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Sleepy Heads: Active and Quiet Sleep in Octopus laqueus
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyIn various vertebrate lineages, sleep is typically composed of two distinct stages: rapid eye movement (REM) and slow-wave sleep (SWS), characterized by patterns of brain activity that are, respectively, wake-like and synchronous. This study presents a parallel in octopuses, sophisticated marine invertebrates diverged from vertebrates roughly 550 million years ago. In octopuses, a state akin to ‘quiet’ sleep is periodically interrupted by approximately one-minute intervals marked by vigorous eye and body movement and transitions in skin patterns and textures. Both these states bear the hallmarks of sleep: they are homeostatically regulated, rapidly reversible, and have a heightened arousal threshold. Using deep neural networks, we analyzed the skin patterns exhibited during active sleep. We found that the skin patterns during active sleep were diversified yet consistent for individual octopuses, bearing a striking resemblance to wakeful skin patterns. Electrophysiological studies of the central brain during active sleep indicate that local field potential (LFP) activity is similar to wakefulness. This activity varies among different brain regions, with the most pronounced activity observed within the superior frontal and vertical lobes—areas linked to learning and memory functions. In contrast, during quiet sleep, these regions exhibit reduced activity, punctuated by oscillatory LFPs that are similar to mammalian sleep spindles in both frequency and duration. The observed parallels in sleep architecture between octopuses and vertebrates hint at an evolutionary convergence, suggesting that two-stage sleep could be a common neural phenomenon associated with sophisticated cognitive functions or vision
The Role of the AP-1 Transcription Factor JunB in the Maintenance and Function of Mature T Helper Cells
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyThe AP-1 transcription factor JunB plays a crucial role in CD4+ T helper (Th) cell responses, including host defense and autoimmune inflammation, such as in multiple sclerosis and colitis. Mice with JunB-deficient CD4+ T cells are fully protected from these diseases. However, the role of JunB in already differentiated, mature Th cells remains unclear. In this thesis, I employed the degradation tag (dTag) system to investigate the function of JunB in mature Th cells. By engineering dTag-JunB knock-in mice, where JunB is fused to an FKBP12F36V dTag sequence, JunB could be rapidly degraded in vitro and ex vivo upon dTAG-V1 ligand addition. During pathogenic Th17 (pTh17) cell polarization, JunB degradation mirrored known effects of JunB deficiency. In mature Th cells, I found that JunB promotes cell survival and the stability of Th cell phenotypes. Unlike during pTh17 polarization, JunB degradation in mature pTh17 cells did not affect the expression of Rorc or IL-17A, but did downregulate Il23r. Additionally, JunB was found to mediate activin A signaling, essential for pTh17 polarization, via Inhba upregulation. While JunB-BATF heterodimers are necessary for pioneering chromatin accessibility at the key Th17 loci Rorc, Il17a and Il23r during pTh17 polarization, I found that JunB is not required to maintain this accessibility in mature cells. However, JunB is needed to sustain the active histone marks H3K4me1 and/or H3K27ac at the Il17a and Il23r loci. For successful autoimmune disease therapy, it is important not only to block the differentiation of pTh17 cells but also to control mature pTh17 cells. As IL-23 signaling is critical for Th17 pathogenicity and pTh17 cell maintenance, the role of JunB in promoting Il23r expression positions JunB as a potential therapeutic target
Physiological and Transcriptomic Response of Early-Life Stage Clownfish to Future Ocean Warming and Marine Heatwaves
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyCoral reef fish are currently under pressure from anthropogenic CO2 emissions which are exacerbating the greenhouse effect and driving long-term changes in mean sea surface temperatures, whilst also increasing the frequency and intensity of marine heatwaves. Increasing temperatures threaten to push coral reef fish beyond their natural thermal limits, with previous studies indicating this will elicit a range of adverse physiological and molecular responses. Nevertheless, the immediate, acclimatory and legacy effects of increasing temperature on early life-stage coral reef fish are largely unclear, with the molecular mechanisms underpinning many of the previously observed phenotypes still unidentified. To address these knowledge gaps, I use a combination of genome sequencing, transcriptomics, and physiological measurements to investigate the response of early life-stage clownfish (Amphiprion ocellaris) to temperatures associated with ocean warming and marine heatwaves. Firstly, I present two high