Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).

<p>Pathogenic bacteria present a critical threat to modern medicine. Therapeutic strategies to target and eliminate resilient bacteria are not advancing at the same rate as the emergence of bacterial resistance. An associated urgent concern regarding antibiotic resistance is the existence and proliferation of intracellular bacteria, which find refuge from bactericidal mechanisms by hiding within mammalian cells. Therefore, many once-successful antibiotics become ineffective through the development of resistance, or through failure to reach intracellular locations in therapeutic concentration. To overcome these challenges, the covalent combination of a conventional antibiotic with an antibiotic, cell-penetrating peptide was explored to develop dual-action antibiotic conjugates. </p> <p>Herein, we utilized a strategy in conjugating the antibiotics by a cleavable linkage to cationic amphiphilic polyproline helices (CAPHs) to improve vancomycin and linezolid antibiotics. This approach enables the conjugate to penetrate cells and deliver two potent monomeric antimicrobial drugs. The vancomycin-CAPH conjugate, <b>VanP14S</b>, showed enhanced mammalian cell uptake compared to vancomycin, a poor mammalian cell-penetrating agent; and <b>VanP14S</b> was capable of cleaving and releasing two antibiotics under mimicked physiological conditions. Enhanced antibacterial activity was observed against a spectrum of Gram-positive and Gram-negative pathogens, including drug-resistant strains. Further investigation revealed that this conjugate’s bactericidal activity was not entirely the result of significant membrane perturbation such as a lytic mode of action. Mammalian cell toxicity and red blood cell lysis were insignificant at relevant bactericidal concentrations below 20 µM. The current results suggest an enhanced binding to the peptidoglycan of bacteria, the target of vancomycin, although more work is needed to justify this claim. Preliminary results on <b>VanP14GAPS</b>, a conjugate with a more rigid CAPH, convey similar activity to <b>VanP14S; </b>however,<b> </b>moderate increases in red blood cell lysis and cytotoxicity were observed. </p> <p>Regarding the <b>LnzP14</b> conjugate, preliminary data reveal that the conjugate has Gram-negative activity against <i>Escherichia coli</i>, whereas linezolid is ineffective in killing Gram-negative bacteria. This conjugate showed significant enhancement in cellular uptake compared to the CAPH, and the release of linezolid and CAPH in physiological conditions was confirmed. Overall, arming a conventional antibiotic with an antimicrobial, cell-penetrating peptide appears to be a powerful strategy in providing novel antibiotic conjugates with the propensity to overcome the limitations in treating challenging pathogens.</p>

京都大学 / 0048 / 新制・論文博士 / 博士(工学) / 乙第13145号 / 論工博第4163号 / 新制||工||1687(附属図書館) / (主査)教授 森 泰生, 教授 浜地 格, 教授 梅田 眞郷 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Surgery-induced myeloid derived suppressor cells (sxMDSC)s mediate postoperative suppression of Natural Killer (NK) cells, which enables postoperative cancer recurrence and metastases. Currently, no therapeutics against sxMDSCs have been developed. Recent research has identified that the myeloid-restricted PI3K isoform (PI3Kγ) mediates MDSC activity. I targeted PI3Kγ in sxMDSCs as a therapeutic to reduce postoperative NK cell suppression and metastatic burden. Additionally, I investigated the efficacy of a sxMDSC-specific antibody-drug conjugate (ADC) with a PI3Kγ inhibitor payload. Pharmacological inhibition of PI3Kγ in sxMDSCs led to reduced AKT phosphorylation and reduced suppression of NK cytotoxicity in human and murine models. PI3Kγ inhibition also reduced postoperative metastatic burden. Despite the novelty of the sxMDSC-specific ADC, it didn’t provide considerable benefits in reducing NK cell suppression compared to the unconjugated PI3Kγ inhibitor. However, this is a “first iteration” in what could be a powerful approach to targeting sxMDSCs, thereby preventing postoperative metastatic burden.

