京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18838号 / 理博第4096号 / 新制||理||1589(附属図書館) / 31789 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 阿形 清和, 教授 杤尾 豪人, 教授 森 和俊 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20233号 / 医博第4192号 / 新制||医||1019(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 斎藤 通紀, 教授 松田 文彦, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Ionic and osmotic homeostasis, and the intricately linked mechanisms of acid-base balance are critical for the survival of fish. To date, the role of the gastrointestinal (GI) tract in these processes has received only limited study, and our knowledge has been gained almost exclusively through studies conducted in fasting animals. The impacts of feeding and digestion, ubiquitous processes in the natural environment, are likely to be significant but have been overlooked. The current thesis addressed these shortfalls in our current understanding. Research focused on the rainbow trout (Oncorhynchus mykiss), a euryhaline species capable of withstanding the opposing challenges of life in seawater (diffusive influx of ions and loss of water) and freshwater (diffusive loss of ions and gain of water), and concentrated on its physiological response to ingestion of a meal of commercial, dry trout food, containing concentrated salts and little water. The net absorption and secretion of ions and water was tracked in each section of the GI tract of the rainbow trout over a detailed time course using an experimental diet that contained a simple inert marker, in the presence of external freshwater or seawater. Additionally, changes in overall blood chemistry were investigated to examine changes in osmotic, ionic and acid-base regulation during digestion. Feeding in freshwater resulted in the loss of endogenous water to the GI tract during digestion. Additionally, the meal provided much needed ions to balance those lost by diffusion; indeed all of the ingested ions were assimilated along the GI tract except for sodium which was absorbed in the stomach, but secreted in the intestine such that overall sodium balance was close to zero. Feeding also created a metabolic base load (an increase in the concentration of base, or HC03- due HCl secretion into the stomach lumen) that alkalinized the blood (i.e. caused a rise in pH), a phenomenon known as an alkaline tide. The base load was subsequently removed from the blood through increasedexcretion of base to the water via the gills. In seawater, the commercial diet again provided an avenue for water loss. This was potentially deleterious to an organism already suffering from diffusive water loss to the environment. Ion absorption from the diet was negligible, except for potassium and calcium, which were readily assimilated. As in freshwater, digestion resulted in an alkaline tide, however the mechanism of acid-base homeostasis differed with the excess base likely being excreted into the intestine. In contrast to freshwater fish, the gills took up additional base from the external environment, prolonging the acid-base disturbance in seawater fish. Overall, feeding was a dynamic process with far reaching systemic physiological effects. The research described highlighted intimate interactions between the processes of feeding and digestion and ion, water and acid-base homeostasis, and elucidated mechanisms that enable fish to inhabit a wide range of environments. / Thesis / Doctor of Philosophy (PhD)

R-loop is a specific nucleic acid structure, and that forms during RNA transcription. It comprises an RNA:DNA hybrid and displaced ssDNA. R-loops are prevalent and dynamic in the mammalian genome occupying up to 5%. R-loops are known to act as modulators of genome dynamics. They regulate a variety of gene transcription mechanisms and influence genomic stability. Dysregulation of R-loop is linked to a variety of human diseases. For this reason, protein factors involved in DNA and RNA metabolism are known to mediate R-loop resolution. Dysfunction of these factors resulted in aberrant R-loop accumulation, often resulting in transcription disruption and genomic instability as reported in tumorigenesis and a number of genetic disorders, including trinucleotide repeat-associated diseases and neurological disorders. Here I describe the roles of Senataxin (SETX) and Replication protein A (RPA) complex in nucleolar R-loop resolution. I demonstrate their function in nucleolar homeostasis and in particular in RNA polymerase I mediated rRNA transcription and the maintenance of nucleolar structure.This dissertation is composed of three sessions. Section 1: I review nucleolar transcription that generate a complex network of RNAs, and their contribution to nucleolar R-loops formation, with multiple roles in maintaining nucleolar homeostasis. Section 2: I describe novel roles of Replication Protein A in nucleolar homeostasis. Senataxin (SETX) mutations are linked to two different neurological disease: Amyotrophic Lateral Sclerosis (ALS4) and Ataxia Oculomotor Apraxia (AOA2) both defective in R-loop resolution. We show that loss of SETX promotes RPA translocation into nucleolus in an R-loop dependent manner where it associates with rDNA. The same nucleolar RPA phenotype is evident in SETX- deficient AOA2 patient cells. We further explored this phenotype under conditions of CPT- induced genotoxic stress, which is coupled with accumulation of nucleolar R loops. Additionally, we show that loss of RPA decreased 47S pre-rRNA levels, but increased “promoter and pre-rRNA antisense” RNA (PAPAS) and promoter RNA (pRNA). Meanwhile, we also showed loss of RPA disrupted nucleolar structure. Section 3: I describe the identification and characterization of nucleolar lncRNA (PAPAS) which encodes a short polypeptide RIEP that plays a role in combating genomic instability and mitochondrial stress. We show that this novel peptide encoded by PAPAS is localized to the nucleolus and mitochondria but is translocated to the peri-nucleolus and peri-nucleus region upon heat shock induced cellular stresses. We also showed that RIEP facilitates SETX protein stability and plays a role in restricting genomic instability possibly through its association with H3K9me3 which maintains a heterochromatin state. Finally, we show that RIEP interacts with CHCHD2 and C1QBP(P32) and may consequently function in mitochondrial stress responses.

