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Neurodevelopmental disorders encompass a spectrum of different conditions with both genetic and environmental etiologies. Although rapid progress has been made in deciphering the genetic landscape of these disorders, in most cases, it remains unclear how mutations undermine neurodevelopmental mechanisms. However, increasing identification of risk genes suggests chromatin remodelling is frequently impacted. For instance, de novo variants encoding the chromatin remodeller CHD4 causes Sifrim-Hitz-Weiss syndrome, which manifests as an overgrowth-intellectual disability syndrome. To further understand Chd4’s role during cortical development, we excised the ATPase domain of Chd4 in the germline or specifically in the developing telencephalon, creating three mouse models. Germline heterozygotes presented a slight decrease in brain weight, cortex area and Ctip2+ cells, with females displaying more overt impairments in learning and memory. Telencephalon-specific conditional heterozygotes exhibited slight changes in white matter, increased repetitive movements and altered social behaviours. Telencephalon-specific conditional knockouts presented with decreased brain size, brain weight, and cortex thickness due to decreased upper layer neurons, and anxiety phenotypes. These data reveal an unexpected complexity in the impacts of Chd4 mutations on neurodevelopmental processes and behaviour.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24504号 / 医博第4946号 / 新制||医||1064(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 溝脇 尚志, 教授 浅野 雅秀, 教授 鈴木 実 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This investigation is concerned with cell surface antigens present on murine AKR/J mice spleen and thymus cells which have been extracted with papain. Isolation of individual proteins was accomplished by granulated gel electrofocusing. Similar patterns recorded by both electrofocusing procedures identified several proteins limited to the AKR/J and C3Heb/FeJ spleen and thymus samples, which represent Murine Leukemia Virus-associated surface proteins.

The objective of this study was to identify peptide ligands, using phage display techniques, which bind sites on mouse embryos, ovaries, cytoplasmic membranes and/or intracytoplasmic components. Specifically, M13 coliphage 7-mer, 12-mer and 15-mer random peptide libraries were used separately for biopanning. Peptides derived from the amplified pools were sequenced and studied. The phage display for in vivo ovary experiments yielded no pool of peptides after two cycles of biopanning and re-amplification. With the same initial concentration of a random 7-mer or 12-mer library, there were repeating sequences derived after three and four biopanning cycles on mouse embryos and unfertilized ova. The sequences were not distinguishable from a control group. Subsequent experimentation using a random 15-mer library to select for internalized phage-peptides yielded two apparent consensus sequences, RNVPPIFNDVYWIAF (9/32 or 28%) and HGRFILPWWYAFSPS (11/32 or 34%). The 15-mer control group yielded no clones. The deduced peptide sequences were compared to known sequences to ascertain their uniqueness. No significant similarities were found, yielding two possible novel motifs. Through this adapted process of phage display and further research, the phage display technology may be used as a tool in the recognition of specific mouse gamete sites. By identifying binding sites of mouse gametes, the peptides might be exploited as a means of studying the embryo cell surface or cytoplasmic components and mouse sperm-egg interactions. Such peptides may also be used for macromolecule delivery in transfection or transgenesis. / Master of Science

Allergic asthma is a chronic inflammatory disease of the airways. Importantly, the chronic nature of this disease imparts specific additional consequences that would not otherwise be observed in a strictly acute setting. The development ofvarious structural alterations to the airway wall, collectively tem1ed airway remodeling, represents one such example. Decades of research have provided a great deal of insight into the acute allergic asthmatic response and the processes that govern it. However, less is known about the impact of protracted allergen exposure and chronic immune-inflammatory responses. To this end, the research presented in this thesis explores the consequences of chronic allergen exposure and persistent airway inflammation on asthma pathogenesis, using a mouse model of allergic airway disease induced by respiratory exposure to house dust mite (HDM) allergens. Specifically examined are: i) the impact of continuous allergen exposure and the resulting immune-inflammatory response on the development of de nova sensitization to newly encountered allergens (Chapter 2) and, ii) the roles oftransforming growth factor (TGF)-~ and eosinophils, two putatively critical components of the allergic inflammatory response, in the generation of airway remodeling (Chapters 3 and 4). Our data show that chronic exposure to HDM facilitates the development of the full 'asthmatic phenotype' towards an innocuous antigen. Moreover, they demonstrate that, unlike what has been previously observed in ovalbumin-based models, neither TGF-~ nor eosinophils are critically required for remodeling to develop in the context of HDM exposure. These findings highlight the importance of the lung microenvironment in influencing the type of immune response that develops upon initial antigen encounter and, furthermore, underscore the notion that the role of a particular cell type or molecule in the asthmatic response is contextual and not necessarily broadly applicable. / Thesis / Doctor of Philosophy (PhD)

