Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23845号 / 薬博第852号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

The influence of silane treated amorphous fumed silica fillers on properties of the cured epoxy was examined in the first part of the study. Silica particles were treated with 3- aminopropyldiethoxymethylsilane (APDS) and 3-aminopropyltriethoxysilane (APTS) coupling agents. The filler and coupling agents decreased the mobility of the polymer chains in the vicinity of the filler leading to an increase in the activation energy for the glass transition and an increase in cooperativity. Fumed silica did not significantly affect moisture diffusion properties. Next, a linear dilatometer was used to investigate the effects of cure conditions, mold types, and the presence of filler in the model epoxy. These studies revealed that there was substantial shrinkage in the cured epoxy on heating it through its glass transition region. The shrinkage was determined to be the result of stress in the epoxy generated during cure and could be minimized by curing at lower temperatures, followed by a postcuring heat treatment. Additional free volume in the sample increased the magnitude of the shrinkage by allowing increased stress release through increased network mobility. Decreasing the polymer mobility by adding fillers decreased the observed shrinkage. The influence of the model epoxy crosslink density was examined by varying the content of 1,4-butanediol in the model system. Addition of 1,4-butanediol led to a decrease in the modulus and glass transition temperature, which resulted in a reduction in residual stress and subsequent shrinkage. Moisture uptake increased with the addition of 1,4-butanediol due to an increase in the free volume of the epoxy. However, even with greater moisture uptake, the addition of 1,4-butanediol to the epoxy increased its adhesion to quartz by promoting lower residual stress and increased energy dissipation. These results indicate that bulk diffusion of water is not the controlling factor in adhesive degradation in this system. / Ph. D.

The main goals of this study were to document the structural and functional recovery of differently restored areas, to understand better the relationship between the two, and to determine which types of measurements are best for assessing restoration success. To address these questions, an experimental system was created through topsoil removal and subsequent restoration in a blocked, completely randomized design using two levels of soil amendment (with or without 10 kg of leaf mulch per m2) and three levels of seeding treatment (no seed, a standard reclamation mix, and an alternative, wildflower dominated reclamation mix). All measurements were designed to document responses due to restoration treatment in comparison to adjacent, undisturbed, reference sites. Vegetation structure in amended sites, as measured by total vegetation cover and species richness, recovered to levels similar to references within the two years of the study. Plant community composition did not develop similarity to references in any experimental treatments. Both soil amendment and seeding type affected cellulose decomposition rates, with amended plots showing higher decomposition rates than unamended, and seeded plots exhibiting higher rates than unseeded. Enzyme activities were largely determined by soil amendment, but the reference plots consistently had higher enzymatic activity. Amended sites exhibited significant increases over time in soil respiration, reaching or surpassing the rates observed in reference areas. Methane oxidation rates were generally increased in disturbed plots compared to undisturbed references due to increased atmospheric diffusion into the soil. Amended areas exhibited depressed rates relative to unamended, and seeding level had no significant effect on methane oxidation. Over all measurements, restoration of ecosystem function was most facilitated by the addition of the soil amendment. Seeding treatment significantly altered the resultant plant community, which may have substantial, long-term consequences for succession. The inclusion of functional parameters into restoration assessment provides for better overall information concerning ecosystem performance and may add to the ability to predict long-term success of restoration efforts. / Ph. D.