Assessing the effect of foam layers on transport phenomena is of significant interest in many industries, so a method for predicting foam layer properties has been developed. A model of the propagation of radiation from an amplitude-modulated laser beam through a non-absorbing foam layer has been developed using diffusion theory. Measurements predicted by diffusion theory were compared to results generated using Monte Carlo methods for a variety of foam layer properties in both the time-domain and the frequency-domain. The properties that were varied include the layer thickness, the scattering coefficient, and the asymmetry parameter. Layer thicknesses between 8.5 mm and 18 cm were considered. Values of the scattering coefficient ranged from about 600 m-1 to 14000 m-1, while the asymmetry parameter varied between 0 and 1. A conjugate-gradient algorithm was used to minimize the difference between simulated Monte Carlo measurements and diffusion theory predicted measurements. A large set of simulated measurements, calculated at various source-detector separations and three discrete frequencies were used to predict the layer properties. Ten blind cases were considered and property predictions were made for each. The predicted properties were within approximately 10% of the actual values, and on average the errors were approximately 4%. Predictions of the reduced scattering coefficient were all within approximately 5% with the majority being within 3%. Predictions of L were all within approximately 10% with the majority being within 7%. Attempts to separate g from the reduced scattering coefficient were unsuccessful, and it was determined that implementation of different source models might make such attempts possible. It was shown that with a large number of measurements, properties could be accurately predicted. A method for reducing the number of measurements needed for accurate property estimation was developed. Starting with a single measurement location, property predictions were made. An approach for updating the optimal detector location, based on the current estimate of the properties, was developed and applied to three cases. Property predictions for the three cases were made to within 10% of the actual values. A maximum of three measurement locations were necessary to obtain such predictions, a significant reduction as compared to the previously illustrated method.

A methodology is formulated for the solution of the inverse problem concerned with the reconstruction of multi-dimensional heat fluxes for film cooling applications. The motivation for this study is the characterization of complex thermal conditions in industrial applications such as those encountered in film cooled turbomachinery components. The heat conduction problem in the metal endwall/shroud is solved using the boundary element method (bem), and the inverse problem is solved using a genetic algorithm (ga). Thermal conditions are overspecified at exposed surfaces amenable to measurement, while the temperature and surface heat flux distributions are unknown at the film cooling hole/slot walls. The latter are determined in an iterative process by developing two approaches. The first approach, developed for 2d applications, solves an inverse problem whose objective is to adjust the film cooling hole/slot wall temperatures and heat fluxes until the temperature and heat flux at the measurement surfaces are matched in an overall heat conduction solution. The second approach, developed for 2d and 3d applications, is to distribute a set of singularities (sinks) at the vicinity of the cooling slots/holes surface inside a fictitious extension of the physical domain or along cooling hole centerline with a given initial strength distribution. The inverse problem iteratively alters the strength distribution of the singularities (sinks) until the measuring surfaces heat fluxes are matched. The heat flux distributions are determined in a post-processing stage after the inverse problem is solved. The second approach provides a tremendous advantage in solving the inverse problem, particularly in 3d applications, and it is recommended as the method of choice for this class of problems. It can be noted that the ga reconstructed heat flux distributions are robust, yielding accurate results to both exact and error-laden inputs. In all cases in this study, results from experiments are simulated using a full conjugate heat transfer (cht) finite volume models which incorporate the interactions of the external convection in the hot turbulent gas, internal convection within the cooling plena, and the heat conduction in the metal endwall/shroud region. Extensive numerical investigations are undertaken to demonstrate the significant importance of conjugate heat transfer in film cooling applications and to identify the implications of various turbulence models in the prediction of accurate and more realistic surface temperatures and heat fluxes in the cht simulations. These, in turn, are used to provide numerical inputs to the inverse problem. Single and multiple cooling slots, cylindrical cooling holes, and fan-shaped cooling holes are considered in this study. The turbulence closure is modeled using several two-equation approach, the four-equation turbulence model, as well as five and seven moment reynolds stress models. The predicted results, by the different turbulence models, for the cases of adiabatic and conjugate models, are compared to experimental data reported in the open literature. Results show the significant effects of conjugate heat transfer on the temperature field in the film cooling hole region, and the additional heating up of the cooling jet itself. Moreover, results from the detailed numerical studies presented in this study validate the inverse problem approaches and reveal good agreement between the bem/ga reconstructed heat fluxes and the cht simulated heat fluxes along the inaccessible cooling slot/hole walls

