京都大学 / 新制・課程博士 / 博士(医学) / 甲第23769号 / 医博第4815号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中本 裕士, 教授 大森 孝一, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Conventional charged-particle imaging techniques--such as autoradiography--provide only two-dimensional (2D) images of thin tissue slices. To get volumetric information, images of multiple thin slices are stacked. This process is time consuming and prone to distortions, as registration of 2D images is required. We propose a direct three-dimensional (3D) autoradiography technique, which we call charged-particle emission tomography (CPET). This 3D imaging technique enables imaging of thick sections, thus increasing laboratory throughput and eliminating distortions due to registration. In CPET, molecules or cells of interest are labeled so that they emit charged particles without significant alteration of their biological function. Therefore, by imaging the source of the charged particles, one can gain information about the distribution of the molecules or cells of interest. Two special case of CPET include beta emission tomography (BET) and alpha emission tomography (𝛼ET), where the charged particles employed are fast electrons and alpha particles, respectively. A crucial component of CPET is the charged-particle detector. Conventional charged-particle detectors are sensitive only to the 2-D positions of the detected particles. We propose a new detector concept, which we call particle-processing detector (PPD). A PPD measures attributes of each detected particle, including location, direction of propagation, and/or the energy deposited in the detector. Reconstruction algorithms for CPET are developed, and reconstruction results from simulated data are presented for both BET and 𝛼ET. The results show that, in addition to position, direction and energy provide valuable information for 3D reconstruction of CPET. Several designs of particle-processing detectors are described. Experimental results for one detector are discussed. With appropriate detector design and careful data analysis, it is possible to measure direction and energy, as well as position of each detected particle. The null functions of CPET with PPDs that measure different combinations of attributes are calculated through singular-value decomposition. In general, the more particle attributes are measured from each detection event, the smaller the null space of CPET is. In other words, the higher dimension the data space is, the more information about an object can be recovered from CPET.

<p>In this thesis the focus is mainly on the development of segmentation techniques for human brain structures and of the visualisation of such structures. The images of the brain are both anatomical images (magnet resonance imaging (MRI) and autoradigraphy) and functional images that show blood flow (functional magnetic imaging (fMRI), positron emission tomography (PET), and single photon emission tomograpy (SPECT)). When working with anatomical images, the structures segmented are visible as different parts of the brain, e.g. the brain cortex, the hippocampus, or the amygdala. In functional images, the activity or the blood flow that be seen.</p><p>Grey-level morphology methods are used in the segmentations to make tissue types in the images more homogenous and minimise difficulties with connections to outside tissue. A method for automatic histogram thresholding is also used. Furthermore, there are binary operations such as logic operation between masks and binary morphology operations.</p><p>The visualisation of the segmented structures uses either surface rendering or volume rendering. For the visualisation of thin structures, surface rendering is the better choice since otherwise some voxels might be missed. It is possible to display activation from a functional image on the surface of a segmented cortex. </p><p>A new method for autoradiographic images has been developed, which integrates registration, background compensation, and automatic thresholding to getfaster and more realible results than the standard techniques give.</p>

<p>The nasal administration of drugs offers advantages over administration by intravenous injection. Drugs can be rapidly absorbed through the nasal mucosa, resulting in a rapid onset of action, and also avoiding degradation in the gastrointestinal tract and first-pass metabolism in the liver. The olfactory receptor cells, which are in direct contact with both the environment and the central nervous system (CNS), are potential routes for drugs into the CNS. The olfactory pathway thus circumvents the blood brain barrier (BBB) which prevents many systemically administered drugs from entering the brain.</p><p>The studies used compounds active in the CNS and the experiments were performed in rodents. The nasal bioavailability of (S)-UH-301, NXX-066 and [³H]-dopamine was investigated in a rat model; uptake into the cerebrospinal fluid (CSF) was compared after nasal and intravenous administration. The concentrations of S-UH-301 and NXX-066 in plasma and CSF were measured with high performance liquid chromatography. The possible transfer of dopamine and neurotensin along the olfactory pathway after nasal administration to mice was studied using brain tissue sampling and autoradiography. The radioactivity content in blood, CSF and dissected brain tissue samples after administration of [³H]-dopamine and [³H]-neurotensin was assessed using liquid scintillation, and thin layer chromatography (TLC) was used to investigate the metabolic fate of [³H]-dopamine.</p><p>The results of this thesis suggest that nasal administration of CNS-active compounds with low oral bioavailability is an interesting and workable alternative to intravenous injection. The small lipophilic compounds (S)-UH-301 and NXX-066 were rapidly and completely absorbed after nasal administration, although hard evidence of direct transfer from the nose remains elusive. Radioactivity measurements in the olfactory bulb following nasal administration of</p><p>[³H]-dopamine and [³H]-neurotensin indicate that transfer occurred. The TLC results showed the presence of unchanged dopamine in the olfactory bulb but it is less clear from initial results with neurotensin, which radioactive products of this molecule reached the olfactory bulb, and further studies are required.</p>

