京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19006号 / 工博第4048号 / 新制||工||1623(附属図書館) / 31957 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 中條 善樹, 教授 赤木 和夫, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20398号 / 工博第4335号 / 新制||工||1672(附属図書館) / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 関 修平, 教授 今堀 博, 准教授 伊藤 彰浩, 教授 白川 昌宏 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Several antibiotics that prevent translation by binding to ribosomal subunits have been shown to also inhibit ribosomal subunit assembly (Champney and Tober 2003). The aminoglycoside hygromycin B was examined in Escherichia coli cells for inhibitory effects on translation and ribosomal subunit assembly. The streptogramin antibiotics quinupristin-dalfopristin and XRP 2868 (NXL 103) were examined for similar effects on these 2 cellular functions in antibiotic-resistant strains of Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. Pulse chase experiments were performed which verified slower rates of ribosomal subunit formation in drug treated cells. Hygromycin B exhibited a concentration dependent inhibitory effect on viable cell number, growth rate, protein synthesis and 30S and 50S subunit formation. 16S rRNA specific probes hybridized to rRNA fragments in cells treated with hygromycin B. RNase II and RNase III deficient strains of E. coli exhibited the most accumulation of 16S rRNA fragments upon treatment with hygromycin B. Examination of total RNA from treated cells showed an increase in RNA corresponding to precursor to the 16S rRNA while 16S rRNA decreased. There was also an increase in small fragment RNA. Hygromycin B was a more effective inhibitor of translation than ribosomal subunit formation in E. coli. Two streptogramin antibiotics were compared for inhibitory effects in antibiotic-resistant Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. IC50 values for XRP 2868 were several fold lower than those of quinupristin-dalfopristin for inhibition of cell viability, protein synthesis, and ribosomal subunit formation. Both antibiotics revealed a concentration dependent inhibitory effect on cellular functions including 50S ribosomal subunit formation in the three organisms examined. XRP 2868 inhibited both 50S ribosomal subunit assembly and translation. XRP 2868 was effective against MRSA and was a better inhibitor in each of the antibiotic resistant strains examined compared with quinupristin-dalfopristin.

Polycyclic aromatic compounds (PACs), which comprise a complex class of condensed multi-ring benzenoid compounds, are important environmental pollutants originating from a wide variety of natural and anthropogenic sources. PACs are generally formed during incomplete combustion of pyrolisis of organic matter containing carbon and hydrogen. Because combustion of organic materials is involved in countless natural processes or human activities, PACs are omnipresent and abundant pollutants in air, soil, and water. Chemical analysis of PACs is of great environmental and toxicological importance. Many of them are highly suspect as etiological agents in human cancer. Because PACs carcinogenic properties strongly depend on molecular structure and differ significantly from isomer to isomer, it is of paramount importance to determine the most toxic isomers even if they are present at much lower concentrations than their less toxic isomers. Gas chromatography (GC), high-resolution GC, and high-performance liquid chromatography (HPLC) are the basis for standard PACs identification and determination. Many cases exist where GC, HPLC, and even HR-GC have not been capable to provide unambiguous isomer identification. The lack of reliable analytical data has lead to serious errors in environmental and toxicological studies. This dissertation deals with the development of novel instrumentation and analytical methods for the analysis of PACs in environmental samples. The developed methodology is based on two well-known high-resolution luminescence techniques, namely Shpol'skii Spectroscopy (SS) and Fluorescence Line Narrowing Spectroscopy (FLNS). Although these two techniques have long been recognized for their capability in providing direct determination of target PACs in complex environmental samples, several reasons have hampered their widespread use for the problem at hand. These include inconvenient sample freezing procedures; questions about signal reproducibility; lengthy spectral acquisition, which might cause severe sample degradation due to prolonged excitation; broadband fluorescence background that degrades quality of spectra, precision of measurements and detection limits; solvent constrains imposed by the need of optically transparent media; and, most importantly, the lack of selectivity and sensitivity for unambiguous determination of closely related PACs metabolites. This dissertation presents significant advances on all fronts. The analytical methodology is then extended to the analysis of fluoroquinolones (FQs) in aqueous samples. FQs are one of the most powerful classes of antibiotics currently used for the treatment of urinary tract infections. Their widespread use in both human and animal medicine has prompted their appearance in aquatic systems. The search for a universal method capable to face this new environmental challenge has been centered on HPLC. Depending on the FQ and its concentration level, successful determination has been accomplished with mass