データ同化手法を用いた多種生体内データの統合による生体内システム再構築の研究

京都大学0048新制・課程博士博士(情報学)甲第18699号情博第549号新制||情||97(附属図書館)31632京都大学大学院情報学研究科知能情報学専攻(主査)教授 阿久津 達也, 教授 鹿島 久嗣, 教授 石井 信学位規則第4条第1項該当Doctor of InformaticsKyoto UniversityDFA

Systems biotechnology of baculovirus-producing insect cells

Dissertation presented to obtain the Ph.D degree in Ciências da Engenharia e Tecnologia, especialidade Biotecnologiacritical step in the biopharmaceutical industry is the capacity to properly manufacture biological products in animal cell cultures. The ability to exploit the full potential of the underlying “cellular factories” is essential to reduce manufacturing costs and improve yield and product quality, thus freeing up resources to pursue other added value activities, such as clinical development and pharmacovigilance of new products. Bioprocess optimization efforts have mostly relied on empirical experience and rational, hypothesis-driven studies, rather than by fully leveraging the knowledge of how complex cells behave in culture. Despite the vast amount of information available on different cellular features, specific productivity improvements have been essentially incremental. The relatively recent developments in Systems Biology and computational methodologies present a paradigm-shifting opportunity to develop biotechnology processes with high efficiencies in a consistent, systematic way. (...

Advancing Knowledge on Cyanobacterial Blooms in Freshwaters

Cyanobacterial blooms are a water quality problem that is widely acknowledged to have detrimental ecological and economic effects in drinking and recreational water supplies and fisheries. There is increasing evidence that cyanobacterial blooms have increased globally and are likely to expand in water resources as a result of climate change. Of most concern are cyanotoxins, along with the mechanisms that induce their release and determine their fate in the aquatic environment. These secondary metabolites pose a potential hazard to human health and agricultural and aquaculture products that are intended for animal and human consumption; therefore, strict and reliable control of cyanotoxins is crucial for assessing risk. In this direction, a deeper understanding of the mechanisms that determine cyanobacterial bloom structure and toxin production has become the target of management practices. This Special Issue, entitled “Advancing Knowledge on Cyanobacterial Blooms in Freshwaters”, aims to bring together recent multi- and interdisciplinary research, from the field to the laboratory and back again, driven by working hypotheses based on any aspect of mitigating cyanobacterial blooms, from ecological theory to applied research

Pollution-induced community tolerance in freshwater biofilms – from molecular mechanisms to loss of community functions