Adipose tissue is a key metabolic organ responsible for maintaining energy homeostasis throughout the body. Healthy adipocytes respond to physiological changes and perform a variety of important functions to regulate glucose and lipid metabolism. Dysregulation of adipose tissue function, on the other hand, is strongly associated with the development of metabolic diseases. Peroxisome Proliferator Activated Receptor gamma (PPARγ) is a key transcription factor that regulates various activities in adipocytes as well as other cell types. A growing body of evidence indicates a more complex role for PPARγ beyond its classical ligand-dependent activity, including the exploration of posttranslational modifications and associated target proteins in non-canonical adipogenic reservoirs and adipocyte-associated cells. The first part of the thesis describes our study identifying Adipsin as a downstream target of PPARγ deacetylation and further uncovers its function within the bone marrow niche. Unlike peripheral adipose tissues, marrow adipose tissue has been shown to be uniquely responsive to nutrient fluctuations, hormonal changes, and metabolic disturbances such as obesity and diabetes mellitus. Expansion of marrow adipose tissue has also been strongly associated with bone loss in mice and humans. However, the regulation of bone marrow plasticity remains poorly understood, as does the mechanism that links changes in marrow adiposity with bone remodeling. We show that Adipsin was robustly induced in the bone marrow during bone loss in mouse and humans, in a manner dependent on PPARγ acetylation. Ablation of Adipsin inhibited marrow adipose expansion and improved skeletal health in bone loss conditions of calorie restriction, thiazolidinedione treatment for insulin resistance, and aging. These effects were mediated by Adipsin’s downstream effector, Complement Component 3, to prime common progenitor cells toward adipogenesis rather than osteoblastogenesis through the inhibition of Wnt/β-catenin signaling. Together, our findings reveal an unknown function of Adipsin, mediated by PPARγ acetylation, to promote adiposity and affect skeletal remodeling in the bone marrow niche. The second part of the thesis addresses another novel role for PPARγ, through acetylation in macrophages, to promote adipose tissue inflammation. Chronic, low-grade inflammation characteristic of obesity and metabolic dysfunction is partially driven by macrophage infiltration of adipose tissue and associated inflammatory signaling. PPARγ plays a critical role in regulating anti-inflammatory, M2 polarization of macrophages. However, the involvement of post-translational modifications, such as acetylation, in macrophages is unknown. Here we generated a macrophage specific, PPARγ constitutive acetylation-mimetic mouse line (K293Qflox/flox;LysMcre, mK293Q) to dissect its role. Upon stimulating macrophage infiltration into adipose tissue by high-fat diet feeding, we assessed the overall metabolic profile and tissue-specific phenotype of the mutant mice. We found that the mK293Q mutant promotes pro-inflammatory macrophage infiltration and subsequent fibrosis specifically in epididymal but not subcutaneous white adipose tissue, driving an impaired metabolic response including decreased energy expenditure, insulin sensitivity, glucose tolerance, and adipose tissue function. These detriments are driven by suppressed anti-inflammatory activation of macrophages. Furthermore, mK293Q mice are resistant to improvements in adipose remodeling by Rosiglitazone treatment. Our study reveals acetylation as a new layer of PPARγ regulation in macrophage activation. These findings highlight the importance of post-translational modifications in determining the function of PPARγ when regulating metabolism and promote the discovery of anti-inflammatory associated therapeutics.

No description available.

No description available.

Regulation of intracellular pH is responsible for many cellular processes, such as metabolism, cell cycle progression, and apoptosis. Many chemotherapeutic agents work by inducing target cells to undergo apoptosis, a cell death process still poorly understood. Previous studies demonstrated that a rise in intracellular pH activated apoptotic proteins leading to cytochrome C release. This "apoptotic alkalinization" occurred upon activation of the plasma membrane protein, sodium hydrogen exchanger-1 (NHE1), whose activity is regulated by the stress kinase p38 MAPK. In previous studies, upon cytokine withdrawal from cytokine-dependent lymphocytes induced the activity of the p38 MAP kinase which then phosphorylated the C-terminus of NHE1. To identify the p38 MAPK phosphorylation sites on NHE1, in vitro p38 MAP kinase assays coupled to deletion analysis of NHE1 and mass spectrometry, identified four possible p38 MAPK phosphorylation sites. To establish that NHE1 causes apoptotic alkalinization and determine whether the identified phosphorylation sites on NHE1 are functionally significant, we used PCR site directed mutagenesis to mutate T717, S722, S725, and S728 on the C-terminus of NHE1. Stable NHE1 deficient cell lines, expressing wild type (WT) NHE or the four mutated sites (F4MUTNHE), were assessed for apoptotic alkalinization using the pH-sensitive fluorescent protein, destabilized YFP. Our results show that NHE1 is required for apoptotic alkalinization, since expression of WT NHE restored alkalinization in an NHE deficient cell line, and that this process requires the phosphorylation of the p38 MAPK target sites, since mutation of all four sites prevented the apoptotic alkalinization response.

