京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18172号 / 医博第3892号 / 新制||医||1003(附属図書館) / 31030 / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 中辻 憲夫, 教授 萩原 正敏, 教授 小西 郁生 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21166号 / 農博第2292号 / 新制||農||1061(附属図書館) / 学位論文||H30||N5140(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 久米 新一, 准教授 南 直治郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Assessment of chemical mutagenicity is essential to protecting human health from genetic disease. Current assays are limited in their ability to provide mechanistic insight into the endogenous and exogenous processes involved in mutagenesis. Duplex Sequencing (DS), a novel error-corrected sequencing technology, overcomes many of the limitations faced by conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. Furthermore, customizable panels enable assessment of the endogenous genomic features that drive mutagenesis. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. The objectives of this study were to demonstrate the potential of DS as a robust in vivo mutagenicity test and to explore its rich data to gain a better understanding of spontaneous and chemically-induced mutagenicity in somatic and germ cells. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) and germ cells of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and tissues were sampled at least 28 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in MF and distinct spectra consistent with the known mutagenic mechanisms of PRC in both tissues. Mouse PRC doses at which significant effects were observed are in range with those used for chemotherapy, suggesting that similar effects may be observed in human patients. This supports the contribution of PRC towards secondary cancers following treatment. DS results were comparable to those obtained using the gold-standard lacZ TGR assay, with DS showing greater sensitivity to detect smaller changes in MF. Analysis of mutation spectra and the genomic features that drive the mutational response revealed intrinsic differences between BM and germ cells that may underlie differences in endogenous mutagenic mechanisms and/or DNA repair pathways. The results suggest that germ cells may have intrinsic mechanisms to reduce mutation burden relative to somatic cells. While historically analysis of germ cell mutagenicity has been neglected in favour of somatic cells, our work supports the independent assessment of germ cell mutagenicity during regulatory testing. Finally, we conducted power analyses to inform the optimal DS study designs for the two tissues. We found that low intra-group variability within BM samples allows a reduction in sample size to three animals per group whilst still maintaining 80% power to detect an effect. In contrast, the relatively high intra-group variability and low background MF in germ cells suggests a minimum of eight animals per group to detect an effect. Overall, our results support the use of DS as a mutagenicity test and highlight many of the advantages it holds over conventional assays. Moreover, our study reveals the potential for mutagenic effects in PRC-treated cancer patients. Further work to test DS with more chemicals and across a wider range of tissues is recommended for future implementation as a mutagenicity test.

The genetic variation among extant life forms reflects the outcomes of evolution. The fodder of evolution – germline mutations – is shaped by the interplay among evolutionary forces – notably natural selection and random genetic drift. In turn, these forces leave footprints recorded in the genetic variation of extant life forms. Characterizing these footprints to understand how evolution works is at the heart of population genetics. To this end, massive datasets of genetic variation have opened new avenues of research, around how mutation rates evolve for instance, and reinvigorated long standing questions in population genetics, notably about the genetic basis of adaptation. In turn, theoretical models of evolution inform what kind of footprints we expect evolution to leave behind in such data. Two theoretical models that investigate open questions in population genetics are described in this thesis. In Chapter 1, I consider the evolution of germline mutation rates, particularly on short evolutionary timescales, and ask if recently observed variation in mutation rates among human lineages could be explained by evolution at genetic modifiers of mutation rates. Genetic modifiers of mutation rates are expected to evolve under purifying selection: mutations at modifiers that increase mutation rates (“mutator alleles”) should be selected against, because they increase the burden of deleterious mutations in individuals who carry them. The frequencies of mutator alleles are also affected by mutation, genetic drift, and demographic processes. We model the evolution of mutator alleles under the interplay of these forces and characterize the dynamics at mutation rate modifiers as a function of the efficacy of selection acting on them. We find that modifiers under intermediate selection have the greatest contribution to variation in mutation rates between distantly related populations, but only variation at strongly selected modifiers turns over fast enough to explain variation in mutation rates among human lineages. We also predict that strongly selected modifiers could be potentially identified in the contemporary datasets of human pedigrees used to study germline mutations. In Chapter 2, I consider a central and enduring question in evolutionary biology: whether adaptation typically arises from few large effect changes or from many small effect changes. Both sides are supported by ample evidence. Yet it is unclear how to translate this evidence into general answers about the genetic basis of adaptation, in part because different methodologies have different limitations and ask different questions. Theory may offer a way out of this quagmire or at least a start. To this end, we reframe the question in terms of traits and ask: how does the genetic basis of adaptation depend on the ecological and genetic attributes of a trait? To start answering this question, I model adaptation in a simple yet highly relevant setting. I consider a trait under stabilizing selection and assume the distribution of trait values in the population is initially at mutation-selection-drift-balance. I then characterize the adaptive response that is elicited by a sudden change in the environment. I find that the adaptive response, and notably the probability that adaptation arises from the fixation of large effect alleles, depends on the size of the environmental change and the genetic architecture of the trait. These attributes are measurable and can be directly related to the disparate evidence that we have about the genetic basis of adaptation. Thus, this kind of modeling may help translate such evidence into general conclusions about how traits evolve. My thesis work was interrupted by the global COVID-19 pandemic, and in response to this pandemic, governments around the world implemented shelter-in-place protocols. However, essential workers were exempt from these protocols, potentially decreasing their efficacy. In Chapter 3, we describe our epidemiological project, aimed at understanding the impact of essential workers on epidemic control. To this end, we model three different archetypes of essential workers under a reasonably realistic SEIR model of the COVID-19 pandemic. We find that the different social interactions that essential workers maintain qualitatively changes their personal risk of infection and the spread of the overall epidemic. These results highlight the utility of not considering essential workers as a monolithic group but instead distinguishing between the impact of different types of essential workers on epidemic control.