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Uncoupling protein, a mitochondrial inner membrane protein found in mammalian brown adipose tissue, functions as an uncoupler of oxidative phosphorylation by serving as a proton carrier when activated, resulting in heat production, the function of the tissue. Unlike most nuclear-encoded mitochondrial proteins, uncoupling protein is not made with a cleavable presequence. With the availability of an uncoupling protein cDNA clone, the region responsible for targeting uncoupling protein to mitochondria was examined using in vitro transcription and translation and import into isolated mitochondria. In order to localize the targeting sequence of uncoupling protein, fusion proteins containing portions of uncoupling protein and uncoupling protein modified by site-directed mutagenesis were constructed and analysed for their ability to be imported. Previously it has been shown that there was a targeting signal within uncoupling protein amino acids 13 to 105 (Liu et al., 1988). However, amino acids 13 to 51 did not target a passenger protein to mitochondria (Liu et al., 1988). Here the role of amino acids 53 to 105 of uncoupling protein in targeting was examined with two new constructs, uncoupling protein amino acids 53 to 105 joined to rat ornithine carbamoyltransferase amino acids 147 to 354 and to mouse dihydrofolate reductase. These two constructs along with uncoupling protein with amino acids 2 to 51 deleted were imported into mitochondria consistent with uncoupling protein amino acids 53 to 105 having a potential targeting role in uncoupling protein. Further, these three constructs were processed upon import. The major processed forms of all three constructs are approximately 20 amino acids smaller than the initial translation product. Both fusion constructs also have an intermediate-sized processed form approximately 14 amino acids smaller than the initial translation product. Processing suggests that at least the amino terminus of these proteins has reached the mitochondrial matrix. The location of the proteins was examined using Na2CO3 extraction. Uncoupling protein and U13-105-OCT (uncoupling protein amino acids 13 to 105 joined to ornithine carbamoyltransferase amino acids 147 to 354) were found in the membrane fraction while the processed forms of Ud2-51 (uncoupling protein with amino acids 2 to 52 deleted) and U53-105-DHFR (uncoupling protein amino acids 53 to 105 joined to dihydrofolate reductase) were found in the aqueous fraction suggesting that uncoupling protein amino acids 2 to 52/53 are involved in membrane localization. Analysis of Ud2-35 (uncoupling protein with amino acids 2 to 35 deleted) revealed that it was associated with both the membrane and aqueous fractions. Analysis of uncoupling protein amino acids 53 to 105 revealed the potential existence of two positively charged amphipathic a-helices. Based on the sizes of processed forms and on the helical wheel projection for the first possible sequence, arginine54 , lysine56 and lysine67 were changed to glutamines, individually and in various combinations using oligonucleotide site-directed mutagenesis. All mutant proteins were imported into mitochondria even when all three basic amino acids were replaced. The results suggest that this portion of uncoupling protein, amino acids 54 to 67, is not a targeting signal in the protein. / Thesis / Master of Science (MS)

