Myocardial infarction (MI) frequently occurs in patients with diabetes resulting in higher mortality and morbidity than non-diabetic patients. We and others have shown that bone marrow-derived endothelial progenitor cells (BM-EPCs) promote cardiac neovascularization and attenuate ischemic injury in animal models. Lately, emerging evidence supports that exosomes (Exo), a family of extracellular vesicles, mediate stem cell therapy by carrying cell-specific biological cargo and by inducing signaling via transferring of bioactive molecules to target cells. Despite promising results of stem cells/Exo in preclinical studies, autologous cell-based therapies yielded modest clinical results, suggesting cellular/Exo reparative function may be compromised by the presence of comorbid diseases including complications associated with diabetes. Recent studies suggest that epigenetic mechanisms, such as histone and DNA modifications for gene silencing, promote diabetes-induced vascular complication. Therefore, we hypothesized that diabetic EPCs produce exosomes of altered and dysfunctional content that compromise their reparative function in ischemic heart disease via epigenetic alterations. We collected EPC-Exo from non-diabetic (db/+) and diabetic (db/db) mice and examined their reparative effect in vitro and on permanent left anterior descending (LAD) coronary artery ligation and ischemia/reperfusion (I/R) myocardial ischemic injuries in vivo. Our data demonstrated that compared to non-diabetic EPC-Exo, diabetic EPC-Exo promoted neonatal rat cardiomyocyte cell apoptosis under hypoxic stress and repressed endothelial tube formation and cell survival. In vivo studies revealed that non-diabetic EPC-Exo treatments improved cardiac function and remodeling while diabetic EPC-Exo significantly depressed cardiac function, reduced capillary density, increased fibrosis in the permanent LAD ligation MI injury. Moreover, in the I/R MI model, we found that non-diabetic EPC-Exo mediated cardio-protection was lost compared with diabetic-EPC-Exo, and diabetic-EPC-Exo increased immune cell infiltration, infarcted area, and plasma cardiac troponin-I. Mechanistically, histone 3 lysine 9 acetylation (H3K9Ac), a gene activating histone modification, expression was decreased in mouse cardiac endothelial cells (MCECs) treated with db/db EPC-Exo compared with db/+ EPC-Exo, suggesting diabetic EPC-Exo inhibits endothelial cell gene expression. The H3K9Ac chromatin immunoprecipitation sequencing (ChIP-Seq) results further revealed that diabetic EPC-Exo reduced H3K9Ac level on angiogenic, cell survival, and proliferative genes in MCECs. Moreover, we found that a small molecular inhibitor of HDACs, valproic acid (VPA), effectively prevented diabetic EPC-Exo-medicated H3K9Ac reduction, indicating VPA may rescue the beneficial gene expression and cell function. Taken together, our results provide evidence that diabetic EPC-Exo reparative function is impaired in the ischemic heart and this may be through HDACs-mediated H3K9Ac downregulation leading to inhibition of beneficial genes in recipient cardiac endothelial cells. Reversing diabetic EPC-Exo function by treating with HDAC inhibitors may provide a new path for autologous exosome therapy for myocardial repair in diabetic patients. However, questions still remain on what the content change of stem cell-derived exosome under diabetic condition is.Emerging evidence support a key role of variety of stem /progenitor cell-secreted Exo as a pivotal paracrine entity to mitigate cardiovascular injury. Beside EPC-, cortical bone stem cell (CBSC)-, and cardiac stem/progenitor cell (CPC)- derived Exo are adequate to enhance cardiac repair and regeneration after injury. As widely acknowledged, the comorbidities such as hyperglycemia is a characteristic of diabetes and a major driving factor in CVD. The functional role of stem/progenitor cell- derived Exo and molecular signature of their secreted Exo cargo under hyperglycemic conditions remain elusive. Therefore, we hypothesize that hyperglycemic stress causes transcriptome changes in stem/progenitor cell- derived Exo that may compromise their reparative function. To identify the content change in Exo under hyperglycemia, we performed an unbiased Exo transcriptome signatures from 3 different aforementioned stem/progenitor cells by next generation exosome RNA sequencing (RNA-seq). The results indicated that the size and number of Exo were not changed from 3 stem/progenitor cells between normal and high glucose groups. Furthermore, analysis revealed differential expression of variety of RNA species in Exo and the portions of different RNA were change under hyperglycemia. Specifically, we identified 241 common-dysregulated mRNAs, 21 ncRNAs and 16 miRNAs in three stem cell-derived Exo. Based on mRNA data, Gene Ontology (GO) revealed that potential function of common mRNAs mostly involved in metabolism and transcriptional regulation. We also provided the detail information of these non-annotated ncRNAs and the potential mRNA targets by miRNA-mRNA prediction. This study not only provides potential candidates for individual stem cell types but also identifies common genes in response to hyperglycemia. These reference data are critical for future biological studies and application of stem/progenitor cell-derived Exo in ischemic heart or other diseases to prevent the adverse effects of hyperglycemia-induced stem/progenitor cell- derived Exo dysfunction. / Biomedical Sciences / Accompanied by five Microsoft Excel files: 1) Supplementary Table 1 2) Supplementary Table 2 3) Supplementary Table 3 4) Supplementary Table 4 5) Supplementary Table 5