This research focused on the characterization of organosilicone surfactants and their effects on sulfonylurea herbicide activity. The project included efficacy tests, rainfastness studies in the greenhouse, radiotracer studies on herbicide uptake, fluorescent dye studies on surface deposition, and various measurements of physico-chemical properties. In measuring physico-chemical properties, a logistic dose response relationship was found between adjuvant concentration and contact angle on parafilm. An AsymSigR relationship existed between adjuvant concentration and surface tension for all the adjuvants. The organosilicones, Silwet L-77, Silwet 408, and Sylgard 309, and Kinetic (a blend of an organosilicone with a nonionic surfactant) gave equilibrium surface tension values around 20 dyne/cm and showed great spreading ability on the foliage of velvetleaf. With the conventional adjuvants, Agri-Dex, methylated soybean oil, Rigo oil concentration, and X-77, and Dyne-Amic (a blend of an organosilicone with a crop oil concentrate), surface tension was rarely below 28 dyne/cm and spreading ability was limited on velvetleaf. In addition, the organosilicone surfactant and Kinetic also lowered dynamic surface tension, which may improve droplet retention on leaf surfaces. The differences in physico-chemical properties between Kinetic and Dyne-Amic confirmed that carefully electing a co-adjuvant for an organosilicone blend is critical to avoid antagonism with trisiloxane molecules and retain the unique physico-chemical properties of organosilicone in the blends. Studies involving structurally-related organosilicones showed that the end structure in the trisiloxane hydrophilic group has little or no effect on surface tension, contact angle, spread pattern, herbicide uptake and translocation, and efficacy of primisulfuron on velvetleaf. It may be suggested that there is not a strict requirement to purify the end structure during the synthesis process, which is time consuming and expensive. When 14C-primisulfuron was combined with organosilicones or the blends, the uptake of 14C at 1 or 2 h after herbicide application was significantly higher than when combined with conventional adjuvants in velvetleaf. In the greenhouse, organosilicone surfactants greatly increased the rainfastness of primisulfuron in velvetleaf. The effect was immediate and dramatic, even when simulated rainfall was applied 0.25 h after treatment. In addition, herbicide efficacy on marginally susceptible weed species, velvetleaf and barnyardgrass, was significantly increased. A very complicated relationship exists between herbicides and adjuvants. The enhancement effects of adjuvants are often herbicide specific, weed species specific, and even environment specific. No one type of adjuvant functions well in all circumstances. Therefore, there is a need to understand the properties and functions of each class of adjuvants and locate the 'right' niche for each individual adjuvant. / Ph. D.

The lack of forage resources in the Sahelian region of Mali is a major constraint to food production and food sufficiency. Madiama commune is located in northern Mali, in the Niger Delta region. Three separate experiments were conducted to investigate ways to improve pasture resources and productivity. The first experiment (2003) was designed to investigate the influence of sheep grazing tethered at two different residual heights on botanical composition, forage biomass and animal performance. Young sheep weighing approximately 18-24 kg were tethered for a certain period of time depending on residual canopy height. Two treatments 3 or 6 cm residual height were each replicated 4 times. Animals were rotated based on canopy height and each tethered animal followed an 8 paddock rotation. Measurements included forage biomass, plant diversity, animal performance, and botanical composition. The forage species found on these pastures were primarily Schoenfeldia gracilis, Panicum laetum, Setaria palludefusca, Eragrostis turgida, Eragrostis tremula, Zornia glauchidiata, Tephrosia pedicellata, and Cynodon spp. Accumulated seasonal forage biomass increased while forage quality declined as the growing season progressed. Treatment had only a slight effect on animal weight gains (1 to 3kg season-1). These results suggest that residual height may not affect livestock gain. The second experiment was designed to investigate the potential of Cassia tora (C. tora) which is an invasive weed in the region as a supplemental feed for livestock. Cassia tora was harvested within the Madiama commune and ensiled with or without additives (water and or honey/sugar) for 60 or 90 days. Harvest occurred at the vegetative stage in year 1 and mature growth stage in year 2. Prior to placing the chopped material in the bags for ensiling, sub-samples of fresh C. tora were obtained for dry matter (DM) and chemical analysis (NDF, ADF, CP, IVDMD and TDN). In year 1, the ensiled material/fresh material across treatments and locations had NDF varying from 48 to 56 %/ 56 to 57%, ADF from 34 to 41 %/40 to 42%, CP from 9 to 10 %/9 to 23%, and IVDMD from 53 to 64 %/52 to 54%. In year 2, CP averaged twice as much as year 1 with significantly less fiber probably due to the fact that harvest occurred at the vegetative stage. Addition of water or sugar/honey improved the nutritive values of the ensiled material. These results suggest that C. tora can be a reliable feed source during the dry season. A greenhouse experiment was conducted using various P sources (Tilemsi phosphate rock (TPR), North Carolina phosphate rock (NCPR), Aluminum phosphate (AlP), Iron phosphate (FeP), and Triple superphosphate (TSP) and rates (0, 20, 40, 60, and 80 mg P kg-1 soil). Plants were grown for 10 wks, harvested and separated into above and below ground plant parts. The root and plant material were dried, ground and analyzed for elemental P. The result showed variable P solubility and uptake by the plant. Overall, addition of P resulted in an increase in above ground biomass as well as root mass compared with the untreated control. Field and greenhouse experiments showed that in the Sahel region of Africa where feed resources are scarce 8 out of 12 months a year, anything we can do to increase pasture resources and animal productivity while maintaining a healthy ecosystem, could improve the quality of life in the community. / Ph. D.

