京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22378号 / 医科博第108号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 羽賀 博典, 教授 松村 由美 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Blood is considered one of the most important types of forensic evidence found at a crime scene. The use of surface-enhanced Raman spectroscopy (SERS) provides a potentially non-destructive and highly sensitive technique for the confirmation of blood and this method can be applied using a portable Raman device with quick sample preparation and processing. Crime scenes are inherently complex and the impact of SERS analysis provides easy use and practical application for in-field sample analysis. SERS is one of the few confirmatory techniques employed for the identification of blood at a crime scene or in the forensic laboratory. This method is able to distinguish between blood and other body fluids by collecting a SERS spectrum from a sample placed on a surface that has been embedded with gold nanoparticles (AuNPs). The AuNPs create an electric field surface enhancement that produces an intense molecular vibrational signal, leading to a SERS enhancement. The SERS enhancement allowed for sensitive blood detection at dilutions greater than 1:10,000. A stain transfer method to the SERS substrate was optimized by extracting dried bloodstains with water, saline, and various acid solutions. Fifty percent aqueous acetic acid solutions was found to be the most efficient in retaining the blood components and releasing the hemoglobin component of blood for detection. The SERS spectrum of blood is a robust signature of hemoglobin that does not significantly change between donors nor over time. Characteristic peaks for the identification of blood are 754, 1513, and 1543 wavenumbers (cm-1), attributed to a pyrrole ring breathing mode (15) and two Cβ-Cβ stretches (11, 38), respectively. These key SERS peaks, high sensitivity, and signal enhancement are favorable when compared to normal Raman spectroscopy. A quick and easy-to-use procedure for on-site sample analysis for the detection of blood on different substrates was developed and applied on a portable Raman device. Various nonporous and porous substrates including glass, ceramic tile, cotton, denim, fleece, nylon, acetate, wool, polyester, wood, and coated wood yielded strong results for identification of bloodstains. In addition, different commercial and in-house SERS substrates were tested to determine effectiveness for the detection and identification of blood. SERS identification of blood for forensic work is a potentially non-destructive and portable tool that can be applied for quick and easy examination of evidence at a crime scene. The high sensitivity and selectivity of SERS provides a robust spectroscopic signature that aids in the confirmation of blood, even when it is not visible to the naked eye. It is a more favorable method when compared to current presumptive and confirmatory tests for blood and can be applied to stains on different SERS substrates and a variety sample surfaces for universal testing.

Conventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Furthermore, for some frequently encountered body fluids, such as saliva or vaginal secretions, no confirmatory technique exists. Terminally differentiated cells, such as blood lymphocytes or epithelial cells lining the oral cavity, have a unique pattern of gene expression, which is evinced by the presence and relative abundance of specific mRNA species. If the type and abundance of mRNAs can be determined in a stain or tissue sample recovered at the crime scene, it would be possible to definitively identify the tissue or body fluid in question. Advantages of an mRNA-based approach, compared to conventional biochemical analysis, include greater specificity, simultaneous and semi-automated analysis though a common assay format, improved timeliness, decreased sample consumption and compatibility with DNA extraction methodologies. In this report, we demonstrate that RNA is stable in biological stains and can be recovered in sufficient quantity and quality for analysis using reverse transcriptasepolymerase chain reaction assay (RT-PCR). We have identified sets of candidate tissuespecific genes for body fluids and tissues of forensic interest, namely blood, saliva, semen, vaginal secretions, menstrual blood, urine, skin, muscle, adipose, and brain. We also report the identification of a new housekeeping gene for use in mRNA based assays. Select body fluid-specific genes have been incorporated into multiplex PCR and real-time PCR assays. These assays allow for the positive identification of blood, saliva, semen,vaginal secretions, and/or menstrual blood in a stain. The final task of this work was the molecular characterization of mRNA degradation patterns in biological stains, which not only has fundamental importance in possibly revealing mRNA degradation pathways in dried biological stains, but may ultimately lead to better assay design strategies for mRNA markers for forensic use. An mRNA-based approach described in this report could allow the facile identification of the tissue components present in a body fluid stain and could conceivably supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.