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23521号 / 農博第2468号 / 新制||農||1087(附属図書館) / 学位論文||R3||N5352(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 准教授 坂本 正弘 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Understanding the biological mechanisms of polymeric DNA delivery is essential to develop vehicles that perform optimally. In this work, the cellular internalization mechanisms of poly(glycoamidoamine) (PGAA) DNA delivery polymers were investigated. Polymer:DNA complexes interact with cell-surface glycosaminoglycans (GAGs) in a manner independent of electrostatic interactions. Desulfation and GAG removal leads to decreased uptake. Individual polyplexes appear to have differing affinities for specific GAGs, as polyplex dissociation occurs in a charge-independent manner, and may influence binding. Internalization occurs through close interactions with GAGs, as GAGs accumulate on polyplex surfaces, resulting in negatively-charged polyplexes and decompaction of intact polyplexes is observed upon interaction with GAG. PGAA polyplexes enter cells via a complex, multifaceted internalization route. Pharmacological inhibition of endocytosis and visualization by confocal microscopy reveal that internalization occurs primarily through an actin and dynamin-dependent mechanism. Caveolae/raft-mediated endocytosis appears to be the predominant internalization mechanism, with clathrin-mediated endocytosis also significantly involved. Internalization occurs to a smaller degree via macropinocytosis and direct membrane penetration. Caveolae-mediated, but not clathrin-mediated, internalization leads to transgene expression, suggesting a targeting opportunity based on uptake mechanisms. PEGylation of PGAA polyplexes was achieved to minimize polyplex aggregation in serum. Polyplex size increased in serum, but PEGylation prevented further polyplex growth over time compared to non-PEGylated polymers. Specific targeting of hepatocytes through end-modification of PEG with galactose was unsuccessful, likely due to inaccessibility of targeting groups. Further hepatocyte targeting efforts focused on malonate-based polymers with clickable linkages for facile linkage of targeting groups. Despite favorable surface presentation of galactose, receptor-specific internalization of polyplexes was unsuccessful, as competitive inhibition in HepG2 cells resulted in significant polyplex internalization derived from nonspecific membrane interactions. Chemical modification of vehicles allows systematic study of structure-function properties leading to efficient intracellular delivery. Increasing G4 molecular weight generally increases toxicity and decreases transgene expression in HeLa cells. Incorporating galactose into a lanthanide-chelating polymer facilitated efficient cellular internalization that was visualized by two-photon microscopy. Increased gene expression was observed that correlated to increasing galactose, suggesting that polymer degradation increases gene expression. Also studied were branched peptides targeted to HIV-1 TAR, which displayed high biocompatibility and favorable internalization profiles in mammalian cells. / Ph. D.

The overall goal of this research was to increase, relative to targeting recommendations, the cost-effectiveness of pollution reduction measures within a watershed. The goal was met through development of an optimization procedure for best management practice (BMP) placement at the watershed level. The procedure combines an optimization component, written in the C++ language, with spatially variable nonpoint source (NPS) prediction and economic analysis components, written in the ArcView geographic information system scripting language. The procedure is modular in design, allowing modifications or enhancements to the components while maintaining the overall theory. The optimization component uses a genetic algorithm to optimize a lexicographic multi-objective function of pollution reduction and cost increase. The procedure first maximizes pollution reduction to meet a specified goal, or maximum allowable load, and then minimizes cost increase. For the NPS component, a sediment delivery technique was developed and combined with the Universal Soil Loss Equation to predict average annual sediment yield at the watershed outlet. Although this evaluation considered only erosion, the NPS pollutant fitness score allows for evaluation of multiple pollutants, based on prioritization of each pollutant. The economic component considers farm-level public and private costs, accounting for crop productivity levels by soil and for enterprise budgets by field. The economic fitness score assigns higher fitness scores to scenarios in which costs decrease or are distributed more evenly across farms. Additionally, the economic score considers the amounts of cropland, hay, and pasture needed to meet feed and manure/poultry litter spreading requirements. Application to two watersheds demonstrated that the procedure optimized BMP placement, locating scenarios more cost-effective than a targeting strategy solution. The optimization procedure identified solutions with lower costs than the targeting strategy solution for the same level of pollution reduction. The benefit to cost ratio, including use of the procedure and implementation of resulting solutions, was demonstrated to be greater for the optimization procedure than for the targeting strategy. The optimization procedure identifies multiple near optimal solutions. Additionally, the procedure creates and evaluates scenarios in a repeated fashion without requiring human interaction. Thus, more scenarios can be evaluated than are feasible to evaluate manually. / Ph. D.