In light of the current opioid epidemic, the past 20 years have made it clear that parental life experiences can significantly impact the behavior and neurobiology of their offspring. Preclinical studies indicate that addiction reflects the interaction of an individual’s environment, genetics, and epigenetic modifications they inherit from their parents. Epigenetic mechanisms - including DNA methylation, histone modification, and small non-coding RNAs – refer to the complex interaction between genes and the environment, which produce heritable changes in germ cells that are transmitted to offspring to ultimately influence the brain development and subsequent vulnerability to develop a substance use disorder. The overarching goal of this dissertation was to characterize the behavioral and neurobiological effects of paternal morphine exposure on addiction-related endpoints in offspring. A highly translational rodent model of paternal morphine self-administration was used to produce first-generation (F1) male and female adolescent and adult offspring. As a reference, offspring derived from morphine-exposed fathers were called morphine-sired offspring, and offspring from saline-exposed fathers were called saline-sired offspring. In chapter 2, we revealed that male morphine-sired progeny are more sensitive over time to the pain-relieving effects of morphine. In the periaqueductal grey, an important pain-related brain region, we identified gene expression changes in regulators of G-protein signaling proteins that could partly account for this phenotype. In chapter 3, we demonstrated that adult morphine-sired male offspring self-administered more morphine; were more motived to earn morphine infusions compared to controls; and had more baseline mu-opioid receptor binding in the ventral tegmental area. Next, in chapter 4, we found that a drug-abstinence period of 90 consecutive days following 60 days of morphine exposure in sires was sufficient to prevent morphine-sired males from self-administering more morphine than controls. In chapter 5, we showed that this addiction-like phenotype did not extend to adolescent male or female offspring. Lastly, in chapter 6, using the incubation of craving paradigm, we found that paternal morphine exposure significantly reduced cue-induced active lever pressing for heroin in morphine-sired males. Taken together, these results add to the growing body of literature that show paternal preconception experiences can impact behavioral and neurobiological endpoints in offspring, perhaps via a(n) epigenetically inherited mechanism(s) in the germline. / Psychology

Several factors direct the placement of specific nucleosomes, which in turn have the ability to regulate DNA accessibility. These factors include, but are not limited to, nucleotide sequence preference, nucleotide modifications, the type of histones present within the nucleosome, and the presence of additional transcription factor or chromatin remodelers. A combination of these and other factors are responsible for tightly controlled efficient transcription within the eukaryotic cell. In order to contribute to the understanding of these complicated processes, three separate hypothesis-driven investigations were carried out. First, we looked into nucleosome positioning and phasing within closely related cells lines. Second, we examined domain level nucleosome occupancy on various portions of the chromosome. Finally, we generated a novel method that significantly reduces data loss in in vitro nucleosome reconstitution experiments caused by nucleosome fragment-end bias. All three of our investigations yielded separate results. First, by examining positions and phasing patterns within similar cell types we find common patterns and minor differences within similar cell types. The presence of minor differences in nucleosome positions may cause unique expression patterns. Secondly, we found that decreased domain level nucleosome occupancy at the chromosome arms is not caused by the presence of a class of nucleosomes, termed fragile nucleosomes. Finally, we found that when our nucleosome recovery method is applied conservatively to our dataset, it is possible to recover 80% of the lost nucleosome reconstitution data.

