From Serendipity to the Rational Design of Protein–Protein Interface Modulators Targeting a tRNA-Modifying Enzyme

Serendipity has played a historically significant role in science and especially in the discovery of new drugs. Chance discoveries have led to important drug developments that characterize the pharmaceutical industry and our current health care system. Over the last decades, the concept of rational drug design has developed into one of the key approaches to the research of new and specific drugs. Nevertheless, the development of inhibitors for protein–protein interactions, especially oligomeric protein contact surfaces, remains challenging. The bacterial enzyme, tRNA-guanine transglycosylase (TGT), is an example of a homodimer protein and serves as model target protein in this work. This thesis summarizes three projects that were created from serendipitous discoveries of previous studies to develop new approaches for the modulation of the TGT homodimer interface and thus lay out the basis for alternative inhibition pathways in oligomeric enzymes. A previous crystallographic study of TGT–inhibitor complexes revealed an unexpected, twisted arrangement of the TGT homodimer. However, data from the obtained X-ray crystal structures can neither exclude crystallographic artifacts resulting from crystal packing forces nor study the transition between the two dimeric end states. Therefore, a method based on electron paramagnetic resonance (EPR) was established to study this ligand-induced rearrangement mechanism of the TGT homodimer in a solution equilibrium (Chapter 2). For this purpose, paramagnetic spin markers were introduced via site-directed cysteine mutations on the protein surface. The following pulsed EPR techniques enabled the observation of inter-spin distance distributions within the TGT homodimer. During the study, a pyranose-substituted lin-benzoguanine inhibitor was identified to be superior in transforming the functional TGT dimer to its twisted state. Thus, the developed method can be used to distinguish between the functional and twisted TGT dimer species upon ligand addition in a solution equilibrium. A second project comprises the search of small molecule fragments that target a newly discovered transient binding pocket at the homodimer interface of TGT (Chapter 3). In an earlier mutational study, a new crystal form of the TGT dimer was discovered, in which the two protomers are covalently linked by an introduced disulfide bridge. This arrangement breaks up the original dimer interface and exposes a small hydrophobic binding pocket through an extended movement of the nearby β1α1-loop motif. Surprisingly, the newly formed pocket was occupied by a dimethyl sulfoxide molecule. In this work, a further stabilized variant of the disulfide-linked dimer was designed for subsequent fragment soaking studies to target the interface binding pocket. In the course of the soaking experiments, the solvent channels of the covalently linked TGT dimer were analyzed in silico to estimate the putative cutoff radii of small molecules that are capable of freely traversing through the protein crystal. The structural characterization of initial fragment hits led to the rational design of compounds with optimized functionalities which are able to modulate the β1α1-loop. Furthermore, fragment binding studies were attempted by nuclear magnetic resonance (NMR) techniques. Although no binding could be detected using a 19F-based method, the binding constants of two fragments were successfully estimated in the micromolar range using diffusion-ordered spectroscopy (DOSY). While DOSY provides binding data on two of the investigated fragments, structure determination by X-ray crystallography combined with soaking experiments proves to be superior in the identification of fragment binding events. This study highlights the difficulties in characterizing weak binders to a transient interface pocket within the TGT homodimer. The last project initiated the design of peptide-based modulators derived from the interface of the TGT homodimer (Chapter 4). The idea for this approach was inspired by a crystal structure from the second project in which N-terminal residues of TGT, which were usually ill-defined in the electron density, were structurally resolved and stabilized by the exposed dimer contact surface of a crystallographic symmetry mate. In the functional dimer, the position of this particular N-terminal tail is occupied by helix αE, which contains two aromatic residues of the dimer-stabilizing hot spot. This served as a starting point for the development of helical peptides, that are capable to compete with the native dimer partner at the contact surface. A peptide microarray was used to determine the binding epitope to TGT. Subsequently, the most promising peptides were synthesized and characterized with respect to their binding properties by fluorescence polarization. An optimization approach by peptide stapling was attempted. In order to determine the modulation of TGT dimerization, two biophysical assays each based on MicroScale thermophoresis and isothermal titration calorimetry were developed to track changes in the dimerization constants of TGT. Furthermore, a Shigella host cell invasion assay was established to investigate the invasion pathogenicity of a Shigella flexneri Δtgt mutant strain. Henceforth, the established in vitro assays can be further used to characterize potential peptides and drug candidates for TGT inhibition. In conclusion, serendipitous discoveries served as a foundation for the projects discussed in this thesis. It was demonstrated that accidental findings in basic research can be used and acted upon to uncover novel mechanisms of action or for the design of new small molecule fragments and peptides as starting points with the common goal of modulating challenging protein–protein interactions