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13566号 / 論医博第2293号 / 新制||医||1068(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 柊, 卓志, 教授 篠原, 隆司, 教授 後藤, 慎平 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Yes / There is a need for improved reproductive toxicology assays that do not require large numbers of animals but are sensitive and informative. Therefore, Staput velocity-sedimentation separation followed by culture of specific mouse testicular cells was used as such a system. The specificity of separation was assessed using immunocytochemistry to identify spermatids, spermatocytes and spermatogonia. The efficacy of the system to detect toxicity was then evaluated by analysing the effects of hydrogen peroxide (H2O2) by the terminal uridine-deoxynucleotide end-labelling (TUNEL) assay to show the rate of apoptosis induced among the different types of germ cells. We found that 2 h of treatment at both 1 and 10 μM induced increases of over ∼10-fold in the percentage of apoptotic cells (p ≤ 0.001), confirming that testicular germ cells are prone to apoptosis at very low concentrations of H2O2. It was also demonstrated for the first time for this compound that spermatogonia are significantly more susceptible than spermatocytes, which are more affected than spermatids. This reflects the proportion of actively dividing cells in these cell types, suggesting a mechanism for the differential sensitivity. The approach should thus form the basis of a useful test system for reproductive and genetic toxicology in the future.

Yes / Anthracyclines such as doxorubicin (Dox), widely used to treat various types of tumours, may result in induced testicular toxicity and oxidative stress. The present investigation was designed to determine whether exposure of isolated and purified mouse germ cells to Dox induces DNA damage in the form of strand breaks (presumably) resulting in apoptosis and to investigate the relative sensitivity of specific cell types. DNA damage was assessed using the Comet assay and the presence of apoptosis was determined by TUNEL assay. Isolated mouse germ cells were treated with different concentrations (0.05, 0.5 and 1 mM, respectively) of Dox, and fixed 1 h after treatment. The incidences of both DNA damage shown by single cell gel-electrophoresis and of apoptosis increased significantly in each specific cell type in a concentration-dependent manner. The DNA damage and apoptosis incidences gradually increased with concentration from 0.05 to 1 mM with Dox. Our results indicate that apoptosis plays a vital role in the induction of germ cell phase-specific toxicity caused by Dox with pre-meiotically and meiotically dividing spermatogonia and spermatocytes respectively as highly susceptible target cells. / Higher Education Funding Council for England (HEFCE)

Primordial germ cells (PGCs) are the embryonic precursors of the germ cell lineage. Segregation of the chicken germ line from somatic cells occurs very early in embryonic development. By day two of incubation chicken PGCs can be isolated from the circulating blood. The in vitro culture of chicken PGCs has significant potential as a tool for the investigation of germ cell development and as a cell-based system for the production of genetically modified chickens. The isolation, culture and manipulation of migratory chicken PGCs reported previously have not been independently validated. Initial attempts to isolate and culture chicken PGCs by reproducing a published protocol proved difficult. Key components of the published culture medium are by their nature variable, including the use of BRL-conditioned medium and animal sera. The protocol also stated that addition of SCF to the culture medium is essential but did not identify the source of SCF used. Several components of the culture conditions were tested including sources and batches of bovine and chicken sera and the growth factors FGF2 and SCF. Chicken PGCs from wild type and GFPexpressing chicken embryos were cultured and several cell lines established, proliferating for more than 100 days in culture. After seventy days in culture a single chicken PGC cell line was shown to retain the potential to develop into functional sperm. This was demonstrated by injection of the cultured chicken PGCs into early chick embryos, which were hatched and produced offspring derived from the injected chicken PGCs. To understand and produce a more robust system for the isolation and propagation of chicken PGCs three signalling pathways, AKT, MAPK and JAK/STAT, were investigated. When any of these signalling pathways were blocked, using chemical inhibitors, chicken PGC proliferation in vitro was significantly inhibited, showing the pathways to be essential for chicken PGC proliferation. Chicken PGCs were treated with individual components of the standard culture medium, FGF2, SCF, animal sera, BRL-conditioned medium, LIF and IGF, and the activation status of the key signalling pathways was assessed by western blot. Individual components of the culture medium induced activation of the AKT and MAPK pathways but not the JAK/STAT pathway. These data increase our understanding of PGC biology and are the first steps towards the development of a feeder- and serum-free medium for the growth of chicken PGCs. Published methods for the genetic manipulation of chicken PGCs are inefficient. To improve the efficiency of stable transgene integration, transposable element-derived gene transfer vectors were assessed for their ability to transpose into the genome of chicken PGCs. Comparison of Tol2 and piggyBac transposable elements, carrying reporter transgenes, demonstrated that both can be used to genetically-modify chicken cells. The incidence of stable transposition achieved was higher when using the Tol2 transposable element in comparison to the piggyBac element. The genetically-modified chicken PGCs formed functional gametes, demonstrated by injection of genetically modified chicken PGCs into host embryos which were hatched and produced transgenic offspring expressing the reporter gene construct.

