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Probing Stimulus Encoding in the Olfactory Bulb Using Synthetic Olfactory Stimuli
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyMitral and tufted cells form the main output of the olfactory bulb. I aim to study the stimulus encoding used by the mitral and tufted cells of the olfactory bulb.
I explored the encoding strategies. Neurons can encode sensory information through both spike rate and spike timing. However, in mitral and tufted cells, which of these coding schemes actually drives sensory decisions remains unclear. To dissect their respective roles, I used a system that can modulate the firing rate and timing of the stimuli. By optogenetically activating mitral and tufted cells, I generated distinct activity patterns that varied either in spike rate or timing. My behavioral experiments revealed that mice could quickly learn to discriminate spike number differences but did not learn to distinguish spike timing variations in the sniff cycle using our paradigm. Also, mice were able to discriminate between synchronous vs staggered activations of the output neurons, with accuracy influenced by the timing of stimulus presentation relative to the sniff cycle.
Together, these results suggest that firing rate and synchrony may be robust features of neural codes for olfactory communication downstream of olfactory bulb. These findings contribute to our understanding of neural coding strategies in the olfactory system. Future directions include further research to determine how these principles relate to downstream decoding mechanisms and how they apply to natural odor processing
Decomposing Predictive Information in Social Dynamics
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophySocial behaviors include some of the most interesting interactions in living systems yet their principled characterization remains unsolved. Here, we explore quantitative abstractions of social dynamics in the context of two male zebrafish engaged in a dominance contest. First, we modeled interactions between individuals based on information theory by adapting partial information decomposition to dissect the information between the contestants’ past and future movements using their 3D velocities. At the end of the contest, we find asymmetries between contestants in redundant, synergistic, and unique information indicative of the emergent dominance relationship. We further applied this approach to mecp2 zebrafish mutants, an autism model, we find that predictive information is reduced overall, but especially for synergistic flows, which is indicative of difficulties in more complex social dynamics. Second, we modeled the joint behavior of the two agents as a single dynamical system and applied transfer operator analysis to extract long-lived predictive structures. This analysis revealed non-stationarity even within the fight phase, influencing a structure associated with a key contest strategy: the escalation of attacks by the future loser. Finally, we discuss how social dynamics can be broadly modeled as a process of mutual prediction between organisms, linking social behavior to communication
Exploring the Properties of Hole-Transport Materials for Perovskite Solar Cells: Toward Scalable and Ultra-Flexible Applications
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyMetal halide perovskites are a promising and cost-effective option for high-efficiency solar cells. However, challenges related to stability and scalability remain. This thesis focuses on optimizing the deposition process and investigating the impact of [2-(9H-carbazol-9-yl)ethyl]phosphonic acid (2PACz) stability and performance of inverted perovskite solar cells for both rigid and ultra-flexible substrates. The morphological and energetic properties of 2PACz films deposited via scalable methods such as spray coating and thermal evaporation, were systematically studied. Comprehensive characterization of 2PACz monolayers and thick films revealed that spray-coated 2PACz can slightly enhance performance. In contrast, thermally evaporated 2PACz requires post-deposition treatment and exhibits lower stability than its solution-processed counterpart. Additionally, proof-of-concept perovskite solar modules were fabricated, demonstrating that spray coating is a promising and scalable technique for 2PACz deposition. Furthermore, this work explores the application of carbazole based self-assembled monolayer (SAM) hole transport materials, specifically 2PACz, in the fabrication of ultra-flexible perovskite solar cells. A dual-hole transport layer architecture was investigated, where 2PACz serves as a passivation layer on nickel oxide (NiOX) nanoparticles, enabling record stability of 1,200 h under inert conditions with negligible power conversion efficiency degradation. Ambient stability was also significantly improved by implementing a flexible gas barrier while maintaining ultra-thin and lightweight properties. Finally, a preliminary characterization of the 2PACz/perovskite interface stability was conducted, comparing photo and thermal decomposition processes with and without 2PACz passivation. The results highlight the necessity of advanced interface-sensitive techniques to probe buried interface characteristics under conditions that closely resemble real device operation conditions
Effects of Exogenous Testosterone on the Estrous Cycle, Impulsivity, and Dopamine Transmission in Female Rats
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyThis thesis investigates the behavioral and neural effects of exogenous testosterone on choice impulsivity and nucleus accumbens (NAc) dopamine release in female rats. In Experiment 1, we developed and validated a rodent model of testosterone-based transgender gender-affirming hormone therapy (tGAHT) by demonstrating consistent suppression of estrous cycling following testosterone administration, providing evidence of systemic androgenic effects. In Experiment 2, we tested the effects of testosterone and DHT on sensitivity to delay of reinforcement in an operant task, which measures a component of choice impulsivity. Despite prior evidence linking gonadal steroids to impulsivity and anecdotal reports from people undergoing tGAHT, exogenous androgen administration did not significantly alter sensitivity to delay of reinforcement. This suggests that testosterone may not directly modulate this aspect of impulsive choice behavior in individuals with ovaries. Experiment 3 used fast-scan cyclic voltammetry (FSCV) to examine whether acute and chronic testosterone administration alters NAc dopamine dynamics. Chronic administration of testosterone, but not its non-aromatizable metabolite dihydrotestosterone (DHT), modestly suppressed the enhancement of dopamine signal by methylphenidate, suggesting that the observed chronic effect of testosterone was produced via aromatization to estradiol. These findings build on our emerging understanding of gonadal steroid-dopamine interactions beyond the oversimplified dichotomy of normative "sex differences"
Exploring and Engineering DNA/RNA Methyltransferases for Development of RNA Labeling Tools
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyRNA methyltransferases (MTases) have the potential to facilitate the development of advanced synthetic biology tools for manipulating RNA methylation states and attaching covalent tags to RNA. However, it is challenging due to limited understanding of structure-function relationships in RNA MTases.
