京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21455号 / 医博第4422号 / 新制||医||1033(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 山下 潤, 教授 湊谷 謙司, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13232号 / 論医博第2172号 / 新制||医||1036(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 山下 潤, 教授 江藤 浩之, 教授 木村 剛 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Research into the molecular pathology of prevalent public health epidemics such as neurodegenerative diseases including frontotemporal dementia (FTD) and Alzheimer’s Disease (AD), non-medical and illicit opioid use (OU), and Human Immunodeficiency Virus-1 (HIV-1) has been hindered by a lack of systems that allow for rapid and high-throughput modeling of the complex multifactorial conditions in a human context. In this thesis we have addressed this challenge using a multi-pronged approach that encompasses single cell RNA sequencing (scRNA-seq) of three-dimensional (3D) human induced pluripotent stem cell (hiPSC) assembloid culture models and patient derived peripheral blood mononuclear cell (PBMC) samples. We describe the development of an iPSC derived neuron-astrocyte assembloid model of tauopathies, including FTD and AD (AstTau), that rapidly recapitulates propagation of toxic human oligomeric tau (oTau) and cell type specific pathology including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. scRNA-seq identified vulnerable excitatory neuron specific inflammatory pathways and a heat shock response in astrocytes, recapitulating transcriptomic signatures of adult neurodegeneration and supporting a hypothesis of cell type specific neuroinflammation in tau pathogenesis. To more completely model AD, we incorporated amyloid precursor protein (APP) mutant iPSCs into the assembloid model. These iPSCs contained the familial AD APP V717I mutation or the isogenic CRISPR corrected control, and were used to derived neurons, astrocytes, and microglia. This advanced combinatorial system (AstAD and MAstAD) enabled selective microglial incorporation, APP mutation expression, and oTau seeding allowing us to identify discrete contributions to AD pathogenesis. Ast/MAstAD developed extracellular amyloid-β (Aβ) and microglial activation in addition to the pathology observed in AstTau. scRNA-seq identified divergent microglial activation in response to Aβ and oTau pathology, with APP V717I mutation and oTau seeding synergistically exacerbating AD phenotypes. These assembloid models enable study of the cellular and molecular inflammatory mechanisms in multifactorial neurodegenerative diseases. To better understand disease signatures at the crossroads of multifactorial OU, HIV-1, and antiretroviral (ART) viral suppression we also produced a scRNA-seq data set of more than 100,000 peripheral blood mononuclear cells (PBMCs) from 75 study participants. We identified chronic immune activation and T cell activation dysfunction driven by interferon transcriptomic signatures that were elevated in people with HIV (PWH) with opioid use as compared to PWH without OU. We also identified a functional natural killer cell subtype that was depleted with OU in PWH. Cessation of OU reduced these potentially deleterious inflammatory transcriptomic profiles, supporting the hypothesis that OU in PWH amplifies a state of chronic immune activation. Taken together, single cell transcriptomic resolution has enabled the identification of cell type specific disease signatures in complex pathophysiologies. These data demonstrate the dynamic range of inflammatory signaling across multifactorial disease states and emphasize the need for disease- and cell- type specific approaches to therapeutic development. / 2025-02-05T00:00:00Z

No description available.

Myelodysplastic syndromes (MDS) encompass a heterogeneous group of age-related hematopoietic disorders characterized by ineffective and incomplete hematopoiesis leading to an increased risk of acute myeloid leukemia (AML). The development of accurate and easily used in vitro models is crucial for understanding the pathogenesis of MDS and identifying potential therapeutic targets. Induced pluripotent stem cells (iPSCs) can be used to study MDS due to their ability to differentiate into any cell type depending on the environment. The main limitation is that the blood progenitors produced by iPSCs are of a fetal state, which hinders modeling of MDS, a disease of older adulthood. This study aimed to optimize the maturation state of blood progenitors derived from iPSCs by induction of the micro-RNA let-7, which, we hypothesize will increase the maturation and adult phenotypic state of hematopoietic progenitors. iPSC lines were generated from healthy controls and samples containing the SRSF2 mutation, a common mutation in MDS, containing a doxycycline-inducible, stabilized let-7 transgene. A stepwise differentiation efficiently drove the iPSCs toward hematopoietic progenitors and, subsequently, other mature lineages. The hematopoietic progenitors were characterized by assessing the expression of specific cell surface markers and functional properties using flow cytometry, colony-forming assays, and multi-lineage differentiation abilities. These findings demonstrate the potential of using iPSC engineering to create a novel model for MDS and other age-biased disorders by inducing let-7 expression in iPSCs and, when differentiating them, exposing them to doxycycline to promote an adult cell phenotype. This approach offers a valuable potential tool for elucidating the molecular mechanisms underlying these disorders and exploring potential therapeutic interventions. / 2026-03-13T00:00:00Z

京都大学 / 新制・論文博士 / 博士(医科学) / 乙第13611号 / 論医科博第12号 / 新制||医科||11(附属図書館) / 九州大学大学院薬学府創薬科学専攻 / (主査)教授 井上 治久, 教授 松田 秀一, 教授 萩原 正敏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25177号 / 医博第5063号 / 新制||医||1071(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 川口 義弥, 教授 山中 伸弥, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