Exposure to herbicides poses a threat to aquatic biofilms by affecting their community structure, physiology and function. These changes render biofilms to become more tolerant, but on the downside community tolerance has ecologic costs. A concept that addresses induced community tolerance to a pollutant (PICT) was introduced by Blanck and Wängberg (1988). The basic principle of the concept is that microbial communities undergo pollution-induced succession when exposed to a pollutant over a long period of time, which changes communities structurally and functionally and enhancing tolerance to the pollutant exposure. However, the mechanisms of tolerance and the ecologic consequences were hardly studied up to date. This thesis addresses the structural and functional changes in biofilm communities and applies modern molecular methods to unravel molecular tolerance mechanisms. Two different freshwater biofilm communities were cultivated for a period of five weeks, with one of the communities being contaminated with 4 μg L-1 diuron. Subsequently, the communities were characterized for structural and functional differences, especially focusing on their crucial role of photosynthesis. The community structure of the autotrophs was assessed using HPLC-based pigment analysis and their functional alterations were investigated using Imaging-PAM fluorometry to study photosynthesis and community oxygen profiling to determine net primary production. Then, the molecular fingerprints of the communities were measured with meta-transcriptomics (RNA-Seq) and GC-based community metabolomics approaches and analyzed with respect to changes in their molecular functions. The communities were acute exposed to diuron for one hour in a dose-response design, to reveal a potential PICT and uncover related adaptation to diuron exposure. The combination of apical and molecular methods in a dose-response design enabled the linkage of functional effects of diuron exposure and underlying molecular mechanisms based on a sensitivity analysis. Chronic exposure to diuron impaired freshwater biofilms in their biomass accrual. The contaminated communities particularly lost autotrophic biomass, reflected by the decrease in specific chlorophyll a content. This loss was associated with a change in the molecular fingerprint of the communities, which substantiates structural and physiological changes. The decline in autotrophic biomass could be due to a primary loss of sensitive autotrophic organisms caused by the selection of better adapted species in the course of chronic exposure. Related to this hypothesis, an increase in diuron tolerance has been detected in the contaminated communities and molecular mechanisms facilitating tolerance have been found. It was shown that genes of the photosystem, reductive-pentose phosphate cycle and arginine metabolism were differentially expressed among the communities and that an increased amount of potential antioxidant degradation products was found in the contaminated communities. This led to the hypothesis that contaminated communities may have adapted to oxidative stress, making them less sensitive to diuron exposure. Moreover, the photosynthetic light harvesting complex was altered and the photoprotective xanthophyll cycle was increased in the contaminated communities. Despite these adaptation strategies, the loss of autotrophic biomass has been shown to impair primary production. This impairment persisted even under repeated short-term exposure, so that the tolerance mechanisms cannot safeguard primary production as a key function in aquatic systems.:1. The effect of chemicals on organisms and their functions .............................. 1 1.1 Welcome to the anthropocene .......................................................................... 1 1.2 From cellular stress responses to ecosystem resilience ................................... 3 1.2.1 The individual pursuit for homeostasis ....................................................... 3 1.2.2 Stability from diversity ................................................................................. 5 1.3 Community ecotoxicology - a step forward in monitoring the effects of chemical pollution? ................................................................................................................. 6 1.4 Functional ecotoxicological assessment of microbial communities ................... 9 1.5 Molecular tools – the key to a mechanistic understanding of stressor effects from a functional perspective in microbial communities? ...................................... 12 2. Aims and Hypothesis ......................................................................................... 14 2.1 Research question .......................................................................................... 14 2.2 Hypothesis and outline .................................................................................... 15 2.3 Experimental approach & concept .................................................................. 16 2.3.1 Aquatic freshwater biofilms as model community ..................................... 16 2.3.2 Diuron as model herbicide ........................................................................ 17 2.3.3 Experimental design ................................................................................. 18 3. Structural and physiological changes in microbial communities after chronic exposure - PICT and altered functional capacity ................................................. 21 3.1 Introduction ..................................................................................................... 21 3.2 Methods .......................................................................................................... 23 3.2.1 Biofilm cultivation ...................................................................................... 23 3.2.2 Dry weight and autotrophic index ............................................................. 23 3.2.4 Pigment analysis of periphyton ................................................................. 23 3.2.4.1 In-vivo pigment analysis for community characterization ....................... 24 3.2.4.2 In-vivo pigment analysis based on Imaging-PAM fluorometry ............... 24 3.2.4.3 In-vivo pigment fluorescence for tolerance detection ............................. 26 3.2.4.4 Ex-vivo pigment analysis by high-pressure liquid-chromatography ....... 27 3.2.5 Community oxygen metabolism measurements ....................................... 28 3.3 Results and discussion ................................................................................... 29 3.3.1 Comparison of the structural community parameters ............................... 29 3.3.2 Photosynthetic activity and primary production of the communities after selection phase ................................................................................................. 33 3.3.3 Acquisition of photosynthetic tolerance .................................................... 34 3.3.4 Primary production at exposure conditions ............................................... 36 3.3.5 Tolerance detection in primary production ................................................ 37 3.4 Summary and Conclusion ........................................................................... 40 4. Community gene expression analysis by meta-transcriptomics ................... 41 4.1 Introduction to meta-transcriptomics ............................................................... 41 4.2. Methods ......................................................................................................... 43 4.2.1 Sampling and RNA extraction................................................................... 43 4.2.2 RNA sequencing analysis ......................................................................... 44 4.2.3 Data assembly and processing................................................................. 45 4.2.4 Prioritization of contigs and annotation ..................................................... 47 4.2.5 Sensitivity analysis of biological processes .............................................. 48 4.3 Results and discussion ................................................................................... 48 4.3.1 Characterization of the meta-transcriptomic fingerprints .......................... 49 4.3.2 Insights into community stress response mechanisms using trend analysis (DRomic’s) ......................................................................................................... 51 4.3.3 Response pattern in the isoform PS genes .............................................. 63 4.5 Summary and conclusion ................................................................................ 65 5. Community metabolome analysis ..................................................................... 66 5.1 Introduction to community metabolomics ........................................................ 66 5.2 Methods .......................................................................................................... 68 5.2.1 Sampling, metabolite extraction and derivatisation................................... 68 5.2.2 GC-TOF-MS analysis ............................................................................... 69 5.2.3 Data processing and statistical analysis ................................................... 69 5.3 Results and discussion ................................................................................... 70 5.3.1 Characterization of the metabolic fingerprints .......................................... 70 5.3.2 Difference in the metabolic fingerprints .................................................... 71 5.3.3 Differential metabolic responses of the communities to short-term exposure of diuron ............................................................................................................ 73 5.4 Summary and conclusion ................................................................................ 78 6. Synthesis ............................................................................................................. 79 6.1 Approaches and challenges for linking molecular data to functional measurements ...................................................................................................... 79 6.2 Methods .......................................................................................................... 83 6.2.1 Summary on the data ............................................................................... 83 6.2.2 Aggregation of molecular data to index values (TELI and MELI) .............. 83 6.2.3 Functional annotation of contigs and metabolites using KEGG ................ 83 6.3 Results and discussion ................................................................................... 85 6.3.1 Results of aggregation techniques ........................................................... 85 6.3.2 Sensitivity analysis of the different molecular approaches and endpoints 86 6.3.3 Mechanistic view of the molecular stress responses based on KEGG functions ............................................................................................................ 89 6.4 Consolidation of the results – holistic interpretation and discussion ............... 93 6.4.1 Adaptation to chronic diuron exposure - from molecular changes to community effects.............................................................................................. 93 6.4.2 Assessment of the ecological costs of Pollution-induced community tolerance based on primary production ............................................................. 94 6.5 Outlook ............................................................................................................ 9

