem to be functional homologues. The molecular interactions mediating the preference for acidic amino acids in position P2´ were further investigated. By site-directed mutagenesis of the human chymase, amino acids Arg143 and Lys192 were concluded to synergistically mediate this preference. Our data show that chymases, of different MC subpopulations, display quite different extended cleavage specificities. However mouse do possess a MC chymase with almost identical cleavage specificity as the human MC chymase indicating a strong evolutionary pressure to maintain this enzyme specificity.

UDP-galactopyranose mutase (EC 5.4.99.9; UGM), the product of the glf gene, is an enzyme that catalyzes the conversion of uridine diphosphate galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM activity is found in bacteria, parasites and fungi, however is absent in higher eukaryotes. This enzyme is essential for the viability of many pathogenic organisms, such as Mycobacterium tuberculosis and Escherichia coli, due to the broad distribution of Galf in crucial structures such as the cell wall or capsular polysaccharide. Not surprisingly, galactofuranose biosynthesis has become an attractive antimicrobial target due to the absence of these sugars in higher eukaryotes. The UGM homologue, UDP-Nacetylgalactopyranose mutase (UNGM), was identified in Campylobacter jejuni 11168, encoded for by the cj1439c gene. UNGM is known to function as a bifunctional mutase, which catalyzes the reversible ring contraction between the pyranose-furanose forms of UDPgalactose (UDP-Gal) and UDP-N-acetylgalactosamine (UDP-GalNAc). UNGM is essential for the virulence of C. jejuni, due to the incorporation of UDP-N-acetylgalactofuranose into the capsular polysaccharide. We report the first structure of UNGM determined by X-ray crystallography, to a resolution of 1.9 Å. Analysis of the dimeric, holoenzyme structure of UNGM has identified that the cofactor flavin adenine dinucleotide is bound within each monomer of the enzyme. Comparative analysis with UGM homologues has confirmed the conserved active site residues involved in the binding of various substrates. Docking studies suggest that UNGM binds its natural substrates in a productive binding mode for catalysis with the flavin cofactor, which is consistent with the proposed mechanism for UNGM. The mobile loops are essential for substrate binding, and we have identified that the conserved arginine residue, Arg169, and the neighboring Arg168, function to stabilize the diphosphate region of UDP, although not concurrently. The non-conserved arginine residue, Arg168, appeared to favor the stabilization of N-acetylated sugars, which is in agreement with the enzyme’s higher binding affinity for UDP-GalNAc over UDP-Gal by a factor of 0.9. We have also identified that the active site Arg59 exists in two conformations in the structure of UNGM, with one conformation directed toward the active site. Arg59 is 2.5 to 3.0 Å from the acetamido moiety of GalNAc, which is favorable for stabilization and is believed to confer specificity for this substrate.

Youden index, a main summary index for the Receiver Operating Characteristic (ROC) curve, is a comprehensive measurement for the effectiveness of a diagnostic test. For a continuous-scale diagnostic test, the optimal cut-point for the positive of disease is the cut-point leading to the maximization of the sum of sensitivity and specificity. Finding the Youden index of the test is equivalent to maximize the sum of sensitivity and specificity for all the possible values of the cut-point. In this thesis, we propose a new non-parametric confidence interval for the Youden index. Extensive simulation studies are conducted to compare the relative performance of the new interval with the existing intervals for the index. Our simulation results indicate that the newly developed non-parametric method performs as well as the existing parametric method but it has better finite sample performance than the existing non-parametric methods. The new method is flexible and easy to implement in practice. A real example is also used to illustrate the application of the proposed interval.

Caspases are important players in programmed cell death. Normal activities of caspases are critical for the cell life cycle and dysfunction of caspases may lead to the development of cancer and neurodegenerative diseases. They have become a popular target for drug design against abnormal cell death. In this study, the recognition of P5 position in substrates by caspase-3, -6 and -7 has been investigated by kinetics, modeling and crystallography. Crystal structures of caspase-3 and -7 in complexes with substrate analog inhibitor Ac-LDESD-CHO have been determined at resolutions of 1.61 and 2.45 Å, respectively, while a model of caspase-6/LDESD is constructed. Enzymatic study and structural analysis have revealed that Caspase-3 and -6 recognize P5 in pentapeptides, while caspase-7 lacks P5-binding residues. D-arginine dehydrogenase catalyzes the flavin-dependent oxidative deamination of D-amino acids to the corresponding imino acids and ammonia. The X-ray crystal structures of DADH and its complexes with several imino acids were determined at 1.03-1.30 Å resolution. The DADH crystal structure comprises a product-free conformation and a product-bound conformation. A flexible loop near the active site forms an “active site lid” and may play an essential role in substrate recognition. The DADH Glu87 forms an ionic interaction with the side chain of iminoarginine, suggesting its importance for DADH preference of positively charged D-amino acids. Comparison of the kinetic data of DADH activity on different D-amino acids and the crystal structures demonstrated that this enzyme is characterized by relatively broad substrate specificity, being able to oxidize positively charged and large hydrophobic D-amino acids bound within a flask-like cavity. Understanding biology at the system level has gained much more attention recently due to the rapid development in genome sequencing and high-throughput measurements. Current simulation methods include deterministic method and stochastic method. Both have their own advantages and disadvantages. Our group has developed a deterministic-stochastic crossover algorithm for simulating biochemical networks. Simulation studies have been performed on biological systems like auto-regulatory gene network and glycolysis system. The new system retains the high efficiency of deterministic method while still reflects the random fluctuations at lower concentration.