I investigated how anthropogenic acidification influences stream metabolism and nitrogen (N) cycling by considering the stress response of microbial compartments responsible for these ecosystem processes. Microcosm incubations of leaf biofilms from streams of differing pH revealed greater rates of fungal biomass-specific respiration (i.e. the stress metric <i>q</i>CO₂) and biomass-specific N uptake (i.e. <i>q</i>N) with increasing acidity. The positive relationship between <i>q</i>CO₂ and <i>q</i>N indicated alternate fates for N other than structural biomass, possibly related to increased exoenzyme production as part of the stress response. Whole-stream ¹⁵N experiments and measurements of respiration and fungal standing crop across the pH gradient resulted in similar patterns in <i>q</i>CO₂ and <i>q</i>N found in microcosm experiments, supporting <i>q</i>CO₂ as an ecosystem-level stress indicator and providing insight towards controls over N cycling across the pH gradient. Fungal biomass and ecosystem respiration declined with increasing acidity while N uptake metrics were not related to pH, which suggested <i>q</i>N in acid streams was sufficiently high to counteract declines in fungal abundance. During spring, chlorophyll <i>a</i> standing crops were higher in more acidic streams despite lower nutrient concentrations. However, N uptake rates and gross primary production differed little between acid and circumneutral streams. Reduced heterotrophy in acid streams was apparent in lower whole-stream respiration rates, less ability to process organic carbon, and little response of N uptake to added carbon resources. Overall, acid-induced stress in streams was found to impair decomposer activity and caused a decoupling of carbon and nitrogen cycles in these systems. / Ph. D.

The objective of this study was to determine the photothermal and photochemical tissue response to carbon nanotube inclusion in laser therapy using experimental and computational methods. In this study, we specifically considered varying types and concentrations (0.01-1 mg/ml) of carbon nanotubes (CNTs), e.g., multi-walled carbon nanotubes (MWNTs), single-walled carbon nanotubes (SWNTs), and single-walled carbon nanohorns (SWNHs). In order to determine the photothermal effect of CNT inclusion, the thermal conductivity and optical properties of tissue representative phantoms with CNT inclusion were measured. Thermal conductivity of tissue phantoms containing CNTs was measured using the hot wire probe method. For identical CNT concentrations, phantoms containing MWNTs had the highest thermal conductivity. Optical properties (absorption and reduced scattering coefficients) of solutions and tissue phantoms containing carbon nanotubes were measured with spectrophotometry and determined by the inverse adding doubling (IAD) method. Inclusion of CNTs in phantoms increased light absorption with minimal effect on scattering and anisotropy. Light absorption of MWNTs was found to be higher than SWNTs and SWNHs. The photochemical response to laser irradiation (wavelength 1064 nm) of CNTs was measured with spin-trap electron paramagnetic resonance (EPR) spectroscopy. Only SWNHs appeared to produce significant levels of ROS production in response to laser excitation in the presence of NADH. We detected the predominant presence of trapped hydroxyl radical (•OH) with a trace of the trapped super oxide (O2•-) radical. These free radicals are highly reactive and could be utilized to cause targeted toxicity to cancer cells. The distribution of CNTs at the cellular level, in phantoms, and in kidney tumors was measured using transmission electron microscopy (TEM) imaging. Samples were imaged following various time periods (2-48h) of incubation and CNTs were observed inside the cell cytoplasm, nucleus, vacuole, and outside cells for the above mentioned time periods. CNTs in phantoms and tumor tissue were randomly and uniformly distributed in the entire volume. Computational model geometries were developed based on CNTs distribution in cells, tissue phantoms, and kidney tumor tissue. In the computational part of this research the temperature response to laser irradiation alone or with CNT inclusion was determined using Penne's bioheat equation which was solved by finite element methods. Experimentally measured thermal conductivity and absorption and reduced scattering coefficients were used as input parameters in Penne's bioheat equation. The accuracy of the model predicted temperature distribution was determined by comparing it to experimentally measured temperature in tissue phantoms and kidney tumors following CNT inclusion and laser therapy. The model determined temperature distribution was in close correspondence with the experimentally measured temperature. Our computational model can predict the effectiveness of laser cancer therapy by predicting the transient temperature distribution. / Ph. D.