Handbook of Marine Model Organisms in Experimental Biology

"The importance of molecular approaches for comparative biology and the rapid development of new molecular tools is unprecedented. The extraordinary molecular progress belies the need for understanding the development and basic biology of whole organisms. Vigorous international efforts to train the next-generation of experimental biologists must combine both levels – next generation molecular approaches and traditional organismal biology. This book provides cutting-edge chapters regarding the growing list of marine model organisms. Access to and practical advice on these model organisms have become aconditio sine qua non for a modern education of advanced undergraduate students, graduate students and postdocs working on marine model systems. Model organisms are not only tools they are also bridges between fields – from behavior, development and physiology to functional genomics. Key Features Offers deep insights into cutting-edge model system science Provides in-depth overviews of all prominent marine model organisms Illustrates challenging experimental approaches to model system research Serves as a reference book also for next-generation functional genomics applications Fills an urgent need for students Related Titles Jarret, R. L. & K. McCluskey, eds. The Biological Resources of Model Organisms (ISBN 978-1-1382-9461-5) Kim, S.-K. Healthcare Using Marine Organisms (ISBN 978-1-1382-9538-4) Mudher, A. & T. Newman, eds. Drosophila: A Toolbox for the Study of Neurodegenerative Disease (ISBN 978-0-4154-1185-1) Green, S. L. The Laboratory Xenopus sp. (ISBN 978-1-4200-9109-0)