The nasal administration of drugs offers advantages over administration by intravenous injection. Drugs can be rapidly absorbed through the nasal mucosa, resulting in a rapid onset of action, and also avoiding degradation in the gastrointestinal tract and first-pass metabolism in the liver. The olfactory receptor cells, which are in direct contact with both the environment and the central nervous system (CNS), are potential routes for drugs into the CNS. The olfactory pathway thus circumvents the blood brain barrier (BBB) which prevents many systemically administered drugs from entering the brain. The studies used compounds active in the CNS and the experiments were performed in rodents. The nasal bioavailability of (S)-UH-301, NXX-066 and [3H]-dopamine was investigated in a rat model; uptake into the cerebrospinal fluid (CSF) was compared after nasal and intravenous administration. The concentrations of S-UH-301 and NXX-066 in plasma and CSF were measured with high performance liquid chromatography. The possible transfer of dopamine and neurotensin along the olfactory pathway after nasal administration to mice was studied using brain tissue sampling and autoradiography. The radioactivity content in blood, CSF and dissected brain tissue samples after administration of [3H]-dopamine and [3H]-neurotensin was assessed using liquid scintillation, and thin layer chromatography (TLC) was used to investigate the metabolic fate of [3H]-dopamine. The results of this thesis suggest that nasal administration of CNS-active compounds with low oral bioavailability is an interesting and workable alternative to intravenous injection. The small lipophilic compounds (S)-UH-301 and NXX-066 were rapidly and completely absorbed after nasal administration, although hard evidence of direct transfer from the nose remains elusive. Radioactivity measurements in the olfactory bulb following nasal administration of [3H]-dopamine and [3H]-neurotensin indicate that transfer occurred. The TLC results showed the presence of unchanged dopamine in the olfactory bulb but it is less clear from initial results with neurotensin, which radioactive products of this molecule reached the olfactory bulb, and further studies are required.

In this thesis the focus is mainly on the development of segmentation techniques for human brain structures and of the visualisation of such structures. The images of the brain are both anatomical images (magnet resonance imaging (MRI) and autoradigraphy) and functional images that show blood flow (functional magnetic imaging (fMRI), positron emission tomography (PET), and single photon emission tomograpy (SPECT)). When working with anatomical images, the structures segmented are visible as different parts of the brain, e.g. the brain cortex, the hippocampus, or the amygdala. In functional images, the activity or the blood flow that be seen. Grey-level morphology methods are used in the segmentations to make tissue types in the images more homogenous and minimise difficulties with connections to outside tissue. A method for automatic histogram thresholding is also used. Furthermore, there are binary operations such as logic operation between masks and binary morphology operations. The visualisation of the segmented structures uses either surface rendering or volume rendering. For the visualisation of thin structures, surface rendering is the better choice since otherwise some voxels might be missed. It is possible to display activation from a functional image on the surface of a segmented cortex. A new method for autoradiographic images has been developed, which integrates registration, background compensation, and automatic thresholding to getfaster and more realible results than the standard techniques give.

AbstractScientists trying to find biological controls for insect pests are hamperedd by the absence of rapid methods for screening organisms such as bacteria for potential pathogenicity. An organism must grow in the gut of the insect to be pathogenic. By using radioisotopes as tracers a quick method of screening potential insect pathogens may be developed.Escherichia coli and Sarcina flava were used as known nonpathogens and Bacillus thuringiensis was used as a known pathogen. In this work an attempt was made to verify the presence of bacteria in specific tissues of the insects.European corn borer larvae were fed 1-C14 palmitic acid and labeled E. coli, S. flava and B. thuringiensis. Parasaggital sections were made of the corn borers and radioautograms were made of the sections. Grain counts over a 2,000 u2 area were made of various tissues and compared.There was a statistically significant difference in the distribution of label in corn borer larvae fed labeled bacteria as compared to corn borers fed 1-C14 palmitic acid. Label tended to incorporate into fatty tissue in the corn borers.If this technique can be used to positively demonstrate the establishment of selected bacteria in the gut of the insect then it may be possible to use such methods to screen for potential insect pathogens and give insight into the mechanisms which result in the death of insects.Ball State UniversityMuncie, IN 47306

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types.

Social conflict is a part of everyday life, and it can be a potent stressor for both humans and other animals. In the laboratory, when two Syrian hamsters (Mesocricetus auratus) compete for territory, a dominance hierarchy is quickly formed. Becoming subordinate is a significant stressor resulting in increased release of adrenocorticotropic hormone, β-endorphin, and cortisol. Defeated hamsters will also subsequently fail to display territorial aggression in future social encounters and will instead display increased submissive behavior, even in the presence of a smaller, non-aggressive intruder. This change in behavior is consistent and long-lasting and has been termed conditioned defeat (CD). Corticotropin releasing factor (CRF) is an important neuropeptide in the control of the hypothalamo-pituitary-adrenal (HPA) axis response to stress. It is also involved in a number of behaviors such as anxiety, stress responding, food intake, learning, and memory. The widespread distribution of CRF, CRF-like peptides, and CRF receptors, particularly in brain sites related to anxiety, fear, and stress responses, suggests a role for CRF and CRF-like peptides in modulating emotional responses other than via HPA axis activity. It has also been shown that CRF may have a role in the acquisition and expression of CD. Non-specific and CRF type 2-specific CRF antagonists reduce the acquisition and expression of CD in male hamsters while injection of a CRF type 1-specific antagonist does not. Therefore, the goal of this dissertation was to investigate the role of CRF type 1 and 2 receptors in CD in hamsters and to identify neuroanatomical locations where CRF may be acting. It was found that non-specific or CRF type 1 receptor specific agonists enhance the expression, but not acquisition, of CD. Further, these agonists appear to enhance aggressive behavior in animals that were not previously defeated, suggesting a modulatory role for CRF type 1 receptors in agonistic behavior that depends on an animal’s previous social experience. Further, localization of CRF receptors was determined in hamster brain in sites thought important for CD and agonistic behavior, but changes in receptor binding following defeat were not observed. Implications of these results and future directions are discussed.