<p> Subcutaneous injection of formalin produced acute sodium deficiency in rats, characterized by marked hypovolemia and hyponatremia, due to an extravascular leakage of plasma and destruction of cells at the injection site. This reduction in intravascular fluid volume elicited both behavioral and physiological mechanisms of fluid restoration: sodium appetite and thirst as well as renal retention of sodium and water. Appetite and retention evolved together but intakes continued well after retention ceased and plasma volume and sodium concentration were restored to normal. These results indicate that appetite alone is not a true indicator or need, and that sodium and water balances (intake - excretion) must be considered in defining the deficient state.</p> / Thesis / Master of Arts (MA)

The overall goal of this study was to identify mineralized scaffolds which can serve as potential alternatives to bone graft substitutes intended for cleft palate repair. The aim of this preliminary study was to evaluate the role of fibrinogen (Fg) and nano-hydroxyapatite (nHA) in enhancing mineralization potential of polydioxanone (PDO) electrospun scaffolds. Scaffolds were fabricated by blending PDO:nHA:Fg in the following weight ratios: 100:0:0, 50:25:25, 50:50:0, 50:0:50, 0:0:100 and 0:50:50. Scaffolds were immersed in different simulated body fluids for 5 and 14 days to induce mineralization. The inclusion of fibrinogen induced sheet-like mineralization while individual fiber mineralization was noticed in its absence. Modified protocols of alizarin red staining and burn-out test were developed to quantify mineral content of scaffolds. After mineralization, 50:50:0 scaffolds were still porous and contained the most mineral. 50:25:25 scaffolds had the highest mineralization potential but lacked porosity. Therefore, it can be anticipated that these mineralized organic-inorganic electrospun scaffolds will induce bone formation.