quality chromosome-scale genomes for the anemonefish Amphiprion ocellaris and Amphiprion clarkii, that will aid future studies of these species. Then, via a series of aquaria-based experiments I show that A. ocellaris exhibit a range of physiological changes at +3°C, as larvae develop faster, and both larvae and juvenile clownfish display elevated metabolic rates. However, I also show that early exposure to +3 °C may have an acclamatory effect, as the effect of acute temperature stress on metabolic rates are reduced with increasing developmental exposure. Furthermore, via stage- and tissue-specific transcriptome sequencing of larvae and juveniles I show widespread changes in gene expression at +3 °C. These genes encompass a range of biological functions including immune response, epigenetic reprogramming, neurotransmission, heat-stress, liver fibrosis and insulin signalling. Overall, these results indicate that A. ocellaris may experience a significantly different developmental regime if they hatch and develop at +3 °C, with these changes likely to impact future population persistence. Moreover, the comprehensive transcriptomic sequencing conducted here, advances our understanding of how coral reef fish will respond to future climate change at the molecular level. Chapters one and two of this thesis have been peer-reviewed and published, and chapter three is currently in preparation for journal submission
Cross-talk between tissues is critical for intergenerational acclimation to environmental change in Acanthochromis polyacanthus
Full text linkOrganisms’ responses to environmental changes involve complex, coordinated responses of multiple tissues and potential parental influences. Here using a multi-tissue approach we determine how variation in parental behavioural tolerance and exposure to elevated CO2 influences the developmental and intergenerational molecular responses of their offspring in the coral reef fish Acanthochromis polyacanthus to future ocean acidification (OA) conditions. Gills and liver showed the highest transcriptional response to OA in juvenile fish regardless of parental OA conditioning, while the brain and liver showed the greatest intergenerational acclimation signals. Developmentally induced signals of OA, such as altered neural function in the brain, were restored to control levels after intergenerational exposure. Intergenerational CO2 exposure also enabled the offspring to adjust their metabolic processes, potentially allowing them to better meet the energetic demands of a high CO2 environment. Furthermore, offspring of OA-exposed parents differentially expressed a new complement of genes, which may facilitate intergenerational acclimatory responses. A genetic component of intergenerational plasticity also played a crucial role, with the parental behavioural phenotype largely determining the offspring’s transcriptional signals. Overall, our results reveal tissue-specific transcriptional changes underlying intergenerational plastic responses to elevated CO2 exposure, enhancing understanding of organismal acclimation to OA throughout the whole body
Dscamb Regulates Zebrafish Cone Mosaic via Filopodium-Mediated Homotypic Recognition
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyCone photoreceptors, the color-detecting neurons in the retina, organize themselves into distinct cellular patterns known as cone mosaics. In zebrafish, red, green, and blue cones bind together to create the characteristic G-R-B-R-G pentameric cone clusters that alternate with UV cones, forming a crystal structure-like mosaic. This beautiful cellular pattern has surprised and baffled researchers for decades. However, to date, no molecule has been revealed to be crucial in the cone mosaic formation. In my thesis research, I discovered that the deficiency of Dscamb, a cell adhesion molecule, led to disrupted cone mosaic. Through time-lapse live imaging, I noticed that red cones extend many filopodia, filamentous cellular protrusions, to probe the surrounding environment at the outer limiting membrane. Interestingly, filopodia were inhibited upon contact with other red cones, mediated by the homophilic interaction of Dscamb. In contrast, filopodia in the dscamb mutants continued to grow after homotypic contact, crawling over the apical region of other red cones. Considering the importance of filopodia in cell migration, I propose that Dscamb-mediated inhibition of filopodia limits the ability of cells to migrate towards each other, thereby promoting homotypic repulsion among cells. Other cone types might also follow similar rules to establish their own regular patterns, contributing to the construction of the cone mosaic
Unraveling Neuronal Cell Assemblies: From Computational Models to Pattern Detection