Ferrofluids are comprised of subdomain particles of magnetite or iron oxide material that can become magnetized in the presence of a magnetic field. These unique liquids are being incorporated into many new applications due to the ability to control them at a distance using magnetic fields. However, although our understanding of the dynamics of ferrofluids has evolved, many aspects of ferrohydrodynamics remain largely unexplored, especially experimentally. This study is the first to characterize the stability and internal dynamics of accumulating or dispersing ferrofluid aggregates spanning the stable, low Reynolds number behavior to unstable, higher Reynolds numbers. The dynamics of ferrofluid aggregates are governed by the interaction between the bulk flow shear stresses acting to wash away the aggregate and magnetic body forces acting to retain them at the magnet location. This interaction results in different aggregate dynamics, including the stretching and coagulation of the aggregate to Kelvin-Helmholtz shedding from the aggregate interface as identified by focused shadowgraphs. Using TRDPIV, the first time-resolved flow field measurements conducted in ferrofluids reveal the presence of a three-stage process by which the ferrofluid interacts with a pulsatile bulk flow. An expanded parametric study of the effect of Reynolds number, magnetic field strength, and flow unsteadiness reveals that the increased field results can result in the lifting and wash away of the aggregate by means of vortex strengthening. In pulsatile flow, different forms of the three-stage interaction occur based on magnetic field, flow rate, and Reynolds number. / Ph. D.

Amphiphilic block copolymers consisting of poly(ethylene oxide) and poly(lactide) have great potential for formulating drug delivery systems. Our approach was to synthesize poly(ethylene oxide-b-D,L-lactide), (PEO-b-PDLLA), block copolymers with controlled molecular weights and good functionality on the poly(ethylene oxide) end for the design of potential core-shell delivery vehicles for HIV drugs. PEO-b-PDLLA block copolymer was used as a polymeric nanocarrier to encapsulate the HIV protease inhibitor, Ritonavir, within magnetite nanoparticles. Well-defined multifunctional polymeric nanoparticles with controlled sizes and size distributions were fabricated by rapid nanoprecipitation using blends of the PEO-b-PDLLA block copolymer with poly(L-lactide), (PLLA) homopolymer. Heterobifunctional PEO oligomers were directly prepared by initiating ethylene oxide with functional alcohols bearing vinylsilane, vinylether and maleimide moieties to provide appropriate end groups for conjugating targeting ligands. The polyethers with narrow molecular weight distributions were utilized as macroinitiators for the synthesis of poly(lactide) block. Heterobifunctional diblock copolymers possessing carboxylic acids were prepared from ene-thiol addition reaction of mercaptoacetic acid across the vinyl group on the PEO end, while preserving the hydroxyl functionality on the other end. Additionally, PDLLAs bearing maleimide functionality with controlled molecular weights were synthesized using maleimide functional initiator. End group modification was performed via  Michael addition using cysteamine hydrochloride to introduce an amino group over the vinyl bond. The resulting carboxylic acid functional PEO-b-PDLLA diblock copolymers, and amino functional PDLLAs are potential biocompatible polymers that can be utilized to encapsulate an array of bioactive molecules, targeting ligands. / Master of Science

The lack of efficacy of existing chemotherapeutic treatments of solid tumors is partially attributed to the limited diffusion distance of therapeutics and the low selectivity of anti-cancer drugs with respect to cancerous tissue, which also leads to high levels of systemic toxicity in patients. Thus, chemotherapy can be enhanced through improving anti-cancer drug carrier selectivity and transport properties. Several strains of gram positive (e.g. Clostridium and Bifidobacterium) and gram-negative (e.g. Salmonella Typhimurium and Escherichia coli) bacteria have been shown to possess the innate ability to preferentially colonize tumor tissues. The overall goal of this dissertation is to characterize the transport and sensing of Bacteria-Enabled Drug Delivery Systems (BEADS) in select relevant environments and to investigate the associated underlying principles. BEADS consist of an engineered abiotic load (i.e. drug-laden micro or nano-particles) and a living component (i.e. bacteria) for sensing and actuation purposes. Findings of this dissertation work are culminated in experimental demonstration of deeper penetration of the NanoBEADS within tumor tissue when compared to passively diffusing chemotherapeutic nanoparticles. Lastly, the transport mechanisms that Salmonella Typhimurium VNP20009 utilize to preferentially colonize solid tumors are also examined with the ultimate goal of engineering intelligent and more efficacious drug delivery vehicles for cancer therapy. / Ph. D.