Prostate cancer is the second-most diagnosed and fatal carcinoma for males in the United States, and better diagnostic markers and potential therapies are needed. microRNAs are small, single-stranded RNA molecules that affect protein expression at the translational level, and dysregulation can dramatically affect cell metabolism. Comparison of 736 microRNA expression levels between the poorly metastatic SV40T immortalized prostate epithelial cell line P69 to its highly tumorigenic and metastatic subline M12 identified 231 miRs that were overexpressed and 150 miRs that showed loss of expression in the M12 cell line. Further evaluation of fourteen identified miRs was accomplished using other prostate cell lines as well as laser-capture microdissected prostate samples. Inhibition of miR-147b was found to affect proliferative, migratory and invasive capabilities of M12 cells, and reduced tumour growth in nude athymic mice. AATF, an activator of the cell-cycle inhibitor p21, was identified as a target. Overexpression of miR-9 was found to affect the epithelial to mesenchymal transition through suppression of e-cadherin, a protein characterized as lost in EMT, as well as suppression of SOCS5, an attenuator of JAK-STAT signaling. Inhibition of miR-9 resulted in reduction of migratory and invasive potential, and significant reduction of tumorigenesis and metastases in male nude athymic mice. miR-17-3p was previously identified as down-regulated in prostate cancer and loss of miR-17-3p shown to cause vimentin transcriptional activation. Reverse phase microarray analysis (RPMA) identified c-KIT as a potential second mRNA target for miR-17-3p. miR-17-3p was shown to modulate not only protein levels, but also messenger RNA levels of c-KIT. Four miR-17-3p binding sites in the c-KIT mRNA were identified. Thus, a number of microRNAs involved in prostate cancer were identified, and their targets found to be highly relevant to tumour progression and could potentially be used as targets for therapy or diagnostics. Stability of microRNAs in forensically relevant biological fluids was evaluated through heat treatment, ultraviolet radiation, and chemical treatment. The dried body fluids showed some susceptibility to harsh treatment, but in most cases microRNAs were still detectable in the samples. microRNAs could represent a highly stable species for body fluid identification methods in forensic science.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-09-21T17:41:19Z No. of bitstreams: 1 LuanaAlves.pdf: 1633592 bytes, checksum: 785a3f9aa7f3fc08d80023603f457b39 (MD5) / Made available in DSpace on 2017-09-21T17:41:19Z (GMT). No. of bitstreams: 1 LuanaAlves.pdf: 1633592 bytes, checksum: 785a3f9aa7f3fc08d80023603f457b39 (MD5) Previous issue date: 2017-07-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão / Bioactive borate glass has presented superior results then bioactive silicate glasses, when compared its conversion rates in apatite and its potential of bioactivity. In this work, new Borate glasses with the basic 60B2O3 - 4P2O5 - 18Na2O – xCaF2 – (18-xCaO), com x = 0, 5 e 10 % wt were synthesized, and investigated the influence of CaF2 addition on bioactivity of samples in vitro, in a period of 28 days in a simulator of the body fluid (SBF). This bioactivity was investigated by means of X-ray diffraction (DRX), Raman Spectroscopy, FTIR and by the measure of pH. DRX measures, prior to immersion, presented wide bands, evidencing the amorphous structure of glasses. The results of density, thermal analysis, XRD, Raman and FTIR show that the addition of CaF2, until 10 % wt, did not cause significant changes in samples network structure. The thermal stability for all samples was calculated from DSC data and all presented values up to 120°C. The results of pH of SBF solution show increase from for 24 hours, which contributes to the dissolution of the outermost layer of the glass and the precipitation of apatite. In XRD data for 60B sample soaked in SBF for 7 days, there was formation of crystalline peaks, at 26° and 32° (2ϴ), in all measures. These peaks correspond to patterns of hydroxyapatite (HA). The XRD spectra in 60B5CaF and 60B10CaF, soaked in SBF for 7 days, presented peaks in 28°(2ϴ) corresponding to fluorapatite (FA), due to the presence of CaF2 in these glasses. Raman and FTIR measurements confirmed what was measured by XRD, showing characteristic peaks of HA and FA for all the samples. Results show that the prepared samples present potential for being used as biomaterials in biomedical applications, such as orthopedics, dentistry and tissue engineering. / Vidros boratos bioativos têm apresentado resultados superiores aos vidros silicatos bioativos, quando comparamos suas taxas de conversão em apatita e seu potencial de bioatividade. Neste trabalho sintetizamos novos vidros boratos com a composição básica 60B2O3 - 4P2O5 - 18Na2O – xCaF2 – (18-xCaO), com x = 0, 5 e 10 % em massa, e investigamos a influência da adição de CaF2 na bioatividade das amostras in vitro, em um período de 28 dias, em um simulador do fluído corporal (SBF). As medidas de DRX, antes da imersão, apresentaram bandas largas, comprovando a estrutura amorfa dos vidros. E verificou-se por meio das análises de Densidade, análise térmica, DRX, Raman e FTIR, que a adição de CaF2 até 10%, em massa, não provocou grandes mudanças estruturais na amostras. Por meio da calorimetria exploratória diferencial, determinamos a estabilidade térmica dos vidros que apresentaram valores acima de 120°C. A bioatividade foi investigada por meio das técnicas de difração de raios-X (DRX), espectroscopia Raman e FTIR, e pela medida do pH da solução SBF. Os resultados obtidos mostraram que, em 24h, houve um aumento no pH da solução SBF, o que contribui para a dissolução da camada mais externa do vidro e sua conversão em apatita. Nos difratogramas (DRX) para as amostras imersas por 7 dias, houve a formação de picos cristalinos, em 26° e 32° (2ϴ), em todas as amostras medidas. Esses picos correspondem aos padrões de hidroxiapatita. A presença de CaF2 na composição das amostras imersas por 7 dias apresentou picos em 28° (2ϴ) correspondendo a fluorapatita. A intensidade destes picos apresentou um aumento em função do tempo de imersão, durante todo período estudado. As medidas de Raman confirmaram os resultados do DRX, apresentando para todas as amostras espectros característicos da hidroxiapatita em 960 cm-1 . As amostras preparadas com CaF2 apresentaram picos em 965 cm-1 , que corresponde a fluorapatita. O FTIR confirmou os resultados apresentados no DRX e Raman, em que todas as amostras apresentaram um pico centrado em 1041 cm-1 para a hidroxiapatita e 1042 cm-1 característico para a fluorapatita. Os resultados demonstram que as amostras preparadas apresentam potencial para serem usados como biomateriais em aplicações biomédicas, como ortopedia, odontologia e engenharia de tecidos.

Apatite was formed on the surface of titanium dioxide (TiO2) powders by a biomimetic process. The deposition was accomplished by immersing TiO2 powders in simulated body fluid (SBF) for 1, 3, 6, 12, and 24 h. SBF used throughout this study had calcium and phosphate ion concentrations 10 times greater than those of human blood plasma. Photocatalytic activity of the apatite-deposited TiO2 powders was investigated in terms of the decomposition of methylene blue solution under ultraviolet (UV) irradiation. It has been shown that apatite deposition enhanced the photocatalytic activity of TiO2. The best photocatalytic performance was acquired on the powders that are immersed in SBF for 3 h. The time required for the complete degradation of methylene blue decreased from 3.5 h to 2 h upon immersion of powders in SBF for 3 h. Photochemical durability of poly(methyl methacrylate) increased when it was mixed with apatite-deposited TiO2 powders.