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyAn intriguing and open question in neuroscience is why neurons maintain activity even when there is no external stimulation or task at hand. This fundamental property of the nervous system, called spontaneous activity, is thought to result from ongoing information processing and maintenance and is essential for cognitive functions including memory, perception and spatial navigation. Of special interest is structured spontaneous activity, which is believed to result from the activity of cell assemblies – groups of strongly connected and functionally related neurons – which are theorized by some to be the fundamental units of memory. Despite the popularity of this view, complete mechanistic understanding of how cell assemblies might implement memory and potentially other cognitive functions, remains elusive. In this thesis I attempt to address this gap by investigating, in a biologically plausible and experimentally constrained recurrent network model, the spontaneous and evoked emergence of cell assemblies as well as their properties that might be relevant for memory function. I specifically model the contribution of various plasticity mechanisms and astrocytes. To complement the theoretical contribution of this thesis, I also propose two methods for the detection of patterned neural activity. The first uses backprop-optimized filters, offering significant improvements in speed and scalability over similar techniques. The second utilizes graph neural networks, presenting an innovative unsupervised approach to pattern detection by representing spike data as graphs. Both the theoretical insights and the proposed pattern detection methods contribute to advancing our understanding of spontaneous neural activity and its role in cognitive functions
Characterization of an Overlooked Kinematical Descriptor in the Second-Gradient Hyperelastic Theory for Thin Shells
Full text linkIn 1978, Murdoch presented a direct second-gradient hyperelastic theory for thin shells in which the strain-energy density associated with a deformation η of a surface S is allowed to depend constitutively on the three kinematical descriptors C, H, and F^⊤ G, where F=〖"Grad" 〗_S η, C=F^⊤ F, H=F^⊤ L_(S^' ) F is the covariant pullback of the curvature tensor L_(S^' ) of the deformed surface S^', and G=〖"Grad" 〗_S F. On the other hand, in Koiter’s direct thin-shell theory, the strain-energy density depends constitutively on only C and H. Due to the popularity of Koiter’s theory, the second-order tensors C and H are well understood and have been extensively characterized. However, the third-order tensor F^⊤ G in Murdoch’s theory is largely overlooked in the literature. We address this gap, providing a detailed characterization of F^⊤ G. We show that for η twice continuously differentiable, F^⊤ G depends solely on C and its surface gradient 〖"Grad" 〗_S C and does not depend on L_(S^' ). For the special case of a conformal deformation, we find that a suitably defined strain measure corresponding to F^⊤ G depends only the conformal stretch and its surface gradient. For the further specialized case of an isometric deformation, this strain measure vanishes. An orthogonal decomposition of F^⊤ G reveals that it belongs to a ten-dimensional subspace of the space of third-order tensors and embodies two independent types of non-local phenomena: one related to the spatial variations in the stretching of S^' and the other to the curvature of S
Development of Catalytic Asymmetric Reactions of Pyruvates as Nucleophiles
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyPyruvates or α-ketoesters, are important molecules in both organic chemistry and biology due to their intrinsic reactivity. Pyruvates can act as nucleophiles (reactions as enolates and enamines) and as electrophiles (reactions at the ketone and ester carbonyl groups). In enzyme catalyzed reactions, pyruvates are often used as nucleophiles. Enzymes can precisely control the dual reactivity of pyruvates. In contrast, non-enzymatic reactions of pyruvates often lean towards reacting as electrophiles, and reactions of pyruvates as nucleophiles are challenging. Especially, simple pyruvates, such as ethyl pyruvate and methyl pyruvate, readily react each other to give self-aldol (homo-aldol) reaction products. In this thesis study, stereoselective (enantioselective and diastereoselective) bond formation reactions of pyruvates as nucleophiles with electrophiles catalyzed by small-molecule amine catalysts were developed. The small molecule amine-catalyzed reactions of pyruvates would be used as alternatives to pyruvate enzyme-catalyzed reactions. Firstly, enantioselective Mannich reactions between simple pyruvates (e.g. ethyl pyruvate and methyl pyruvate) and cyclic imines catalyzed by a primary amine-acid-based catalyst system were developed. The application of the methodology was demonstrated by transformations of the Mannich product to various functionalized molecules including amino acid and glycolic acid derivatives. Secondly, investigations were performed to understand the mechanisms of the developed Mannich reactions catalyzed by the amine-acid catalyst system, including to understand the effect of acids