Cancer therapies are often limited by bulk and cellular barriers to transport. Nanoparticle or chemotherapeutic compound intracellular transport has implications in understanding therapeutic effect and toxicity. The scope of this thesis was to study the intracellular transport of carbon nanohorns and to improve the efficacy of various chemotherapeutic agents through increased intracellular transport. In the first study, fluorescent probes (quantum dots) were conjugated to carbon nanohorns to facilitate the optical visualization of the nanohorns. These hybrid particles were characterized with transmission electron microscopy, electron dispersive spectroscopy and UV-VIS/FL spectroscopy. Their cellular uptake kinetics, uptake efficiencies, and intracellular distribution were determined in three malignant cell lines (breast – MDA-MB-231, bladder – AY-27, and brain – U87-MG) using flow cytometry and confocal microscopy. Intracellular distribution did not vary greatly between cell lines; however, the uptake kinetics and efficiencies were highly dependent on cell morphology. In the second study, the efficacy of various chemotherapeutic agents (i.e., doxorubicin, cisplatin, and carboplatin) was evaluated in AY-27 rat bladder transitional cell carcinoma cells. In the future, severe hyperthermia and chemothermotherapy (chemotherapy + hyperthermia) will also be evaluated. Doxorubicin and cisplatin compounds were more toxic compared to carboplatin. Hyperthermia has previously shown to increase the cellular uptake of chemotherapeutic agents; therefore, chemothermotherapy is expected to have synergistic effects on cell death. This work can then be translated to carbon nanohorn-based laser heating to generate thermal energy in a local region for delivery of high concentrations of chemotherapeutic agents. Although these two concepts are small pieces of the overall scope of nanoparticle-based therapies, they are fundamental to the advancement of such therapies. / Master of Science

Histophilus somni is an etiologic agent of shipping fever pneumonia, myocarditis, and other systemic diseases in bovines, although nonpathogenic commensal strains also exist. Virulence factors that have been identified in H. somni include biofilm formation, lipooligosaccharide phase variation, immunoglobulin binding proteins, survival in phagocytic cells, and many others. To identify genes responsible for virulence, an efficient mutagenesis system is needed. Mutagenesis of H. somni using allelic exchange is difficult due to its tight restriction modification system. Mutagenesis by natural transformation in Haemophilus influenzae is well established and may be enhanced by the presence of uptake signal sequences (USS) within the genome. We hypothesized that natural transformation occurs in H. somni because its genome is over-represented with USS and contains all the necessary genes for competence, except that ComD and ComE are mutated. For natural transformation, H. somni was grown to exponential phase, and then transferred to a non-growth defined medium to induce competence. H. somni strain 2336 was successfully transformed with homologous linear DNA (lob2A) containing an antibiotic marker gene, but at low efficiency. Shuttle vector pNS3K was also naturally transformed into H. somni at low efficiency. To attempt to improve transformation efficiency, comD and comE from H. influenzae were cloned into shuttle vector pNS3K to generate the plasmid pSScomDE. Although introduction of pSScomDE into H. somni was expected to increase the number and breadth of mutants generated by natural transformation, multiple attempts to electroporate pSScomDE into H. somni were unsuccessful. A native plasmid (pHS649) from H. somni strain 649 may prove to be a more efficient shuttle vector. Due to inefficiency in generating mutants by allelic exchange, transposon (Tn) mutagenesis with EZ / Master of Science