<p>This study has been triggered by the fact that stainless steel is being increasingly used in new applications, where possible environmental effects may be a matter of concern. When stainless steel is exposed to a given environment, a key issue is the release of small amounts of the main alloying elements iron, chromium, nickel and molybdenum. Published release rate data of these elements turned out to be sparse. Furthermore, only little was known about the role of different parameters that may affect the release rate, such as degree of alloying, exposure time and surface finish. Hence, the aim of this study was to develop methodological means and to provide accurate metal release rates of alloying constituents from different grades of stainless steels- austenitic, ferritic and duplex- when exposed to selected environments: artificial rain and synthetic body fluids. The results and discussion have been summarised in this thesis by formulating and answering ten questions, all believed to be crucial for the understanding of possible environmental effects of stainless steels.</p><p>Some common conclusions could be drawn, independent of stainless steel grade and exposure condition. Iron was always preferentially released, and the release rates of chromium, nickel and molybdenum (when measured) were significantly lower than of iron, also when considering the bulk proportion of these elements. The release rate of all elements was initially high and decreased with exposure time, mainly because of an observed enrichment of chromium in the passive film formed.</p><p>The release rates of iron (2 μgcm^-2week^-1) and nickel (0.08 μgcm-²week-¹) from stainless steel from grades 304 and 316 exposed to artificial rain were much lower than corresponding rates for the pure metals (750 μgcm-²week^-1 released Fe and 15 μgcm^-2week^-1 released Ni), whereas chromium exhibited similar release rates from stainless steel and the pure metal (0.1 μgcm^-2week^-1). This implies that the common procedure to calculate release rates, based on the pure metals and the nominal steel composition, significantly overestimates release rates of iron and nickel from stainless steel, but not of chromium.</p><p>Total release rates from seven stainless steel grades in synthetic body fluid were found to decrease with increasing alloy content in the following release rate order: grade 409 >> grade 430 > grades 316L ≈ 201 ≈ 2205 ≈ 304 > grade 310. The release rate was highly sensitive to pH of the synthetic body fluid but only slightly sensitive to stainless steel surface finish.</p>

This work extends a previously developed finite-volume overset-grid fluid flow solver to enable the characterisation of rigid-body-fluid interaction problems. To this end, several essential components have been developed and blended together. The inherent time-dependent nature of fluid-solid interaction problems is captured through the laminar transient incompressible Navier-Stokes equations for the fluid, and the Euler-Newton equations for rigid-body motion. First and second order accurate time discretisation schemes have been implemented for the former, whereas second and third order accurate time discretisation schemes have been made available for the latter. Without doubt the main advantage the overset-grid method offers regarding moving entities is the avoidance of the time consuming grid regeneration step, and the resulting grid distortion that can often cause numerical stability problems in the solution of the flow equations. Instead, body movement is achieved by the relative motion of a body fitted grid over a suitable background mesh. In this case, the governing equations of fluid flow are formulated using a Lagrangian, Eulerian, or hybrid flow description via the Arbitrary Lagrangian-Eulerian method. This entails the need to guarantee that mesh motion shall not disturb the flow field. With this in mind, the space conservation law has been hard-coded. The compliance of the space conservation law has the added benefit of preventing spurious mass sources from appearing due to mesh deformation. In this work, two-way fluid-solid interaction problems are solved via a partitioned approach. Coupling is achieved by implementing a Picard iteration algorithm. This allows for flexible degree of coupling specificationby the user. Furthermore, if strong coupling is desired, three variants of interface under-relaxation can be chosen to mitigate stability issues and to accelerate convergence. These include fixed, or two variants of Aitken’s adaptive under-relaxation factors. The software also allows to solve for one-way fluid-solid interaction problems in which the motion of the solid is prescribed. Verification of the core individual components of the software is carried out through the powerful method of manufactured solutions (MMS). This purely mathematically based exercise provides a picture of the order of accuracy of the implementation, and serves as a filter for coding errors which can be virtually impossible to detect by other means. Three instances of one-way fluid-solid interaction cases are compared with simulation results either from the literature, or from the OpenFOAM package. These include: flow within a piston cylinder assembly, flow induced by two oscillating cylinders, and flow induced by two rectangular plates exhibiting general planar motion. Three cases pertaining to the class of two-way fluid-interaction problems are presented. The flow generated by the free fall of a cylinder under the action of gravity is computed with the aid of an intermediate ‘motion tracking’ grid. The solution is compared with the one obtained using a vorticity based particle solver for validation purposes. Transverse vortex induced vibrations (VIV) of a circular cylinder immersed in a fluid, and subject to a stream are compared with experimental data. Finally, the fluttering motion of a rectangular plate under different scenarios is analysed.