<p>Alzheimer’s disease (AD) continues to plague the healthcare community as a serious healthcare crisis. Currently there fails to be an effective FDA approved drug that can treat the underlying mechanisms of the disease. Pathologically the disease is characterized by the accumulation of neurotoxic amyloid-b(Ab) plaques within a diseased patient’s brain. These plaques are widely accepted to be generated by the sequential proteolytic cleavage of amyloid precursor protein (APP) by b-secretase (BACE1, memapsin 2) and g-secretase. Numerous biochemical markers suggest that BACE1 is a viable target for AD drug development. Since the cloning and expression of BACE1 there has been an explosion of drug development efforts that have consisted of peptidomimetic-based and non-peptide-based inhibitors. These efforts have led to 13 BACE1 drug candidates some of which have made it to advanced stages of clinical trials. Unfortunately, an effective and tolerable BACE1 drug candidate continues to be rather elusive to the medicinal chemistry community. GRL-8234 is a potent BACE1 inhibitor that has been extensively shown to be tolerable during several short-term and long-term <i>in vivo </i>studies. The scaffold of GRL-8234 provides a suitable template for further lead development. Namely the P1’ 3-methoxy-benzylamine is of particular interest due to the advantageous interactions in the flap region of BACE1 that can be achieved by incorporation of substitution at the benzylic position. In the same way, the <i>meta</i>-substituent of the benzylamine P1’ ligand was investigated to probe the hydrophobic interactions in the S1’-S2’ binding pocket. A novel class of BACE1 inhibitors containing P1’ spirocyclic benzylamine derivatives were designed, synthesized and evaluated for their inhibitory activity against BACE1. In the process of preparing the aforementioned BACE1 inhibitors a method was established to be able to incorporate the desired 3,5-difluorophenyl methyl transition state isostere by utilizing an ester-derived Ti-enolate to access optically pure <i>syn</i>- and <i>anti</i>-aldol adducts as a key step. Additionally, a novel stereoselective method was established to afford heterospirocyclic benzylamines by utilizing a diastereoselective allyl grignard addition to optically pure a-silyloxy <i>N</i>-sulfinyl ketimines as a key step. Biological evaluation of these novel BACE1 inhibitors has been fruitful and continues to be ongoing. </p>

It has been well established that both carbohydrate-loading before and carbohydrate ingestion during exercise can enhance endurance performance by supplying carbohydrate for oxidation. However, the precise mechanism(s) underlying the proposed ergogenic effects of these procedures remain to be established. The studies in this thesis were therefore designed to examine the effects of carbohydrate-loading and carbohydrate ingestion on fuel substrate kinetics.

As part of a project designed to utilize common dairy waste products profitably, reconstituted dry whey permeate and delactosed whey were tested as growth substrates for mycelia of the edible mushroom Lentinus edodes. This mushroom was chosen because it is possible to profitably cultivate it due to its popular culinary appeal and perceived medical benefits. Growth experiments were performed in petri dishes containing either reconstituted dry whey permeate or delactosed whey as a growth substrate, and the measured response was the size of the growing mycelia colony. When reconstituted dry whey permeate was utilized as a growth substrate, the factors of substrate concentration, pH, and growth temperature were controlled in an effort to determine the optimal growth conditions for the mushroom mycelia. These conditions were determined by applying an analytical method known as response surface methodology (RSM). RSM is a collection of mathematical techniques that is able to determine optimal values for many variables run simultaneously in an experiment. Mycelia were also grown on delactosed whey at different substrate concentrations in an effort to determine if this substrate would be suitable for the growth of mushroom mycelia. Results: RSM was successfully utilized to determine the optimal growth conditions for L. edodes when grown on reconstituted dry whey powder. These conditions were 40 g/L substrate concentration, pH 4 .97, and temperature 23.6°C Delactosed whey was successfully utilized as a growth substrate for L. edodes. However, delactosed whey concentrations above 40% v/v were lethal to the mushroom mycelia, suggesting a possible use for delactosed whey as a fungicide.