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Immunotherapy offers a promising approach to cancer treatment by harnessing a patient’s own immune system to fight malignant cells. However, the clinical application of immunotherapy has been hindered by the immunosuppressive tumor microenvironment generated by cancer cells as a mechanism to impede immune function and evade immune detection. Clinically used immunotherapies, such as immune checkpoint inhibitors and adoptive cell therapy, aim to overcome the immunosuppressive tumor microenvironment by blocking key regulatory pathways and exogenously activating immune cells. While effective against some cancers, these therapies are still limited by systemic toxicity, poor delivery kinetics, and continuous tumor adaptation that leads to immune escape. Herein, we propose the synthesis of nanoscale branching DNA architectures, known as dendrons, to (1) encode and deliver a DNA sequence, termed G3YSD, capable of activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway; and (2) deliver epigenetic modifiers to reprogram immunosuppressive cues in tumor cells. This solution exploits the modularity, programmability, and ease of control over DNA synthesis to generate architectures that exhibit improved delivery kinetics and favorable presentation of cargo to enhance immunomodulatory effects. Our proposed solution directly targets immunosuppressive mechanisms in tumor cells to sensitize them to immune attack and make them more easily recognized by the immune system. Delivery of G3YSD-encoding dendrons to murine B16 melanoma significantly increased the expression of major histocompatibility complex I (MHC I) and programmed cell death-ligand 1 (PD-L1) surface-bound receptors, which are critical for immune signaling pathways. The chemical conjugation of romidepsin, a histone deacetylase inhibitor, to G3YSD-encoding dendrons resulted in more than a 2-fold increase in MHC I expression compared to unconjugated G3YSD sequences and free romidepsin, indicating that the spatial arrangement and presentation of romidepsin has a synergistic impact on cGAS-STING signaling. In addition, pretreatment of B16 melanoma cells with zebularine, a DNA methyltransferase inhibitor, followed by G3YSD-encoding dendrons significantly increased levels of cytotoxic T lymphocyte-mediated lysis in a physiologically relevant co-culture. Developing novel architectures capable of interacting with tumor cells to remodel and overcome immunosuppressive cues will lead to significant advances in the field of immunotherapeutic design and cancer treatment. / 2026-05-23T00:00:00Z

This dissertation presents applications of machine learning and statistical approaches to infer protein-DNA bindings in the presence of epigenetic modifications. Epigenetic modifications are alterations to the DNA resulting in gene expression regulation where the structure of the DNA remains unaltered. It is a heritable and reversible modification and often involves addition or deletion of certain chemical compounds to the DNA. Histone modification is an epigenetic change that involves alteration of the histone proteins – thus changing the chromatin (DNA wound around histone proteins) structure – or addition of methyl-groups to the Cytosine base adjacent to a Guanine base. Epigenetic factors often interfere in gene expression regulation by promoting or inhibiting protein-DNA bindings. Such proteins are known as transcription factors. Transcription is the first step of gene expression where a particular segment of DNA is copied into the messenger-RNA (mRNA). Transcription factors orchestrate gene activity and are crucial for normal cell function in any organism. For example, deletion/mutation of certain transcription factors such as MEF2 have been associated with neurological disorders such as autism and schizophrenia. In this dissertation, different computational pipelines are described that use mathematical models to explain how the protein-DNA bindings are mediated by histone modifications and DNA-methylation affecting different regions of the brain at different stages of development. Multi-layer Markov models, Inhomogeneous Poisson analyses are used on data from brain to show the impact of epigenetic factors on protein-DNA bindings. Such data driven approaches reinforce the importance of epigenetic factors in governing brain cell differentiation into different neuron types, regulation of memory and promotion of normal brain development at the early stages of life. / Doctor of Philosophy / A cell is the basic unit of any living organism. Cells contain nucleus that contains DNA, self replicating material often called the blueprint of life. For sustenance of life, cells must respond to changes in our environment. Gene expression regulation, a process where specific regions of the DNA (genes) are copied into messenger RNA (mRNA) molecules and then translated into proteins, determines the fate of a cell. It is known that various environmental (such as diet, stress, social interaction) and biological factors often indirectly affect gene expression regulation. In this dissertation, we use machine learning approaches to predict how certain biological factors interfere indirectly with gene expression by changing specific properties of DNA. We expect our findings will help in understanding the interplay of these factors on gene expression.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第25151号 / 理博第5058号 / 新制||理||1721(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 平田 聡, 教授 三谷 曜子 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Transgenerational epigenetic inheritance in mamals is a widely discussed topic in today's biology. Epigenetic modifications are molecules that play a crucial role in regulation of gene transcription. Epigenetic modifications regulate another epigenetic modification's establishment. The extrinsic and the intrinsic cellular or organismal environment is involved within the establishment of epigenetic state. The molecules involved in epigenetic processes are able to regulate gene transcription in reaction to the environment and therefore these molecules partly shape the phenotype. Most importantly, epigenetic processes are affected by cellular or organismal history. A question emerges: Are these molecules able to transfer information through germline to subsequent generations? Does transgenerational epigenetic inheritance in mammals exist? Experimental data show it is so. What consequences this can mean in our understanding of evolution? Powered by TCPDF (www.tcpdf.org)

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