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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23750号 / 医博第4796号 / 新制||医||1055(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 村川 泰裕, 教授 斎藤 通紀, 教授 藤渕 航 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In this study, we have used genetic, cell biological and transcriptomic methods in the nematode C. elegans as a model to examine the impact of glucose supplementation during development. We show that a glucose-supplemented diet slows the rate of developmental progression (termed "glucose-induced developmental delay" or GIDD) and induces the mitochondrial unfolded protein response (UPRmt) in wild-type animals. Mutation in the insulin receptor daf-2 confers resistance to GIDD and UPRmt in a daf-16-dependent manner. We hypothesized that daf-2(e1370) animals alter their metabolism to manage excess glucose. To test this, we used RNA-sequencing which revealed that the transcriptomic profiles of glucose-supplemented wildtype and daf-2(e1370) animals are distinct. From this, we identified a set of 27 genes which are both exclusively upregulated in daf-2(e1370) animals fed a glucose-supplemented diet and regulated by daf-16, including a fatty acid desaturase (fat-5), and two insulin-like peptides (ins-16 and ins-35). Mutation of any of these genes suppresses the resistance of daf-2(e1370) to GIDD. Additionally, double mutation of ins-16 and ins-35 in a daf-2(e1370) background results in an increase in constitutive dauer formation which is suppressed by glucose supplementation. Further investigation of the insulin-like peptides revealed that ins-16 mutation in a wild-type background results in upregulation of ins-35 and DAF-16 nuclear translocation regardless of diet; however, unlike daf-2(e1370), this translocation is not associated with resistance to GIDD. Taken together, these data suggest that glucose-supplemented daf-2(e1370) animals maintain developmental trajectory in part through upregulation of specific insulin-like peptide genes and fatty acid desaturation and contribute to a deeper understanding of the mechanisms underlying the resistance of daf-2(e1370) animals to GIDD. We also showed another fascinating aspect of GIDD: it becomes more pronounced in subsequent generations exposed to a glucose-supplemented diet, suggesting that the parental glucose diet has an impact on the developmental progression of their offspring.

A cell, the building block of all life, stores a plethora of information in its genome, epigenome, and transcriptome which needs to be analyzed via various Omic studies. The heterogeneity in a seemingly similar group of cells is an important factor to consider and it could lead us to better understand processes such as cancer development and resistance to treatment, fetal development, and immune response. There is an ever growing demand to be able to develop more sensitive, accurate and robust ways to study Omic information and to analyze subtle biological variation between samples even with limited starting material obtained from a single cell. Microfluidics has opened up new and exciting possibilities that have revolutionized how we study and manipulate the contents of the cell like the DNA, RNA, proteins, etc. Microfluidics in conjunction with Next Gen Sequencing has provided ground-breaking capabilities for handling small sample volumes and has also provided scope for automation and multiplexing. In this thesis, we discuss a number of platforms for developing low-input or single cell Omic technologies. The first part talks about the development of a novel microfluidic platform to carry out single-cell RNA-sequencing in a one-pot method with a diffusion-based reagent swapping scheme. This platform helps to overcome the limitations of conventional microfluidic RNA seq methods reported in literature that use complicated multiple-chambered devices. It also provides good quality data that is comparable to state-of-the-art scRNA-seq methods while implementing a simpler device design that permits multiplexing. The second part talks about studying the transcriptome of innate leukocytes treated with varying levels of LPS and using RNA-seq to observe how innate immune cells undergo epigenetic reprogramming to develop phenotypes of memory cells. The third part discusses a low-cost alternative to produce tn5 enzyme which low-cost NGS studies. And finally, we discuss a microfluidic approach to carrying out low-input epigenomic studies for studying transcription factors. Today, single-cell or low-input Omic studies are rapidly moving into the clinical setting to enable studies of patient samples for personalized medicine. Our approaches and platforms will no doubt be important for transcriptomic and epigenomic studies of scarce cell samples under such settings. / Doctor of Philosophy / This is the era of personalized medicine which means that we are no longer looking at one-size-fits-all therapies. We are rather focused on finding therapies that are tailormade to every individual’s personal needs. This has become more and more essential in the context of serious diseases like cancer where therapies have a lot of side-effects. To provide tailor-made therapy to patients, it is important to know how each patient is different from another. This difference can be found from studying how the individual is unique or different at the cellular level i.e. by looking into the contents of the cell like DNA, RNA, and chromatin. In this thesis, we discussed a number of projects which we can contribute to advancement in this field of personalized medicine. Our first project, MID-RNA-seq offers a new platform for studying the information contained in the RNA of a single cell. This platform has enough potential to be scaled up and automated into an excellent platform for studying the RNA of rare or limited patient samples. The second project discussed in this thesis involves studying the RNA of innate immune cells which defend our bodies against pathogens. The RNA data that we have unearthed in this project provides an immense scope for understanding innate immunity. This data provides our biologist collaborators the scope to test various pathways in innate immune cells and their roles in innate immune modulation. Our third project discusses a method to produce an enzyme called ‘Tn5’ which is necessary for studying the sequence of DNA. This enzyme which is commercially available has a very high cost associated with it but because we produced it in the lab, we were able to greatly reduce costs. The fourth project discussed involves the study of chromatin structure in cells and enables us to understand how our lifestyle choices change the expression or repression of genes in the cell, a study called epigenetics. The findings of this study would enable us to study epigenomic profiles from limited patient samples. Overall, our projects have enabled us to understand the information from cells especially when we have limited cell numbers. Once we have all this information we can compare how each patient is different from others. The future brings us closer to putting this into clinical practice and assigning different therapies to patients based on such data.