The collection of crop yield data has become much easier with the introduction of technologies such as the Global Positioning System (GPS), ground-based yield sensors, and Geographic Information Systems (GIS). This explosive growth and widespread use of spatial data has challenged the ability to derive useful spatial knowledge. In addition, outlier detection as one important pre-processing step remains a challenge because the technique and the definition of spatial neighbourhood remain non-trivial, and the quantitative assessments of false positives, false negatives, and the concept of region outlier remain unexplored. The overall aim of this study is to evaluate different spatial outlier detection techniques in terms of their accuracy and computational efficiency, and examine the performance of these outlier removal techniques in a site-specific management context. In a simulation study, unconditional sequential Gaussian simulation is performed to generate crop yield as the response variable along with two explanatory variables. Point and region spatial outliers are added to the simulated datasets by randomly selecting observations and adding or subtracting a Gaussian error term. With simulated data which contains known spatial outliers in advance, the assessment of spatial outlier techniques can be conducted as a binary classification exercise, treating each spatial outlier detection technique as a classifier. Algorithm performance is evaluated with the area and partial area under the ROC curve up to different true positive and false positive rates. Outlier effects in on-farm research are assessed in terms of the influence of each spatial outlier technique on coefficient estimates from a spatial regression model that accounts for autocorrelation. Results indicate that for point outliers, spatial outlier techniques that account for spatial autocorrelation tend to be better than standard spatial outlier techniques in terms of higher sensitivity, lower false positive detection rate, and consistency in performance. They are also more resistant to changes in the neighbourhood definition. In terms of region outliers, standard techniques tend to be better than spatial autocorrelation techniques in all performance aspects because they are less affected by masking and swamping effects. In particular, one spatial autocorrelation technique, Averaged Difference, is superior to all other techniques in terms of both point and region outlier scenario because of its ability to incorporate spatial autocorrelation while at the same time, revealing the variation between nearest neighbours. In terms of decision-making, all algorithms led to slightly different coefficient estimates, and therefore, may result in distinct decisions for site-specific management. The results outlined here will allow an improved removal of crop yield data points that are potentially problematic. What has been determined here is the recommendation of using Averaged Difference algorithm for cleaning spatial outliers in yield dataset. Identifying the optimal nearest neighbour parameter for the neighbourhood aggregation function is still non-trivial. The recommendation is to specify a large number of nearest neighbours, large enough to capture the region size. Lastly, the unbiased coefficient estimates obtained with Average Difference suggest it is the better method for pre-processing spatial outliers in crop yield data, which underlines its suitability for detecting spatial outlier in the context of on-farm research.

Abstract After the industry of Taiwan's bicycle and its parts were brought to the top in 1980, due to the change of economic environment of Taiwan, the proprietors of bicycle and its parts that belong to the conventional industries face the predicament of wages upsurge, the invariable of human resource and lands. The industry of Taiwan's bicycle and its parts has been played the role of manufacturing division supply chain of the system of global industry division and faced the conflicts of liberalization, globalization and low price for a long time. In order to operate in the situation of global industry division and its congregation plus the channel of marketing was controlled by others, how to get the cheaper human resource and lands to lower the producing cost to satisfy the marketing runners about the managed trend of low price. In early 1990, part of manufacturers started to have industrial relocation in Mainland China and sustained their business life by thinking the reason of economic open of Mainland China, cheap human resource and lands. Under the management of people who are from Taiwan and other countries and the advantage of lands for 10 years, the industry of Taiwan's bicycle and its parts still play an important role in global supply chain. With good economic situation of China after year 2000, its conventional industries extended rapidly, because of the competitive advantage of it, this advantage was used on the role of global manufacturing division and became a base of operations of global conventional industries. The strategy of Taiwan's bicycle and its parts that used cheap manpower, land resource as a step of global export was successful in early period, but it faces the cruel managed situation of foreign companies expends its business, overproduced and some conventional industries of China grow up and seize this big global market now. The industry of Taiwan's bicycle and its parts is part of medium-sized enterprises mostly. In this thesis, we found that this industry of medium-sized enterprises was the main managed factor or growing up in international division in early period, and it could enter China's market fast and keep the benefit and life of enterprises management safe then expended the situation of it continually. In this thesis, we found that the main points of industry of Taiwan's bicycle and its parts in the strategy of international division are, first, Mainland China was the main foothold for foreign companies to go and expended their business. Second, foreign companies all got together in Shenzhen area which was near Hong Kong in early period and used the step of position of Hong Kong as a free port of delivering and supplying this convention to the whole world. Third, after the management became stable in late 1990, the foreign company owners moved their business to Shanghai area, which was the core place of the industry of China's bicycle and competed for this global market with conventional industries of China. Fourth, most business runners started to occupy the inland market of China after everything got steady. The last point was though all the business areas were still controlled by Taiwan, the manufacturing had a recession gradually or even stopped producing totally. What's the next step of industry of Taiwan's bicycle and its parts after it gets its manufacturing base of operations? As for the industrial situation of the whole world, goods price is still low, but the capital of manufacturing tends to get high. Managed environment has been successful for 10 years from now because of the open strategy of Mainland China and makes the competition harder and harder. Businessmen of Taiwan should think about how to become better in the aspect of creating new stuffs and knowledge management to enlarge the competitive difference between Taiwan and China and still keeps the competitive core. Mainland China is a place where full of people, if we want to use the operating way of Taiwan's bicycle and its parts by medium-sized enterprises, only management is not strong enough to cover the whole market in China. Only using strategy of congregation and alliance can have an efficient overall arrangement in Mainland China and selecting strategy of diversity marketing to different markets. The conventional industry of Taiwan under the baptism of global division leads to agile managed way or our convention still has to face the managed predicament of loosing superiority one day if the purpose of industrial relocation is just getting low human resource and land cost.