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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20243号 / 医博第4202号 / 新制||医||1020(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 齊藤 博英, 教授 原田 浩, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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<p>Microorganisms are the most abundant living things on the planet and they drive many important environmental processes. They can do this by coupling reduction – oxidation (redox) reactions. In such reactions, the oxidation of reduced organic matter is coupled with the reduction of another compound, which serves as the electron acceptor. All microbes contain lipids in their cells; phospholipids are the main components of the cell membrane where they make up a consistent component of cell mass. Therefore, in situations where direct cell count is unrealistic, lipid analysis can be used to provide information on microbial communities. Because they hydrolyze shortly after cell death,PLFAs indicate only viable cell biomass, and PLFA analysis provides valuable insight on cell density distribution across a site. One application of PLFA analysis is within this thesis, where it was used to investigate the microbial community at Mildred Lake, Syncrude’s primary tailings settling basin. At Mildred Lake, Syncrude is constructing a freshwater fen over the deposited composite tailings (CT) as part of their reclamation process. Understanding the microbial biogeochemical cycling associated with these reclamation activities is an important component for management decisions affecting the site and thus, inform future reclamation activities.</p> <p>PLFA analysis on samples from the site showed variable concentrations equivalent to estimated cell densities on the order of 107 decreasing to 106 in the CT.These cell density ranges are expected for oligotrophic systems. Phospholipids can also be biomarkers if they are indicative of a specific group of microbes. The study at Mildred Lake identified biomarkers for sulfate reducing bacteria (SRBs). The presence of these biomarkers provided a basis for the hypothesis that sulfide detected at the site was potentially from SRBs.</p> <p>This thesis provides information on the fundamental concepts of lipids and the application of lipid analysis on the environmental samples from the Mildred Lake site to understand its microbial community and cycling of sulfur to prevent potential environmental issues associated with the generation of sulfide.</p> / Master of Science (MSc)

Syngas fermentation via gasification is a two-stage process, which contains gasification of feedstock into syngas and syngas bio-methanation by anaerobic microorganisms. This project is a study on syngas fermentation. The gasification feedstock can be difficult-to-degrade solid waste so; waste volumes are reduced while green energy is produced. The main target of this thesis was to study novel configurations of reverse membrane bioreactor (RMB) in order to retain microbial cells inside the digester and thereafter increase methane production. In the first experiment, microbial cells encased in PVDF sachets were proved to perform efficiently in batch mode in comparison to free cells at optimum temperature, 55 oC. Moreover, encased cells in co-digestion of syngas and organic waste exhibited higher methane amounts compared to pure syngas treatment. Encased cells were then tested in thermophilic semi-continuous process and showed better performance compared to the free cell reactor. The RMB containing encased cells retained successfully the cells during the 154 days of the experiment, while free cells were washed-out. The highest amounts of methane from RMB and the free cell reactor were produced during the 126th - 130th day (6 and 1.5 mmol/day, respectively). In the last experiment, a RMB containing 13 membrane layers of enclosed cells was studied and compared to a conventional reactor of free cells. The RMB performed successfully in syngas bio-methanation under semi-continuous conditions during 49 days. The highest methane amount produced was 10 mmol/day in both RMB and free cell reactor. / Program: Industriell bioteknik

Lactococci are widely used in the cheese industry as a starter culture. Starter cultures face carbohydrate starvation due to the absence of a fermentable carbohydrate in the cheese curd after pressing. Starvation leads to a decreased ability to synthesize ATP, generate a proton motive force, and accumulate nutrients necessary to maintain viability. The aim of this work was to investigate the culturability of lactococci grown with and without lactose in a chemically defined medium, and to define the metabolic changes that occur during carbohydrate starvation. Lactose metabolism provided energy for logarithmic phase growth and greater cell density in L. lactis ssp. lactis ML3 and L. lactis ssp. cremoris S2. However, the rate of lactose metabolism was strain dependent in that L. lactis ssp. lactis 11454 did not metabolize lactose as rapidly as did ML3 and S2. In the absence of lactose the cells became nonculturable on agar. In addition to becoming nonculturable, the aminopeptidase and lipase/ esterase activity became nonmeasurable after 21 d, and cellular metabolism was altered because of carbohydrate starvation. Nevertheless, the cells remained viable for up to 42 d in spent media as measured by fluorescent viability stains and intracellular ATP content. Fluorescent viability staining demonstrated that the cells maintained an intact cell membrane to contain their DNA, as well as to contain enzymes and ATP necessary to maintain viability and metabolic activity. With the addition of arginine to the basal medium, the survival time, cell number, and ATP concentration increased. Amino acids, including arginine, provided energy after carbohydrate exhaustion. At the onset of lactose exhaustion, the extracellular concentrations of arginine, glycine/valine, glutamate, and glutamine decreased in the media when energy was present for their transport. There was a significant increase in serine and methionine concentrations in the spent media over the same time period. These data indicated lactococci remained viable and metabolically active, but were nonculturable in response to carbohydrate starvation. Additionally, amino acids are in a dynamic state during carbohydrate starvation, and utilization of amino acids, such as arginine and serine, could facilitate lactococcal cells in maintaining viability in harsh environments such as ripening cheese.

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The large size of the datasets produced by medical imaging protocols contributes to the success of supervised discriminative methods for semantic labelling of images. Our study makes use of a general and efficient emerging framework, discriminative random forests, for the detection of brain lesions in multi-modal magnetic resonance images (MRIs). The contribution is three-fold. First, we focus on segmentation of brain lesions which is an essential task to diagnosis, prognosis and therapy planning. A context-aware random forest is designed for the automatic multi-class segmentation of MS lesions, low grade and high grade gliomas in MR images. It uses multi-channel MRIs, prior knowledge on tissue classes, symmetrical and long-range spatial context to discriminate lesions from background. Then, we investigate the promising perspective of estimating the brain tumor cell density from MRIs. A generative-discriminative framework is presented to learn the latent and clinically unavailable tumor cell density from model-based estimations associated with synthetic MRIs. The generative model is a validated and publicly available biophysiological tumor growth simulator. The discriminative model builds on multi-variate regression random forests to estimate the voxel-wise distribution of tumor cell density from input MRIs. Finally, we present the "Spatially Adaptive Random Forests" which merge the benefits of multi-scale and random forest methods and apply it to previously cited classification and regression settings. Quantitative evaluation of the proposed methods are carried out on publicly available labeled datasets and demonstrate state of the art performance.