A tranferência nuclear de células somáticas (TNCS) está sendo utilizada para produzir cavalos de elite. No entanto, durante este procedimento pode ocorrer a perfuração da zona pelúcida, levando, ocasionalmente, à secção da massa celular interna, e conseqüente derivação de gêmeos monozigóticos. Além de serem relatadas alterações no processo de imprinting genômico, que conduzem ao desenvolvimento de doenças. Com a descoberta da possibilidade de reprogramar as células somáticas a um estado de pluripotência (iPSCs), estas células passaram a ser muito utilizadas em pesquisas de neurociência. Contudo, também ocorrem modificações epigenéticas durante esta reprogramação celular. Portanto, nossas hipóteses são que os gêmeos eqüinos gerados pela TNCS podem levar às irregularidades no desenvolvimento do sistema nervoso. O padrão de metilação do SNRPN nas estruturas dos fetos muares clonados, e as células iPSCs são diferentes dos padrões encontrados nos muares analisados. A expressão dos genes SNRPN, Necdin e UBE3A são maiores no cérebro, enquanto a expressão do H19 é maior nas membranas extra-embrionárias. Em nosso estudo, obtivemos duas gestações gemelares equinas derivadas da TNCS, que foram interrompidas com 40 e 60 dias de gestação, e comparados com gestações eqüinas únicas de idade similar. Diferenças no comprimento entre os embriões gêmeos foram observadas aos 40 (2.0 e 2.2 cm 10%) e aos 60 (6,5 e 8,5 cm 24%) dias de gestação. Somente o plexo coróide do quarto ventrículo apresentou-se mais desenvolvido nos fetos com maior comprimento. Ao analisarmos fetos muares clonados em diferentes idades gestacionais e compará-los com muares, nos períodos embrionário, fetal e adulto, não foi observada diferença no padrão de metilação do gene SNRPN. No entanto, na décima passagem das células iPSC o padrão de metilação alterou, em relação aos muares estudados e ao padrão observado nos fibroblastos. Ao analisarmos os fetos clonados nas diferentes idades gestacionais observou-se no cérebro menor expressão dos gene H19 e UBE3A, e maior expressão do gene SNRPN. Contudo, a expressão do gene Necdin variou entre as estruturas estudadas. Em conclusão, apesar dos gêmeos eqüinos provenientes de TNCS diferirem quanto ao tamanho, morfologicamente são iguais. Dentre as estruturas cerebrais o plexo coróide se apresentou mais desenvolvido nos fetos de maior comprimento. Os fetos muares clonados não apresentaram diferença no padrão de metilação do gene SNRPN. No entanto, as iPSCs apresentaram alteração no padrão de metilação deste gene na décima passagem. Embora os genes SNRPN, Necdin e UBE3A sejam expressos no cérebro, o SNRPN apresentou-se prevalente nessa estrutura / The nuclear transfer of somatic cells (SCNT) is being used to produce elite horses. However, during this procedure can occur drilling of the zona pellucida, leading occasionally to the section of the inner cell mass, and subsequent derivation of monozygotic twins. Besides being related changes in genomic imprinting process, leading to the development of diseases. With the discovery of the possibility to reprogram somatic cells to a pluripotent state (iPSCs), these cells have become widely used in neuroscience research. However, also occur epigenetic changes during this cellular reprogramming. Therefore, our hypothesis is that equine twins caused by equine ART could lead to developmental irregularities of the nervous system. The patterns of SNRPN methylation in the structures of cloned mule fetuses and in iPSCs are different from the patterns found in the analyzed mules. And the expression of SNRPN, Necdin and UBE3A genes are higher in the brain, while the higher expression of H19 gene occurs in the extraembryonic membranes. In our study we derived two equine twin SCNT pregnancies that were interrupted at 40 and 60 days of gestation and compared to singleton fetuses of similar age. Differences in lengths between twin embryos were observed at both 40 (2.0 and 2.2 cm 10%) and 60 (6.5 and 8.5 cm 24%) days of gestation. Only the choroid plexus in the fourth ventricle more developed in the twins with the greatest length. Analyzing mules cloned fetuses at different gestational ages, and compare them with mules at embryonic, fetal and adult period; there was no difference in the pattern of methylation in SNRPN gene. However, in the tenth passage of the iPSCs the methylation pattern was altered in relation to the studied mules and the pattern observed in fibroblasts. When the cloned fetuses at different gestational ages were analyzed, the brain presented lower expression of H19 and UBE3A genes, and higher expression of SNRPN gene. However, the expression of Necdin gene varied among the structures studied. In conclusion, despite the twin horses from SCNT differ in size, they are morphologically identical. Among the brain structures the choroid plexus performed more developed in the fetuses of greater length. Cloned mules fetuses showed no difference in the pattern of methylation SNRPN gene. However, iPSCs have changes in the pattern of methylation of this gene in the tenth passage. Although SNRPN, Necdin and Ube3A genes are expressed in the brain, SNRPN is prevalent in this structure