An accelerated pace of discovery in biological sciences is made possible by a new generation of computational biology and bioinformatics tools. In this dissertation we develop novel computational, analytical, and high performance simulation techniques for biological problems, with applications to the yeast cell division cycle, and to the RNA-Sequencing of the yellow fever mosquito. Cell cycle system evolves stochastic effects when there are a small number of molecules react each other. Consequently, the stochastic effects of the cell cycle are important, and the evolution of cells is best described statistically. Stochastic simulation algorithm (SSA), the standard stochastic method for chemical kinetics, is often slow because it accounts for every individual reaction event. This work develops a stochastic version of a deterministic cell cycle model, in order to capture the stochastic aspects of the evolution of the budding yeast wild-type and mutant strain cells. In order to efficiently run large ensembles to compute statistics of cell evolution, the dissertation investigates parallel simulation strategies, and presents a new probabilistic framework to analyze the performance of dynamic load balancing algorithms. This work also proposes new accelerated stochastic simulation algorithms based on a fully implicit approach and on stochastic Taylor expansions. Next Generation RNA-Sequencing, a high-throughput technology to sequence cDNA in order to get information about a sample's RNA content, is becoming an efficient genomic approach to uncover new genes and to study gene expression and alternative splicing. This dissertation develops efficient algorithms and strategies to find new genes in Aedes aegypti, which is the most important vector of dengue fever and yellow fever. We report the discovery of a large number of new gene transcripts, and the identification and characterization of genes that showed male-biased expression profiles. This basic information may open important avenues to control mosquito borne infectious diseases. / Ph. D.

Current sequencing technologies allows researchers unprecedented insight into our biology, and how these biological mechanisms can become distorted and lead to disease. These aberrant mechanisms can be brought about by many causes, but some occur as a result of genetic mutations or external factors through the epigenome. Here, we used our microfluidic technology to profile the epigenome and transcriptome to study such aberrant mechanisms in three different diseases and illnesses: breast cancer, chronic inflammation, and mental illness. We profiled the epigenome of breast tissue from healthy women with the BRCA1 mutation to understand how the mutation may facilitate eventual breast cancer. Epigenomic changes in breast cells suggest that cells in the basal compartment may differentiate into a different cell type, and perhaps become the source of breast cancer. Next, we compared the epigenome and genome of murine immune cells under low-grade inflammation and acute inflammation conditions. We found that low-grade inflammation preferentially utilizes different signaling pathways than in acute inflammation, and this may lead to a non-resolving state. Finally, we analyzed the effect of the maternal immune activation on unborn offspring, and how these changes could cause later mental illness. The insights we made into these diseases may lead to future therapies. / Doctor of Philosophy / Despite advances in medical and scientific research, there is still a dearth of information on how diseases affect the expression of our genes, such as breast cancer, chronic inflammation, and influenza. Mutation in the BRCA1 gene is probably the most well-known mutation that can lead to breast cancer. We know the overarching reason that mutation in BRCA1 can lead to cancer, as BRCA1 is responsible for repairing damage in the DNA, so mutations can compound and create cancerous cells. However, we do not know the exact mechanisms by which this actually happens. Another widespread problem is chronic inflammation, which can promote or lead to diseases such as diabetes, cancer, Alzheimer's, Rheumatoid arthritis, and heart disease. In addition, there are many causes of chronic inflammation that many people have experienced at some point in time, including stress, insomnia, being sedentary, poor eating habits, and obesity. Despite this, we still do not fully understand why chronic inflammation differs from normal inflammation, which is a healthy process, or why it does not resolve. There are also other connections that are surprising, and many are not aware of. If a pregnant woman gets the flu during her second trimester, her baby has much higher odds of developing schizophrenia later in its lifetime. Given the prevalence of the flu, there is a very real chance that an expecting mother will be infected during her pregnancy.