An axi-symmetric shock-tube model has been developed to simulate the shock-wave propagation and reflection in both non-reactive and reactive flows. Simulations were performed for the full shock-tube geometry of the high-pressure shock tube facility at Texas A&M University. Computations were carried out in the CFD solver FLUENT based on the finite volume approach and the AUSM+ flux differencing scheme. Adaptive mesh refinement (AMR) algorithm was applied to the time-dependent flow fields to accurately capture and resolve the shock and contact discontinuities as well as the very fine scales associated with the viscous and reactive effects. A conjugate heat transfer model has been incorporated which enhanced the credibility of the simulations. The multi-dimensional, time-dependent numerical simulations resolved all of the relevant scales, ranging from the size of the system to the reaction zone scale. The robustness of the numerical model and the accuracy of the simulations were assessed through validation with the analytical ideal shock-tube theory and experimental data. The numerical method is first applied to the problem of axi-symmetric inviscid flow then viscous effects are incorporated through viscous modeling. The non-idealities in the shock tube have been investigated and quantified, notably the non-ideal transient behavior in the shock tube nozzle section, heat transfer effects from the hot gas to the shock tube side walls, the reflected shock/boundary layer interactions or what is known as bifurcation, and the contact surface/bifurcation interaction resulting into driver gas contamination. The non-reactive model is shown to be capable of accurately simulating the shock and expansion wave propagations and reflections as well as the flow non-uniformities behind the reflected shock wave. Both the inviscid and the viscous non-reactive models provided a baseline for the combustion model iii which involves elementary chemical reactions and requires the coupling of the chemistry with the flow fields adding to the complexity of the problem and thereby requiring tremendous computational resources. Combustion modeling focuses on the ignition process behind the reflected shock wave in undiluted and diluted Hydrogen test gas mixtures. Accurate representation of the Shock - tube reactive flow fields is more likely to be achieved by the means of the LES model in conjunction with the EDC model. The shock-tube CFD model developed herein provides valuable information to the interpretation of the shock-tube experimental data and to the understanding of the impact the facility-dependent non-idealities can have on the ignition delay time measurements.

Specifically addressing cell surface molecules on cancer cells facilitates targeted cancer therapies that offer the potential to selectively destroy malignant cells, while sparing healthy tissue. Thus, undesired side-effects in tumor patients are highly reduced. Peptide-binding receptors are frequently overexpressed on cancer cells and therefore promising targets for selective tumor therapy. In this review, peptide-binding receptors for anti-cancer drug delivery are summarized with a focus on peptide ligands as delivery agents. In the first part, some of the most studied peptide-binding receptors are presented, and the ghrelin receptor and the Y1 receptor are introduced as more recent targets for cancer therapy. Furthermore, nonpeptidic small molecules for receptor targeting on cancer cells are outlined. In the second part, peptide conjugates for the delivery of therapeutic cargos in cancer therapy are described. The essential properties of receptor-targeting peptides are specified, and recent developments in the fields of classical peptide-drug conjugates with toxic agents, radiolabeled peptides for radionuclide therapy, and boronated peptides for boron neutron capture therapy are presented.

Conjugate heat transfer analysis has been carried out on an 89kN thrust chamber in order to evaluate whether combined discrete film cooling and regenerative cooling in a rocket nozzle is feasible. Several cooling configurations were tested against a baseline design of regenerative cooling only. New designs include combined cooling channels with one row of discrete film cooling holes near the throat of the nozzle, and turbulated cooling channels combined with a row of discrete film cooling holes. Blowing ratio and channel mass flow rate were both varied for each design. The effectiveness of each configuration was measured via the maximum hot gas-side nozzle wall temperature, which can be correlated to number of cycles to failure. A target maximum temperature of 613K was chosen. Combined film and regenerative cooling, when compared to the baseline regenerative cooling, reduced the hot gas side wall temperature from 667K to 638K. After adding turbulators to the cooling channels, combined film and regenerative cooling reduced the temperature to 592K. Analysis shows that combined regenerative and film cooling is feasible with significant consequences, however further improvements are possible with the use of turbulators in the regenerative cooling channels.

Cancer became recently the leading cause of death in industrialized countries. Even though standard treatments achieve significant effects in growth inhibition and tumor elimination, they cause severe side effects as most of the applied drugs exhibit only minor selectivity for the malignant tissue. Hence, specific addressing of tumor cells without affecting healthy tissue is currently a major desire in cancer therapy. Cell surface receptors, which bind peptides are frequently overexpressed on cancer cells and can therefore be considered as promising targets for selective tumor therapy. In this review, the benefits of peptides as tumor homing agents are presented and an overview of the most commonly addressed peptide receptors is given. A special focus was set on the bombesin receptor family and the neuropeptide Y receptor family. In the second part, the specific requirements of peptide-drug conjugates (PDC) and intelligent linker structures as an essential component of PDC are outlined. Furthermore, different drug cargos are presented including classical and recent toxic agents as well as radionuclides for diagnostic and therapeutic approaches. In the last part, boron neutron capture therapy as advanced targeted cancer therapy is introduced and past and recent developments are reviewed.