spectrometry, room-temperature fluorescence (RTF) or UV absorption spectrometry. Unfortunately, no single detection mode has shown the ability to detect all FQ at the concentration ratios found in environmental waters. We provide a feasible alternative based on FLNS. On the instrumentation side, we present a single instrument with the capability to collect multidimensional data formats in both the fluorescence and the phosphorescence time domains. We demonstrate the ability to perform luminescence measurements in highly scattering media by comparing the precision of measurements in optically transparent solvents (Shpol'skii solvents) to those obtained in "snow-like" matrixes and solid samples. For decades, conventional low-temperature methodology has been restricted to optically transparent media. This restriction has limited its application to organic solvents that freeze into a glass. In this dissertation, we remove this limitation with the use of cryogenic fiber-optic probes. Our final efforts deal with low-temperature absorption measurements. Recording absorption spectra via transmittance through frozen matrixes is a challenging task. The main reason is the difficulty to overcome the strong scattering light reaching the detector. This is particularly true when thick samples are necessary for recording absorption spectra of weak oscillators. In the case of strongly fluorescent compounds, additional errors in absorbance measurements arise from the emission reaching the detector, which might have comparable intensity to that of the transmitted light. We present a fundamentally different approach to low-temperature absorption measurements as the sought-for-information is the intensity of laser excitation returning from the frozen sample to the intensified-charge coupled device (ICCD). Laser excitation is collected with the aid of a cryogenic fiber optic probe. The feasibility of our approach is demonstrated with single-site and multiple-site Shpol'skii systems. 4.2K absorption spectra show excellent agreement to their literature counterparts recorded via transmittance with closed cycle cryogenators. Fluorescence quantum yields measured at room-temperature compare well to experimental data acquired in our lab via classical methodology. Similar agreement is observed between 77K fluorescence quantum yields and previously reported data acquired with classical methodology. We then extend our approach to generate original data on fluorescence quantum yields at 4.2K.

Polycyclic aromatic hydrocarbons (PAH) are a class of environmental pollutants consisting of a minimum of two fused aromatics rings originating from the incomplete combustion of organic matter and/or anthropogenic sources. Numerous possible anthropogenic and natural sources make the presence of PAH ubiquitous in the environment. The carcinogenic nature of some PAH and their ubiquitous presence makes their chemical analysis a topic of environmental and toxicological importance. Although environmental monitoring of PAH is an important step to prevent exposure to contaminated sites, it provides little information on the actual uptake and subsequent risks. Parent PAH are relatively inert and need metabolic activation to express their carcinogenicity. Covalent binding to DNA appears to be the first critical step in the initiation of the tumor formation process. To this end, the determination of short term biomarkers – such as monohydroxy-PAH metabolites (OH-PAH) - fills an important niche to interpret actual PAH exposure levels, prevent extreme body burdens and minimize cancer risk. One would certainly prefer an early warning parameter over a toxicological endpoint – such as DNA-adducts – indicating that extensive damage has already been done. Several methods have been developed to determine OH-PAH in specific tissue or excreta and food samples. The general trend for the analysis of OH-PAH follows the pattern of sample collection, sample clean-up and pre-concentration, chromatographic separation and quantification. Popular approaches for sample clean-up and preconcentration include liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Chromatographic separation and quantification has been based on high-performance liquid iv chromatography-room temperature fluorescence detection (HPLC) and gas chromatographymass spectrometry (GC-MS). Although chromatographic techniques provide reliable results in the analysis of OHPAH, their experimental procedures are time consuming and expensive. Elution times of 30-60 minutes are typical and standards must be run periodically to verify retention times. If the concentrations of target species are found to lie outside the detector’s response range, the sample must be diluted and the process repeated. On the other end of the concentration range, many samples are “zeroes,” i.e. the concentrations are below detection limits. Additional problems arise when laboratory procedures are scaled up to handle thousands of samples under mass screening conditions. Under the prospective of a sustainable environment, the large usage of organic solvents is one of the main limitations of the current chromatographic methodology. This dissertation focuses on the development of a screening methodology for the analysis of OH-PAH in urine and milk samples. Screening techniques capable of providing a “yes or no” answer to OH-PAH contamination prevent unnecessary scrutiny of un-contaminated samples via conventional