Malnutrition, including both undernutrition and obesity, affects millions of people globally and is persistently on the rise. Obesity affects ~13% of adults globally and was identified as a risk factor for worse disease outcomes after the H1N1 influenza pandemic of 2009 and has since been shown to aggravate disease outcomes of respiratory viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mosquito-borne viruses like West Nile Virus (WNV), chikungunya virus (CHIKV) and Mayaro virus (MAYV) and reduce the vaccine efficacy for influenza and SARS-CoV-2. Obesity is associated with a chronic state of inflammation and dysregulated immune response which has been proposed to be one of the mechanisms driving the severity of coronavirus disease 2019 (COVID-19). These altered signatures or biomarkers might be associated with disease outcome and prognosis. Therefore, animal models reflecting the clinical outcomes and natural immune responses observed in humans are crucial to identifying reliable biomarkers. Using mouse hepatitis virus 1 (MHV-1) as a model for SARS-CoV-2, we established obesity as a risk factor and identified biomarkers and pathways associated with worse disease outcomes. Obesity rates in low and middle-income countries (LMICs) are approaching levels found in high-income countries (HICs). Mosquito-borne viral diseases like dengue, chikungunya, and Zika pose a significant threat to LMICs and cause huge health and economic losses. Obesity was shown to worsen alphavirus pathogenesis, but interestingly, it also reduced their transmission by mosquitoes. Given the global prevalence of obesity and mosquito-borne viruses, it is critical to understand how obesity drives reduced alphavirus transmission. Using a natural transmission cycle between lean and obese mice and mosquitoes, we confirmed that obesity reduced the transmission potential of alphaviruses like CHIKV and MAYV and activated the Toll pathway in mosquito midguts. Various genes and other pathways were also altered in response to obese bloodmeal at various time-points post-bloodmeal; however, one gene, AAEL009965, was downregulated in the mosquito midguts 1-day-post-bloodmeal and its knockdown led to reduced infection rates and titers in mosquitoes. Through this thesis, we aimed to utilize obesity as a tool to identify biomarkers to predict coronavirus disease outcomes and design effective alphavirus transmission control strategies. / Doctor of Philosophy / Obesity, a global epidemic associated with chronic systemic inflammation, has emerged as an independent risk factor following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The impact of obesity on COVID-19 severity, largely based on epidemiological data or research with non-natural hosts of SARS-CoV-2, necessitates the use of natural models of obesity to study the host's response to coronaviruses. Here we used mouse models of diet-induced obesity and infected them with a mouse coronavirus to account for the host's natural immune response against the virus. We showed that obesity exacerbates disease outcomes and identified various genes associated with disease severity in obese mice that correspond to the gene expression patterns in severe COVID-19 patients. These findings underscore the critical role of obesity in disease outcomes and pave the way for further research in this area. Obesity also worsens the disease outcome following infection with mosquito-borne alphaviruses like chikungunya (CHIKV) and Mayaro (MAYV), however, its impact on the vector's transmission potential is poorly understood. Using a natural transmission cycle between lean and obese mice and mosquitoes, we showed that obesity reduced alphavirus transmission by mosquitoes. We found altered expression of specific genes and pathways, in mosquito midguts exposed to alphavirus-infected obese bloodmeal compared to the lean ones and their knockdown led to altered infection rates in mosquitoes suggestive of their role in viral infection of mosquitoes. We also established insulin as a potential antiviral factor in obese bloodmeal. Therefore, these studies provide the basic framework for understanding the impact of obesity on viral transmission and pathogenesis by using relevant animal models. Furthermore, these studies utilize obesity as a tool to identify predictors of severe disease outcomes and identify genes that could be used as potential transmission control strategies for various mosquito-borne viruses.