In this dissertation, I explore the sequence space of the bacterial DNA methyltransferase EcoGII (M.EcoGII), a promising target for protein engineering due to its lack of sequence specificity and its residual activity on RNA by using ancestral sequence reconstruction. I thereby created SUPREM, a non-site-specific RNA MTase with better expression, thermostability, and RNA methylation activity than M.EcoGII. The SUPREM showed improved RNA methylation activity in mammalian cells and methylated a larger number of RNA methylation than M.EcoGII. Phylogenetic and mutational analysis suggested a residue that enhances the RNA methylation activity of SUPREM.
Next, to expand the scope of the M.EcoGII and SUPREM applications, I investigated DNA-selective M.EcoGII homologs, spatiotemporal control of SUPREM, and the use of synthetic cofactor. A DNA-specific homolog was identified via sequence similarity network analysis. Targeted intracellular localization of SUPREM increased methylation at specific sites. A split SUPREM variants were reconstituted to functional forms in vitro and in E. coli. Our result suggested that SUPREM needs further engineering for the use of synthetic cofactors.
Collectively, this study developed SUPREM and its M.EcoGII derivative enzymes which hold promise as a versatile tool for in vivo DNA and RNA methylation
Information-Theoretical Analysis of Team Dynamics in Football Matches
Full text linkTeam dynamics significantly influence the outcomes of modern football matches. This study employs an information-theoretical approach, specifically causal emergence, combined with graph theory to explore how team-level dynamics arise from complex interactions among players, utilizing tracking data from 34 J-League matches. We focused on how collective behaviors arise from the interdependence of individual actions, examining team coordination and dynamics through player positions and movements to identify emergent properties. Specifically, we selected relative distance to the field’s center, center of mass (CoM) and clustering coefficients based on velocity similarity and inverse distance as macroscopic features to capture the key aspects of team structure, coordination, and spatial relationships. Relative distance and CoM represent the collective positioning of the team, while clustering coefficients provide insights into localized cooperation and movement similarity among the players. The results indicate that average causal emergence with relative distance and CoM as a macroscopic feature across entire games shows a strong correlation with differences in ball possession rate between home and away teams. In contrast, clustering coefficients based on inverse distance and velocity similarity showed moderate to weak correlations with ball possession rate, indicating that these metrics may capture localized interactions that are less directly tied to team-level emergent behavior compared to CoM. Additionally, relative distance and CoM as macroscopic features yield higher causal emergence in attacking phases than in defending phases before shooting, suggesting that the collective positioning of players may play a more significant role in facilitating successful attacks than in defensive stability. This study offers a novel perspective on team coordination in football, suggesting that effective team coordination may be characterized by emergent patterns arising from collective positioning. These findings have practical implications for understanding coordinated team behaviors and inform coaching and performance analysis focused on enhancing team dynamics
Time-resolved Proteomic Analysis of Plasma Membrane Damage-dependent Senescence
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyCellular senescence is a state of stable cell cycle arrest that mediates several physiological and pathological processes, including wound healing, tumor suppression, and aging. We have previously shown that transient plasma membrane damage (PMD) induces premature cellular senescence in normal human fibroblasts. The plasma membrane damage-dependent senescent cells (PMD-Sen cells) exhibit the majority of the senescent cell characteristics. PMD-Sen is primarily induced via the Ca2+-p53 regulatory axis, but not via the best-studied senescence pathway, ATM/ATR-p53. However, the proteomic profile of PMD-Sen and its potential functions in vivo remain largely unknown. To address these questions, we used normal human fibroblasts to perform time-resolved mass spectrometry-based proteomic profiling using PMD-Sen cells, DNA damage response-dependent senescent cells (DDRSen cells), and replicative senescent cells (Rep-Sen cells). These analyses identified CCN1, CCN2, CDH2, NDUFAF2, and DIP2C as proteins specifically upregulated in PMD-Sen cells. Bioinformatics revealed that, after PMD, the extracellular matrix remodeling and wound healing pathways are activated. In contrast, at the late stage of senescence, DNA replication, DNA repair, RNA splicing, and chromatin organization pathways were uniformly downregulated in PMD-Sen cells, DDR-Sen cells, and Rep-Sen cells. We also identified nobiletin as a novel senolytic and senescence prevention agent. These findings will elucidate the mechanisms underlying PMD-Sen and ultimately contribute to the utilization of PMD-Sen-specific biomarkers for in vivo analyses