used with the amine catalyst in controlling the enantioselectivity of the Mannich products and to understand the selective formation of the Mannich products over self-aldol products of pyruvates. The key role of the acid of the amine-acid catalyst system in catalyzing the Mannich reaction as well as in controlling the enantioselectivity was uncovered. Selection of the acid of the amine-acid catalyst system was important to minimize self-aldol reactions of pyruvates. Thirdly, diastereo- and enantioselective Mannich reactions of substituted pyruvates (i.e., 2-oxobutanoates and larger α-ketoester derivatives) with imines (cyclic and acyclic) were enabled by the use of a tertiary-amine-based catalyst bearing a thiourea group and a sulfonamide group. Last, aminecatalyzed enantioselective aldol reactions of simple pyruvates as nucleophiles with aldehydes and ketones as electrophiles were developed. A diamine-based catalyst having a primary amine group and a sulfonamide group controlled the cross-aldol reactions of simple pyruvates. The developed methodology serves a way for the synthesis of enantioenriched γ- hydroxy-α-amino esters from pyruvates
Dynein Light Chain Roadblock 1 Regulates FMRP Axonal Transport and Degradation
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyThe fragile X messenger ribonucleoprotein 1 (FMRP) is a multifunctional RNA binding protein (RBP) implicated in human neurodevelopmental and neurodegenerative disorders. FMRP mediates the localization and activitydependent translation of its associated mRNAs through the formation of phase separated condensates that are trafficked by microtubule-based motors in axons. Axonal transport and localized mRNA translation are critical processes for longterm neuronal survival and are closely linked to the pathogenesis of neurological diseases. FMRP dynein-mediated axonal trafficking is still largely unexplored, but likely to constitute a key process underlying FMRP spatiotemporal translational regulation. Here, we show that roadblock 1 (Dynlrb1), a subunit of the dynein complex, is a critical regulator of FMRP function in sensory neurons. In axons, FMRP associates with the dynein complex and is retrogradely trafficked in a Dynlrb1-dependent manner. Moreover, Dynlrb1 silencing induced FMRP granules accumulation and repressed the translation of Map1b, one of its primary mRNA targets. Our findings suggest that Dynlrb1 regulates FMRP function through the control of its transport and degradation
Effects of Mean Flow and Turbulence on Planktivory by Anchored Garden Eels and Site-Attached Fish
Full text linkOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyWater flow is a key environmental factor that affects fish in coral reefs. Changes in flow properties, such as mean flow speed and turbulent fluctuation, have been expected to affect the feeding of zooplanktivorous fish through its effects on the motions of both prey and predator. Flows are critical not only for freely swimming fish but also for anchored fish, such as garden eels, that feed while anchored to the sandy bottom by keeping the posterior parts of their bodies inside a burrow.
The objective of this study is to comparatively examine effects of flows on freely swimming reef fish and anchored garden eels to understand ecological traits, such as adaptation, habitat selection, and prey-predator interaction. To address this objective, I combined flow measurements in the field and flume experiments designed to examine effects of mean flow speed and small-scale turbulence on feeding and energy cost of reef damselfish (Chromis viridis) and garden eels (Heteroconger hassi). In situ flow measurements by acoustic Doppler velocimeter (ADV) indicated faster flow speed and stronger turbulence at damselfish habitat above corals compared with garden eel habitat on a flat sandy bottom. Based on the in situ flow measurement, a range of mean flow speed and small-scale turbulence in dissipation range was reproduced in flumes to examine fish responses by labelfree tracking of body points and 3D movement analysis. The relationship between feeding rate and flow speed showed an adaptation of damselfish to faster flow speed compared to garden eels. The energy cost and benefit model also indicated that the energetically optimal range of flow speed of damselfish was faster than that of garden eels. Detailed motion analysis revealed a unique strategy of garden eels to flow speed, leading to the development of first foraging model for this group of fish. The anchored and site-attached fish also differed in their responses to small-scale turbulence: strong turbulence caused a decrease in feeding rates under slow flows for damselfish and under fast flows for garden eels. The turbulence effect was associated with a reduction of the foraging area for damselfish and an increase of the search time for garden eels. By combining field flow measurements and flume experiments, my study advanced the understanding of adaptations to hydrodynamic conditions in fish