methods, reduce analysis cost and expedite the turnaround time for decision making purposes. The proposed methodology is based on capillary zone electrophoresis (CZE) and synchronous fluorescence spectroscopy (SFS). Metabolites extraction and pre-concentration is achieved with optimized SPE, LLE and/or QuEChERS (quick, easy, cheap, effective, rugged and safe) procedures. The small sample and extracting solvent volumes facilitate the simultaneous extraction of numerous samples via an environmentally friendly procedure, which is well-suited for routine monitoring of numerous samples. Sample stacking is successfully implemented to improve CZE limits of detection by two orders of magnitude. The unique electrophoretic pattern of positional isomers of OH-PAH demonstrates the potential of CZE for v the unambiguous determination of metabolites with similar chromatographic behaviors and virtually similar fragmentation patterns. The direct determination of OH-PAH without chromatographic separation is demonstrated via SFS. The non-destructive nature of SFS provides ample opportunity for further metabolite confirmation via chromatographic techniques

In the search for the conformation of extant or extinct life in an extraterrestrial setting the detection of organic molecular species which may be considered diagnostic of life is a key objective. These molecular targets comprise a range of distinct chemical species, with recognisable spectroscopic features. This project aims to use these features to develop an in-situ molecular specific Raman spectroscopic methodology which can provide structural information about the organic–inorganic interface. The development of this methodology identified a surface enhanced Raman spectroscopic technique, that required minimal sample preparation, allowed for the detection of selected organic species immobilised on an inorganic matrix and was effective for quantities below those which conventional dispersive Raman spectroscopy would detect. For the first time spectral information was gained which allowed analysis of the organic–inorganic interface to be carried out, this gave an insight into the orientation with which molecules arrange on the surfaces of the matrices. Additionally a method for the detection of organic residues intercalated into the interlamellar space of smectite type clays was developed. An evaluation of the effectiveness of uni and multivariate methods for the analysis of large datasets containing a small number of organic features was also carried out, with a view to develop an unsupervised methodology capable of performing with minimal user interaction. It has been shown that a novel use of the Hotellings T2 test when applied to the principal component analysis of the datasets combined with SERS allows identification of a small number of organic features in an otherwise inorganic dominated dataset. Both the SERS and PCA methods hold relevance for the detection of organic residues within interplanetary exploration but may also be applied to terrestrial environmental chemistry.

<p> Coal tar is a rich source of polycyclic aromatic compounds (PAC) which include hydrocarbons (PAH), sulfur-containing aromatics (PASH), nitrogen-containing aromatics and high molecular mass PAH. The separation of coal tar into four fractions was carried out on activated (170°C, 48 hrs) neutral alumina. Low mass aromatics and aliphatics were eluted with hexane in fraction A1 (2.2% recovery), while PAH/PASH were eluted by benzene in fraction A2 (25%). High molecular mass PAH were eluted by dichloromethane in fraction A3 (5.5%) and nitrogen-containing aromatics were eluted by methanol in fraction A4 (21%). These fractions were characterized by normal phase liquid chromatography, GCMS, probe mass spectrometry (for fractions A3 and A4) and LC-MS (for fraction A3).</p> <p> While ratio of PASH to PAH in fraction A2 was rather unfavorable (25:1), the separation of PASH from PAH was attempted using two methods: an oxidation/reduction method and a ligand exchange method. The oxidation/reduction method of Lee was a complete failure. The PdCl2-silica method of Nishioka was partially successful. Of the 25 thia-arenes identified in fraction A2, 12 eluted with the PAH while the other 13 were either partially (5) or fully (8) retained by the PdCl2-silica gel column and then eluted later to give a sulfur-enriched fraction. This fraction was further separated using normal phase HPLC to afford sulfur-containing compounds with molecular masses between 184 amu and 258 amu which exhibited almost no contamination due to PAH. Thus, it would be possible to purify about one-half of the PASH from a complex mixture such as coal tar.</p> <p> Fraction A3 which contained high mass PAH was subjected to semi-preparative normal phase HPLC to afford several high molecular mass PAH fractions. The 326 amu PAH fraction was selected because it contributed to 10% of the genotoxic of the mixture. This fraction was further separated by normal phase HPLC and the subfractions were analyzed by reversed-phase HPLC with diode-array detection. Thirty-five peaks were collected from the reversed-phase HPLC analysis and were characterized by probe mass spectrometry and fluorescence spectroscopy (in part). Of the 35 peaks, 8 peaks were eluted in the mutagenic active range; these eight compounds will be evaluated using the Ames assay to determine which are active mutagens.</p> / Thesis / Master of Science (MSc)