ANFIS: Establishing and Applying to Managing Online Damage

Fuzzy logic (FL) and artificial neural networks (ANNs) own individual advantages and disadvantages. Adaptive neuro-fuzzy inference system (ANFIS), a fuzzy system deployed on the structure of ANN, by which FL and ANN can interact to not only overcome their limitations but also promote the ability of each model has been considered as a reasonable option in the real fields. With the vital strong points, ANFIS has been employed well in many technology applications related to filtering, identifying, predicting, and controlling noise. This chapter, however, focuses mainly on building ANFIS and its application to identifying the online bearing fault. First, a traditional structure of ANFIS as a data-driven model is shown. Then, a recurrent mechanism depicting the relation between the processes of filtering impulse noise (IN) and establishing ANFIS from a noisy measuring database is presented. Finally, one of the typical applications of ANFIS related to online managing bearing fault is shown

The determinants of merger arbitrage return: an impirical analysis in the UK context

This thesis explores the magnitude and the determinants of the return to the merger arbitrage strategy in the UK context. We perform empirical analysis of the three hypotheses namely the risk-based hypothesis, the limited arbitrage hypothesis and the arbitrageurs' role hypothesis. First, in the risk-based hypothesis, using a sample of 1105 UK cash and stock mergers from 1987 to 2007, we find that the strategy generates significant positive return in excess of the systematic risk adjustment benchmark. The result is robust to a range of methods to control for systematic risk. The finding is consistent with the existing evidence from other markets. As for the risk-return characteristics of the strategy, in contrast to the US evidence, we find little evidence supporting the non-linear pattern. This finding is in line with the restrictions on bidder's ability to abandon the bid imposed by UK Takeover Code. This finding, combined with the evidence in the US market (strong non-linearity) and the Australian market (no nonlinearity), demonstrates the impact of takeover regulation on the risk-return characteristics of the strategy. Second, in the limited arbitrage hypothesis, we test the impact of different types of risks, costs and constraints (other than systematic risk) on the arbitrage return. We find that transaction costs are one of the important drivers of the cross-sectional variation of the arbitrage return. The result is robust to 4 different proxies for transaction costs, that is, firm size, price level, dollar trading volume, and frequency of zero return days. Holding costs are found to be an important determinant of the return. Idiosyncratic risk, the most important type of holding costs, contributes significantly to the source of the arbitrage return. We find that short-sale constraints appear to be another important holding cost that the arbitrageurs concern about. The result about the impact of shortsale constraints is, however, still inconclusive due to the small sample size. We also test the agency-based model of limited arbitrage hypothesis proposed by Shleifer and . Vishny (1997) but find no supporting evidence. Third, in the arbitrageurs' role hypothesis, utilizing a manually collected dataset to identify arbitrageurs and their holding of target stocks, we examine how different roles that arbitrageurs play in the takeover process help explain the source of the return to the strategy. We find that arbitrage holding is significantly related to arbitrage return after a host of factors that can determine the bid outcome and the market's assessment of the bid outcome are controlled for. This finding shows that the arbitrageurs are better than the average investors in the market in picking better takeover bids, the investment in which yields higher risk-adjusted return. In contrast to the US evidence, arbitrage holding is found to be negatively related to bid premium and has no impact on the probability of bid success. The difference between this finding and the US evidence may be attributable to the much more stringent UK disclosure rule during the takeover period compared to the US counterpart. Overall, our study, while providing evidence broadly in support of significant return to the merger arbitrage strategy, also highlights the importance of recognizing the impact of the takeover regulation on such return

Reclamation of Marine Chitinous Materials for Chitosanase Production via Microbial Conversion by Paenibacillus macerans