A tranferência nuclear de células somáticas (TNCS) está sendo utilizada para produzir cavalos de elite. No entanto, durante este procedimento pode ocorrer a perfuração da zona pelúcida, levando, ocasionalmente, à secção da massa celular interna, e conseqüente derivação de gêmeos monozigóticos. Além de serem relatadas alterações no processo de imprinting genômico, que conduzem ao desenvolvimento de doenças. Com a descoberta da possibilidade de reprogramar as células somáticas a um estado de pluripotência (iPSCs), estas células passaram a ser muito utilizadas em pesquisas de neurociência. Contudo, também ocorrem modificações epigenéticas durante esta reprogramação celular. Portanto, nossas hipóteses são que os gêmeos eqüinos gerados pela TNCS podem levar às irregularidades no desenvolvimento do sistema nervoso. O padrão de metilação do SNRPN nas estruturas dos fetos muares clonados, e as células iPSCs são diferentes dos padrões encontrados nos muares analisados. A expressão dos genes SNRPN, Necdin e UBE3A são maiores no cérebro, enquanto a expressão do H19 é maior nas membranas extra-embrionárias. Em nosso estudo, obtivemos duas gestações gemelares equinas derivadas da TNCS, que foram interrompidas com 40 e 60 dias de gestação, e comparados com gestações eqüinas únicas de idade similar. Diferenças no comprimento entre os embriões gêmeos foram observadas aos 40 (2.0 e 2.2 cm 10%) e aos 60 (6,5 e 8,5 cm 24%) dias de gestação. Somente o plexo coróide do quarto ventrículo apresentou-se mais desenvolvido nos fetos com maior comprimento. Ao analisarmos fetos muares clonados em diferentes idades gestacionais e compará-los com muares, nos períodos embrionário, fetal e adulto, não foi observada diferença no padrão de metilação do gene SNRPN. No entanto, na décima passagem das células iPSC o padrão de metilação alterou, em relação aos muares estudados e ao padrão observado nos fibroblastos. Ao analisarmos os fetos clonados nas diferentes idades gestacionais observou-se no cérebro menor expressão dos gene H19 e UBE3A, e maior expressão do gene SNRPN. Contudo, a expressão do gene Necdin variou entre as estruturas estudadas. Em conclusão, apesar dos gêmeos eqüinos provenientes de TNCS diferirem quanto ao tamanho, morfologicamente são iguais. Dentre as estruturas cerebrais o plexo coróide se apresentou mais desenvolvido nos fetos de maior comprimento. Os fetos muares clonados não apresentaram diferença no padrão de metilação do gene SNRPN. No entanto, as iPSCs apresentaram alteração no padrão de metilação deste gene na décima passagem. Embora os genes SNRPN, Necdin e UBE3A sejam expressos no cérebro, o SNRPN apresentou-se prevalente nessa estrutura / The nuclear transfer of somatic cells (SCNT) is being used to produce elite horses. However, during this procedure can occur drilling of the zona pellucida, leading occasionally to the section of the inner cell mass, and subsequent derivation of monozygotic twins. Besides being related changes in genomic imprinting process, leading to the development of diseases. With the discovery of the possibility to reprogram somatic cells to a pluripotent state (iPSCs), these cells have become widely used in neuroscience research. However, also occur epigenetic changes during this cellular reprogramming. Therefore, our hypothesis is that equine twins caused by equine ART could lead to developmental irregularities of the nervous system. The patterns of SNRPN methylation in the structures of cloned mule fetuses and in iPSCs are different from the patterns found in the analyzed mules. And the expression of SNRPN, Necdin and UBE3A genes are higher in the brain, while the higher expression of H19 gene occurs in the extraembryonic membranes. In our study we derived two equine twin SCNT pregnancies that were interrupted at 40 and 60 days of gestation and compared to singleton fetuses of similar age. Differences in lengths between twin embryos were observed at both 40 (2.0 and 2.2 cm 10%) and 60 (6.5 and 8.5 cm 24%) days of gestation. Only the choroid plexus in the fourth ventricle more developed in the twins with the greatest length. Analyzing mules cloned fetuses at different gestational ages, and compare them with mules at embryonic, fetal and adult period; there was no difference in the pattern of methylation in SNRPN gene. However, in the tenth passage of the iPSCs the methylation pattern was altered in relation to the studied mules and the pattern observed in fibroblasts. When the cloned fetuses at different gestational ages were analyzed, the brain presented lower expression of H19 and UBE3A genes, and higher expression of SNRPN gene. However, the expression of Necdin gene varied among the structures studied. In conclusion, despite the twin horses from SCNT differ in size, they are morphologically identical. Among the brain structures the choroid plexus performed more developed in the fetuses of greater length. Cloned mules fetuses showed no difference in the pattern of methylation SNRPN gene. However, iPSCs have changes in the pattern of methylation of this gene in the tenth passage. Although SNRPN, Necdin and Ube3A genes are expressed in the brain, SNRPN is prevalent in this structure

No description available.