Em mamíferos, a espermatogênese é mantida pela autorrenovação e diferenciação das células-tronco espermatogoniais (SSC). Apesar da grande importância do SSC para a fertilidade masculina, em Bos taurus pouco se sabe sobre a sua identificação e biologia celular. Para roedores, mais de 30 marcadores para células germinativas indiferenciadas já foram descritos. No entanto, ainda não é conhecido um marcador específico apenas para SSC. Quase todos são também expressos por gonócitos, espermatogônias mais diferenciadas ou mesmo células somáticas. Yin Yang 2 (YY2) é um factor de transcrição expresso nas células com a morfologia de gonócitos e SSC, sendo um candidato a marcador de SSC. Assim, a identificação de novos marcadores para SSC e factores que afectam a sua expressão, tais como a idade, são fundamentais para o desenvolvimento da biotecnologia como transgenia e tratamento de infertilidade, nos quais as SSC poderiam ser ferramentas biológicos importantes. Assim, nesta tese temos duas hipóteses principais: 1) a idade do dador afeta a expressão de marcadores moleculares específicos de SSC bovinas assim como potencial de células-tronco dessas células e que as sequências de DNA em que se associa YY2 regulam a expressão génica de SSC em camundongos. Os objetivos específicos, organizados em 4 artigos científicos, foram: identificar a melhor plaqueamento diferencial para enriquecer SSC bovina (artigo 1), verificar se a expressão de marcadores moleculares de SSC bovina difere entre adultos pré-púberes (artigo 2 e 3), identificar novos marcadores específicos para SSC em Bos taurus (artigo 3), verificar que a idade afeta o potencial de célula-tronco de SSC bovinas (artigo 3), descrever YY2 como um marcador específico para SSC em camundongos e verificar se as sequências DNA associadas YY2 são loci de importância para SSC. Assim, definimos o melhor plaqueamento diferencial para o enriquecimento de SSC bovinas, que idade afeta a expressão marcadores já estabelecidos assim como genes específicos do transcriptoma de SSC bovinas e que idade também afeta o seu potencial de células-tronco (ensaio de repopulação). Concluímos também que YY2 é um marcador para SSC de camundongo em cultivo, em animais adultos e que as sequências do genoma que se associam YY2 possivelmente tem capacidade de regulação génica em SSC murina. / Mammalian spermatogenesis is sustained by self-renewal and differentiation of spermatogonial stem cells (SSC). Despite the importance of the SSC for male fertility, in Bos taurus líttle is known about their identification identity and cell biology. For rodents, more than thirty markers for undifferentiated germ cells have already been described. However, none of these represents a marker specific for SSC, as most are also expressed in gonocytes, differentiated spermatogonia or even somatic cells. Yin Yang 2 (YY2) is a transcription factor specifically expressed in cells with the morphology of gonocytes and SSC in the mouse, being a candidate marker for SSC. For the use of SSC as an important biological tool in the development of biotechnology such as transgenesis and the treatment of infertility, it is important to identify new markers for SSC and factors that affect their expression, such as the age of donors. Therefore, the experimental work described in this thesis was based on two main hypothesis: (1) the donor age affects the expression of specific molecular markers in SSC as well their potential as stem cells and 2) YY2 exerts important functions in SSC and genomic targets correspond to loci relevant for gene regulation or genome management in SSC. The specific goals have been organized in 4 manuscripts as follows: optimization of differential plating to enrich for bovine SSC (Article 1), check if the expression of molecular markers of bovine SSCs differs between prepubertal and adult donors (Article 2 and 3), identify new markers specific for SSC in Bos taurus (Article 3), continuation of the initial description of YY2 as a specific marker for SSC in mice and check if sequences bound by YY2 in vivo harbor the capacity to influence gene expression in SSC. As conclusions, we present an optimized differential plating protocol for the enrichment of bovine SSC, we conclude that age effects the expression of SSC markers, the expression of specific genes of bovine SSC and that age also affects their potential as stem cells (measured in repopulation assays). We also contribute to the description of the restricted expression of YY2 in prepuberal and adult SSC in mice and we show that YY2 binding sites represent genomic sequences relevant for control of gene expression.