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Indiana University-Purdue University Indianapolis (IUPUI) / The role of mechanical forces in the fate and function of adherent cells has been revealed to be a pivotal factor in understanding cell biology. Cells require certain physical cues to be present in their microenvironment or the cell will begin apoptosis. Mechanical signals from the environment are interpreted at the cellular level and biochemical responses are made due to the information from outside the cell, this process is known as mechanotransduction. Misinterpretation of physical cues has been indicated in many disease states, including heart disease and asthma. When a cell is bound to the ECM, proteins such as integrins are engaged at static and stable adhesion sites. These tight and static anchoring points found at the ECM exist in stark contrast to the dynamic conditions seen at intercellular junctions. Intercellular junctions, such as gap and adherens junctions, are formed between cells to act as a mechanism to relay information and exchange material. Due to the important role intercellular junctions play in processes of wound healing, epithelial-mesenchymal transition and cancer metastasis developing more sophisticated levels of understanding of these mechanisms would provide valuable insight. Complex biological processes, including immune cell signaling and cellular ECM adhesions, have been effectively replicated in model systems. These model systems have included the use of solid supported lipid bilayers and polymeric hydrogels that display cell adhesion molecules. Studies of cellular mechanotransduction at ECM adhesion sites has also been completed with covalently functionalized polymeric substrates of adjustable elasticity. However, developing model systems that allow the accurate reproduction of properties seen at intercellular junctions, while also allowing the investigation of cellular mechanosensitivity has proven to be a difficult task. Previous work has shown that polymer-tethered lipid bilayers (PTLBs) are a viable material to allow the replication of the dynamics and adhesion seen at intercellular junctions. Although efforts have been made to produce PTLBs with different mechanical properties, there is currently not a material with sufficient tunable elastic properties for the study of cellular mechanotransduction. To establish a system that allows the study of stiffness effects across a biologically relevant range (~0.50 – 40 kPa) while maintaining the dynamic properties seen at cell-to-cell junctions, polymer gel-tethered bilayers (PGTBs) were developed. A fabrication strategy was established to allow the incorporation of a hydrogel support with easily tunable stiffness and a tethered lipid bilayer coating, which produced a powerful platform to study the effects of stiffness at intercellular junctions. Careful attention was given to maintain the beneficial properties of membrane diffusion, and it was shown that on different linking architectures lipid bilayers could be established and diffusion was preserved. Microscopy-based FCS and FRAP methodology were utilized to measure lipid diffusion in these systems, while confocal microscopy was used to analyze cell spreading and adhesion. Three distinct architectures to link the lipid membrane to the underlying polyacrylamide hydrogel were pursued in this work, a non-covalent biotin-streptavidin system, a covalently linked design with fibronectin, and a direct covalent linkage utilizing crosslinker chemistry. In this work, it was shown that cells were able to spread and adhere on these substrates, with cell adhesion zones visualized under plated cells that demonstrate the capability of the cell to rearrange the presented linkers, while maintaining a stable material. Also confirmed is the tunability of the polymer hydrogel across a wide range of stiffness, this was shown by quantitative changes in cell spreading area in response to polymer properties.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19743号 / 工博第4198号 / 新制||工||1647(附属図書館) / 32779 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 田畑 泰彦, 教授 秋吉 一成, 教授 木村 俊作 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Cell culturing on three-dimensional structures has increased the possibilities in tissue engineering and bioreactor research. These structures enable cells to differentiate, proliferate, mobilize, and function in a conformation that more accurately mimics in vivo conditions. Computer generated models aid in development and rapid alteration of three-dimensional cell substrates, defining their internal structure as well as their external morphology. The rapid transition from substrate design to a viable culture is imperative to quickly advance research in biomedical and tissue engineering applications. The aim of this thesis is to investigate the feasibility of a rapid prototyping process by selectively cross-linking and assembling biocompatible films. This investigation revealed that selectively cross-linking and layering gelatin films could produce a three-dimensional substrate with a defined structure after dissolving uncross-linked gelatin. The study also revealed that freeze-drying aided in the rapid dissolution of uncross-linked gelatin. The line width resolution obtained during tests was .5 mm using a template treatment method and was limited by the template construction resolution. Finally, alteration in treatment time, rinsing agitation, and rinsing temperature yielded stable films that better retained their size and shape compared to films produced in previous processes.