Iron homeostasis is a vital process that balances access to free iron to fuel physiological processes with iron storage to reduce the deleterious aspects of excess free iron. Dysfunctional iron homeostasis can lead to iron overload, in which excess free iron can promote cellular injury, ferroptosis, and eventual organ damage as a result. The cellular consequences of long-term iron overload including the cellular pathways that lead to pathological changes or those that may provide protection against damage are incompletely defined. Here, we use dietary and genetically engineered mouse models of iron overload combined with mass spectrometry-based quantitative proteomics to determine the iron overload liver proteome. We identify and quantify over 8,000 proteins representing the most in-depth iron overload proteome evaluation to date. Using bioinformatics, we identify conserved upregulated pathways including ‘response to oxidative stress’ and downregulated pathways including ‘steroid homeostasis.’ Furthermore, we identify an unexpected role for NCOA4, an autophagy adaptor that targets ferritin for autophagic degradation during iron deprivation, during iron overload. This work highlights the importance of further investigation into NCOA4 and its role in diseases of iron overload. / 2025-01-30T00:00:00Z

A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree Master of Science. / Sprint interval training (SIT) is a time-efficient strategy to improve cardiorespiratory fitness; however, most protocols have been studied in a laboratory setting and require specialized equipment. We investigated the efficacy of brief intense stair climbing as a practical model of SIT to improve cardiometabolic health, with a key measure being cardiorespiratory fitness as indicated by peak oxygen uptake (VO2peak). Two separate studies, each consisting of an acute and chronic phase, were conducted in a total of 31 sedentary women (age=24±10 y; BMI=23±4 kg•m-2). The acute phase of Study 1 established that the heart rate (HR), blood lactate concentration (BLa), and rating of perceived exertion (RPE) responses were similar when participants (n=8) performed a SIT protocol that involved 3x20-s “all-out” efforts of either continuous stair climbing or cycling, interspersed with 2 min of recovery. The chronic phase demonstrated that when participants (n=12) performed the 3x20-s stair climbing protocol 3 d•wk-1 for 6 wk, absolute and relative VO2peak increased by 12%, or ~1 metabolic equivalent (1.80±0.25 to 2.02±0.27 L•min-1, p<0.001), as there were no changes in body mass (p=0.35), fat free mass (FFM; p=0.09) or % body fat (p=0.42). There were also no changes in resting systolic and diastolic blood pressure (BP; p=0.82 and p=0.97, respectively), resting HR (p=0.62), and fasting insulin sensitivity (p=0.52). The acute phase of Study 2 established that the HR and RPE responses were similar when participants (n=11) performed three different stair climbing protocols. The protocols investigated include the 3x20-s continuous ascent model used in Study 1 (protocol 1), as well as 3x60-s bouts of ascending and descending either one or two flights of stairs, with 60-s of recovery (protocol 2 and 3, respectively). The chronic phase demonstrated that when the same group of subjects performed the 3x60-s 1-flight protocol 3 d•wk-1 for 6 wk, absolute and relative VO2peak increased by 8 and 7%, respectively (1.79±0.36 to 1.93±0.39 L•min-1, p=0.001; 31.2±4.6 to 33.3±5.3 mL•kg-1•min-1; p=0.01). Despite no changes in % body fat (p=0.10), there was an increase 3% increase in FFM (p<0.001). There was no change in systolic (p=0.50) and diastolic BP (p=1.00), but resting HR improved by 8% after training (p=0.03). The change in insulin sensitivity derived from an OGTT was 7.1±11 mg I2•mmol-1•mIU-1•min-1 (p=0.056). These findings demonstrate that brief intense stair climbing is a practical, time-efficient strategy to improve cardiorespiratory fitness in previously untrained women. / Thesis / Master of Science (MSc) / Sprint interval training (SIT), involving brief bouts of very intense exercise separated by short periods of recovery, is a time-efficient alternative to traditional endurance training for improving fitness. This has largely been established in laboratory settings using specialized equipment, which is impractical for many individuals. This project examined whether brief intense stair climbing was a practical model of SIT to elicit adaptations previously shown with cycling protocols. Subjects performed either three 20-s ascents interspersed with 2 min recovery periods, or three 60-s bouts of ascending and descending one or two flights of stairs, with 60-s recovery periods. Both protocols were 10 min in duration including warm-up and cool-down, and subjects trained three days per week for six weeks. The main finding was that stair climbing is a practical, time-efficient model to improve fitness in previously sedentary individuals.