La malattia di Johne o paratubercolosi è un’enterite cronica granulomatosa provocata dal Mycobacterium avium subsp paratubercolosis (MAP), che colpisce i ruminanti ed in particolare i bovini da latte ed ha un grande impatto economico a livello mondiale. Il MAP sembra anche avere un ruolo nella malattia umana di Crohn. Tale patogeno è capace di sopravvivere molto bene all’interno dei macrofagi dell’ospite dove previene la loro attivazione, blocca l’acidificazione e la maturazione del fagosoma, e interferisce con la presentazione degli antigeni al sistema immunitario. Al fine di analizzare la complessa interazione tra l’ospite e il patogeno, è stata valutata la risposta dopo 2h, 6h, e 24h di macrofagi derivati da monociti bovini (MDM), coltivati in vitro e infettati con il ceppo L1 di MAP utilizzando un approccio di RNA-Seq. L’analisi statistica dei dati di sequenza ha mostrato un aumento del numero di geni differenzialmente espressi durante l’esperimento in risposta all’infezione. Inoltre i geni sottoespressi negli MDM infettati sono stati individuati solo a 24h post-infezione. L’analisi dei pathway ha evidenziato tre network che sono associati alla risposta immunitaria e al processo infiammatorio. Inoltre lo studio dei geni sottoespressi a 24h ha mostrato il ruolo centrale del complemento e del complesso maggiore di istocompatibilità nella patogenesi della malattia. / Johne’s disease (paratuberculosis) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp paratubercolosis (MAP), affecting ruminants worldwide with a significant economic impact. MAP has also been speculated as a cause of human Crohn’s disease. MAP is a pathogen highly adapted for survival within host macrophages due to the organism's capacity to prevent macrophage activation, block phagosome acidification and maturation, and attenuate presentation of antigens to the immune system. The consequence is a very long silent infection and subclinical phases. To decipher the complex interaction between host and MAP, the response of in vitro bovine monocyte-derived macrophages (MDM) after 2h, 6h and 24h of infection with L1 strain of MAP was explored using RNA-Seq approach. Statistical analysis of sequence data revealed an increasing number of differentially expressed genes in MDM following infection through the three time points analysed. Furthermore down-regulated genes were only found at 24 h post-infection. Ingenuity Pathways Analysis of differentially expressed genes showed that “cell-mediated immune response” was the most significant network related to 2hpi dataset, “immune cell trafficking” for 6hpi, and “inflammatory response” for 24hpi. Finally the analysis of down-regulated genes at 24hpi confirmed the role of complement and major histocompatibility complex (MHC) in the pathogenesis of MAP in cattle.

Le chlorométhane (CH3Cl) est un composé organique volatile responsable de plus de 15 % de la dégradation de l’ozone stratosphérique due aux composés chlorés. Il est produit majoritairement par les plantes vivantes ou en décomposition. Les bactéries capables d’utiliser le CH3Cl comme source de carbone pour leur croissance peuvent jouer un rôle de filtre dans les émissions de CH3Cl vers l'atmosphère. Ce processus biologique reste à quantifier dans l'environnement, notamment pour les sols forestiers considérés comme un puits majeur de ce composé.Dans les études environnementales, le gène cmu A est utilisé comme biomarqueur de la dégradation bactérienne du CH3Cl. Il code une chlorométhane méthyltransférase essentielle à la croissance bactérienne avec le CH3Cl parla voie cmu (pour chloromethane utilisation), la seule caractérisée à ce jour. Mon projet de thèse avait un double objectif : i) l’approfondissement des connaissances de l’adaptation au CH3Cl chez une bactérie méthylotrophe modèle, Methylobacterium extorquens CM4; ii) l’exploration de la diversité des bactéries CH3Cl-dégradantes de sols forestiers. L’étude RNAseq chez la souche CM4 a montré que la croissance avec le CH3Cl s'accompagne de différences dans la transcription de 137 gènes de son génome (6.2 Mb) par rapport à sa croissance sur le méthanol (CH3OH). Les gènes de la voie cmu, ainsi que d’autres gènes impliqués dans le métabolisme de cofacteurs essentiels à l’utilisation du CH3Cl par cette voie et eux aussi portés par le plasmide pCMU01 de la souche, en font partie. Les paralogues de ces gènes localisés sur le chromosome