<p> The variation in sensitivity of L60T cells to gamma rays has been studied as a function of position in the cell division cycle. For a dose range of 0-12,000 rads, no significant variation was found for mitotic delay. Such was not the case for sensitivity to cell killing, which was found to increase as the cells passed from G1 through S to G2 of the division cycle. The results of mitotic delay are in disagreement with results published by other workers although the survival data agree with previous reports for a similar cell line. Results reported in connection with cell cycle determinations and mitotic delay suggest that the existence of a repair cycle operating concurrently with the normal cell cycle may be postulated. The theoretical treatment of mitotic delay given by Lea is examined and is not found to describe adequately the present results. Finally, the evidence reported here suggests that mitotic delay and radiation lethality are not separate manifestations of the same phenomenon. Experimental materials for further investigation into the repair processes involved are suggested.</p> / Thesis / Master of Science (MSc)

Parkinson’s Disease (PD), the second most common neurodegenerative disease worldwide, pathologically presents with the inclusion of Lewy bodies and dopaminergic cell loss in the brain. Lewy bodies are composed of aggregated a-synuclein protein, and although essential to our understanding of PD, not much is known about the native, pre-synaptic state of a-synuclein (a-syn). Due to its mostly synaptic local, immunostaining results in diffuse signal, ultimately providing little insight into the types of a-syn-resident cells. As a result, insight into a-syn expression driven cellular vulnerability has been difficult to ascertain. Using a knockin mouse model that localizes a-syn to the nucleus of cells by insertion of a nuclear localization signal into the a-syn gene locus (SncaNLS), we overcome visualization issues and map out the topography and cells-of-origin of a-syn in mice. I performed immunohistochemistry on SncaNLS mouse tissue to map out the endogenous distribution of a-synuclein in the brain. Using ilastik machine learning analysis, I determined regions with high a-syn expression, which were subsequently co- stained with cell-type specific markers to gain further topographical granularity. a-syn showed high expression in the olfactory bulb, hippocampus, cerebral cortex, substantia nigra and cerebellum. Within these structures, there was a high level of expression of a-syn in granule cells, pyramidal cells, mitral cells, and dopaminergic neurons. Taken together, the SncaNLS mouse serves as a tool to define an atlas of a-syn topography, potentially providing insight into cellular vulnerability in PD.

Fetal Alcohol Spectrum Disorders (FASDs), are caused by maternal alcohol consumption during pregnancy [3]. FASD encompasses a wide variety of cardiac and neural anomalies, while also associated with improper limb development, abnormal craniofacial features, problems within the central nervous system (CNS), and disabilities in learning and communication. Gene-regulating FASDs have not been well studied during the crucial phases of early embryonic development. Genes within the Wnt/Beta-catenin pathway control a vast amount of embryonic developmental processes. Among them is the Wnt7a gene, a significant downstream gene regulator which positively controls neural stem cell proliferation and cardiomyocyte differentiation on a large scale during early embryonic development. This project will serve to provide potential insight into the genes involved in FASD. We hypothesize that ethanol administration to early embryonic mice will suppress Wnt7a expression in the heart and brain, leading to FASD development. RNA-sequencing (RNA-Seq) and real-time quantitative PCR (qPCR) were used to measure Wnt7a gene expression within the early embryonic mouse heart and brain. After evaluation of RNA-Seq data and a comparative analysis using the 2-ΔΔCTmethod, it is evident Wnt7a is present in embryonic mouse age E10.5 heart and brain samples, and Wnt7a is suppressed at age E10.5 in embryonic mouse heart, but not brain, when induced with ethanol.

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