[[abstract]]: Chitinous materials from marine byproducts elicit great interest among biotechnologists for their potential biomedical or agricultural applications. In this study, four kinds of marine chitinous materials (squid pens, shrimp heads, demineralized shrimp shells, and demineralized crab shells) were used to screen the best source for producing chitosanase by Paenibacillus macerans TKU029. Among them, the chitosanase activity was found to be highest in the culture using the medium containing squid pens as the sole carbon/nitrogen (C/N) source. A chitosanase which showed molecular weights at 63 kDa was isolated from P. macerans cultured on a squid pens medium. The purified TKU029 chitosanase exhibited optimum activity at 60 ◦C and pH 7, and was stable at temperatures under 50 ◦C and pH 3-8. An analysis by MALDI-TOF MS revealed that the chitosan oligosaccharides (COS) obtained from the hydrolysis of water-soluble chitosan by TKU029 crude enzyme showed various degrees of polymerization (DP), varying from 3–6. The obtained COS enhanced the growth of four lactic acid bacteria strains but exhibited no effect on the growth of E. coli. By specialized growth enhancing effects, the COS produced from hydrolyzing water soluble chitosan with TKU029 chitinolytic enzymes could have potential for use in medicine or nutraceuticals.[[sponsorship]]MOST[[notice]]補正完

Thermoresistance of p-Type 4H–SiC Integrated MEMS Devices for High-Temperature Sensing

There is an increasing demand for the development and integration of multifunctional sensing modules into power electronic devices that can operate in high temperature environments. Here, the authors demonstrate the tunable thermoresistance of p‐type 4H–SiC for a wide temperature range from the room temperature to above 800 K with integrated flow sensing functionality into a single power electronic chip. The electrical resistance of p‐type 4H–SiC is found to exponentially decrease with increasing temperature to a threshold temperature of 536 K. The temperature coefficient of resistance (TCR) shows a large and negative value from −2100 to −7600 ppm K−1, corresponding to a thermal index of 625 K. From the threshold temperature of 536–846 K, the electrical resistance shows excellent linearity with a positive TCR value of 900 ppm K−1. The authors successfully demonstrate the integration of p–4H–SiC flow sensing functionality with a high sensitivity of 1.035 μA(m s−1)−0.5 mW−1. These insights in the electrical transport of p–4H–SiC aid to improve the performance of p–4H–SiC integrated temperature and flow sensing systems, as well as the design consideration and integration of thermal sensors into 4H–SiC power electronic systems operating at high temperatures of up to 846 K

Temporal force governs the microbial assembly associated with Ulva fasciata (Chlorophyta) from an integrated multi-trophic aquaculture system

Ulva spp., one of the most important providers of marine ecosystem services, has gained substantial attention lately in both ecological and applicational aspects. It is known that macroalgae and their associated microbial community form an inseparable unit whose intimate relationship can affect the wellbeing of both. Different cultivation systems, such as integrated multi-trophic aquaculture (IMTA), are assumed to impact Ulva bacterial community significantly in terms of compositional guilds. However, in such a highly dynamic environment, it is crucial to determine how the community dynamics change over time. In the current study, we characterized the microbiota associated with Ulva fasciata grown as a biofilter in an IMTA system in the Gulf of Aqaba (Eilat, Israel) over a developmental period of 5 weeks. The Ulva-associated microbial community was identified using the 16S rRNA gene amplicon sequencing technique, and ecological indices were further analyzed. The Ulva-associated microbiome revealed a swift change in composition along the temporal succession, with clusters of distinct communities for each timepoint. Proteobacteria, Bacteroidetes, Planctomycetes, and Deinococcus-Thermus, the most abundant phyla that accounted for up to 95% of all the amplicon sequence variants (ASVs) found, appeared in all weeks. Further analyses highlighted microbial biomarkers representing each timepoint and their characteristics. Finally, the presence of highly abundant species in Ulva microbiota yet underestimated in previous research (such as phyla Deinococcus-Thermus, families Saprospiraceae, Thiohalorhabdaceae, and Pirellulaceae) suggests that more attention should be paid to the temporal succession of the assembly of microbes inhabiting macroalgae in aquaculture, in general, and IMTA, in particular. Characterizing bacterial communities associated with Ulva fasciata from an IMTA system provided a better understanding of their associated microbial dynamics and revealed this macroalgae's adaptation to such a habitat