ne sont quant à eux pas différentiellement exprimés. En revanche, d’autres gènes du chromosome, potentiellement impliqués dans l’excrétion de protons produits lors de la déshalogénation (hppA), la régénération du NADP+ (pnt), ou le métabolisme du cofacteur tétrahydrofolate(gènes gcvPHT), le sont. L’étude de la diversité des bactéries CH3Cl-dégradantes de sol forestier de la réserve naturelle de Steigerwald (Allemagne) a été réalisée sur des microcosmes par une approche de « Stable Isotope Probing ». Les microorganismes capables d’assimiler le CH3Cl marqué au [13C] incorporent cet isotope lourd du carbone dans leur ADN. L'analyse des séquences amplifiées par PCR des gènes codant l’ARN 16S des fractions d'ADN enrichies en [13C] a permis de mettre en évidence de nouveaux phylotypes, du genre Methylovirgula et de l’ordre des Actinomycetales, distincts de ceux auxquelles les souches dégradant le CH3Cl isolées jusqu'ici sont affiliées. En revanche, les séquences du gène cmuA et d’autres gènes du métabolisme méthylotrophe obtenues par PCR à partir de l'ADN enrichi en [13C] sont très proches de celles des souches CH3Cl-dégradantes connues. Les résultats obtenus suggèrent ainsi que des bactéries ayant acquis par transfert horizontal les gènes de dégradation de la voie cmu ou ne possédant pas de gène cmuA contribuent au filtrage biologique du CH3Cl des sols forestiers. A l'avenir, le couplage de différentes méthodes moléculaires et des approches culturales visera à découvrir de nouvelles voies microbiennes de l’utilisation du CH3Cl, et à caractériser l’abondance et la diversité des métabolismes impliqués dans la dégradation du CH3Cl dans les sols et d'autres compartiments environnementaux. / Chloromethane (CH3Cl) is a volatile organic compound responsible for over 15% of stratospheric ozone degradation due to chlorinated compounds. It is mainly produced by living and decaying plants. Bacteria utilizing CH3Cl as sole carbon and energy source for growth were shown to be involved in the filtering of CH3Cl emissions to the atmosphere. This biological process remains to be quantified in the environment, especially for forest soil, a major CH3Cl sink. The cmuA gene is used as a biomarker of bacterial CH3Cl degradation in environmental studies. It encodes a CH3Cl methyltransferase essential for bacterial growth by the cmu (chloromethane utilization) pathway for growth with CH3Cl and the only one characterized so far. My thesis project had a double aim: i) In depth studies of CH3Cl adaptation of a model methylotrophic bacterium, Methylobacterium extorquens strain CM4; ii) Exploration of bacterial CH3Cl-utilizers in forest. An RNAseq study of strain CM4 has shown that growth with CH3Cl leads to a difference of transcription of 137 genes in its 6.2 Mb genome compared to growth with methanol (CH3OH). Among those, genes of the cmu pathway and other genes involved in the metabolism of essential cofactors for CH3Cl utilization by this pathway, are all plasmid pCMU01-encoded. Paralogous genes located on the chromosome were not differentially expressed. On the other hand, other chromosomal genes potentially involved in extruding protons generated during CH3Cl deshalogenation (hppA), NADP+ regeneration (pnt), or in the cofactor tetrahydrofolate metabolism (gcvPHT) were differentially expressed. The diversity of CH3Cl-degrading bacteria in forest soil of the German natural park of Steigerwald was studied in microcosms using stable isotope probing. Microorganisms able to assimilate labeled [13C]- CH3Cl incorporate this heavy carbon isotope in their DNA. Sequence analysis of the PCR-amplified 16S RNA encoding gene from [13C]-DNA fractions uncovered phylotypes of the genus Methylovirgula and of the order of the Actinomycetales, which were not associated with bacterial CH3Cl degradation so far. In contrast, PCR-amplified sequences of cmuA and other genes of methylotrophic metabolism were closely related to known CH3Cl-degrading isolates. These results suggest that bacteria containing genes of the cmu pathway acquired by horizontal gene transfer as well as bacteria lacking the cmu pathway contribute to biological filtering of CH3Cl in forest soil. Future experiments coupling molecular and culture methods will aim to discover new CH3Cl-degrading pathways and to characterize the abundance and diversity of CH3Cl-degradation metabolism in soil and other environmental compartments.