Epigenetic Regulation of Cold Stress Response and Memory in Arabidopsis thaliana
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyPlants have developed various strategies to cope with environmental stress, including the ability to “remember” previous stress events, which allows them to produce a more efficient response upon re-exposure – a phenomenon known as stress priming. In Arabidopsis thaliana, cold stress priming is associated with transcriptomic and metabolomic changes that confer increased cold tolerance. While transcriptional responses to cold priming – such as accelerated or amplified gene expression, gene sensitization, and downregulation – are well documented, the epigenetic mechanisms underlying these responses remain poorly understood.
In this study, I examined the transcriptome and DNA methylation dynamics in primed and nonprimed A. thaliana under cold stress, using wild-type and DNA methylation-deficient mutants to investigate the epigenetic regulation of cold tolerance and priming. I identified 500 cold memory genes in the wild-type plants which were redistributed in the epigenetic mutants. Cold stress induced widespread DNA methylation changes across all cytosine contexts (CG, CHG, CHH). In addition, cold stress memory genes were significantly hypo-methylated compared to the background genes, suggesting that DNA hypo-methylation can be a hallmark of cold stress memory in A. thaliana. Cold stress memory genes were also enriched for active histone marks, such as H3K4me3 and H3K36me3, and the histone variants H2A.Z and H3.3. The key regulators of cold stress response, including CBF/DREB1 and COR genes, were over-expressed in the met1 DNA methylation mutant, suggesting the role of CG methylation in cold stress response regulation. Finally, I identified cold-responsive and cold-primed non-coding RNAs (ncRNAs) and transposable elements (TEs) that might be involved in cold acclimation and memory. These findings suggest that DNA methylation dynamics contribute to transcriptional memory of cold stress, highlighting the significance of epigenome regulation in stress tolerance and adaptation in plants
Maternal Immune Activation Alters Fetal Cerebellar Development and Motor Behavior in Mouse Offspring via Type-I Interferon
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of Philosoph
Dendritic Voltage Signaling in Cerebellar Purkinje Neurons during Associative Motor Learning
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyThe cerebellar cortex is critical for many forms of associative motor learning. Purkinje neurons (PNs), the sole output of the cerebellar cortex, arborize into massive dendritic trees allowing a wide range of possibilities for integration of input signals to produce an instructive output to downstream neurons. The output pattern of PNs is altered during cerebellar learning, furthermore, it has been reported that changes in the PN dendritic calcium activity are correlated with the strength and duration of a given air puff stimulus in mice. The changes in the postsynaptic voltage response of PN dendrites during learning, however, remains to be explored. The aims of my thesis project are to understand the diverse motor responses during the learning process, which reveal different patterns of conditioned responses that were not previously identified, and to investigate the corresponding changes in dendritic voltage activity during motor learning. I use the voltage sensitive dye ANNINE-6plus combined with 2-photon imaging to study high frequency dendritic complex spikes, spikelets, and dendritic spikes, as well as subthreshold post synaptic potential during delay eyeblink conditioning in habituated awake mice, as they learn for multiple days, with minimal disruption to the neuronal circuits. In addition, I established sophisticated analysis tools to analyze the high dimensional behaviour-voltage data using advanced signal extraction, signal detection, and kmeans clustering algorithms. This is the first study to investigate the gradual changes in the postsynaptic dendritic voltage responses with millisecond temporal resolution, for multiple days, during a learning task