京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第21874号 / 人博第903号 / 新制||人||215(附属図書館) / 2018||人博||903(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 小林 純也, 教授 宮下 英明, 准教授 三浦 智行 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13360号 / 論農博第2891号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5299(農学部図書室) / (主査)教授 中﨑 鉄也, 教授 白岩 立彦, 教授 土井 元章 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

abstract: Anoxia tolerance is strongly correlated with tolerance to heat, desiccation, hyperosmotic shock, freezing, and other general stressors, suggesting that anoxia tolerance is broadly related to stress tolerance. Age affects the capacity of many animals to survive anoxia, but the basis to this ontogenic variation is poorly understood. We exposed adult Drosophila, 1, 3, 5, 7, 9, and 12 days past eclosion, to six hours of anoxia and assessed survival 24-hours post-treatment. Survival of anoxia declined strongly with age (from 80% survival for one-day-old flies to 10% survival for 12 day-old-flies), a surprising result since adult fly senescence in Drosophila is usually observed much later. In anoxia, adenosine triphosphate (ATP) levels declined rapidly (< 30 min) to near-zero levels in both 1 and 12-day old adults; thus the higher anoxia-tolerance of young adults is not due to a better capacity to keep ATP elevated. Relatively few physiological parameters are reported to change over this age range in D. melanogaster, but gut bacterial content increases strongly. As a partial test for a causal link between bacterial load and anoxia tolerance, we replaced food daily, every third day, or every sixth day, and assayed survival of six hours of anoxia and bacterial load at 12 days of age. Anoxia tolerance for 12-day old flies was improved by more food changes and was strongly and negatively affected by bacterial load. These data suggest that increasing bacterial load may play an important role in the age-related decline of anoxia tolerance in Drosophila. / Dissertation/Thesis / Masters Thesis Biology 2020

This experiment attempted to study the effectiveness of biofeedback training on an individual's peak performance while performing a stress-provoking task. In a between subjects design 30 individual college students were divided into 2 groups. One group received auditory biofeedback and relaxation training, and the other group received no training. Both groups met the researcher for 1 hour a week for seven weeks. During each session the participant completed a ten-minute Competition and Coaction (C & C) computer software program (Shea, 1992). The biofeedback equipment recorded the physiological state of each of the participants while he/she performed the C & C task. Both groups' physiological values and C & C scores were compared using the SPSS software. The biofeedback group had statistically lower stress values than the non-biofeedback group (Schwartz, 1995). There was no statistical difference between the 2 groups' C & C scores. The STAI Y-1 and Y-2 anxiety inventories were given to each of the participants at the 1st and 7th session to examine the anxiety differences between the 2 groups. There were no statistical differences between the 2 groups' STAI scores. This study's findings show that individuals can be taught to lower their stress response while performing a computer task.

Indiana University-Purdue University Indianapolis (IUPUI) / Human skin, consisting of the outer epidermis and inner dermis, serves as a barrier that protects the body from an onslaught of environmental stresses. Keratinocytes in the stratified epidermis undergo sequential differentiation that consists of multiple layers of cells differing in structure and function. Therefore, keratinocytes must not only combat environmental stress, but need to undergo massive changes in gene expression and morphology to form a proper barrier. One mode by which cells cope with stress and differentiation is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P), which causes global inhibition of protein synthesis coincident with preferential translation of select gene transcripts. Translational repression allows stressed cells to conserve energy and prioritize pro-survival processes to alleviate stress damage. Since eIF2α kinases are each activated by distinct types of stress, this pathway is referred to as the Integrated Stress Response (ISR). We sought to identify the roles of the ISR in the keratinocyte response to the stresses associated with differentiation and ultraviolet B (UVB) irradiation. In this thesis, we show that both general and gene-specific translational control in the ISR are activated following differentiation or UVB irradiation of human keratinocytes. ISR deficiency through genetic modifications or pharmacological interventions caused severe divergence from the appropriate keratinocyte response to differentiation or UVB. Differentiation genes were selectively translated by eIF2α-P, and inhibition of the ISR diminished their induction during differentiation. Furthermore, loss of the eIF2α kinase GCN2 (EIF2AK4) adversely affected the ability of keratinocytes to stratify in three dimensional cultures. Our analysis also revealed a non-canonical ISR response following UVB irradiation, in which downstream factors ATF4 (CREB2) and CHOP (DDIT3/GADD153) were poorly expressed due to repressed transcription, despite preferential translation in response to eIF2α-P. The ISR was cytoprotective during UVB and we found that eIF2α-P was required for a UVB induced G1 arrest, cell fate determination, and DNA repair via a mechanism involving translational control of human CDKN1A (p21 protein) transcript variant 4 mRNA. Collectively, this thesis describes novel roles for the ISR in keratinocyte differentiation and response to UVB, emphasizing the utility of targeting translational control in skin disease therapy.

付記する学位プログラム名: 京都大学大学院思修館 / 京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20424号 / 農博第2209号 / 新制||農||1047(附属図書館) / 学位論文||H29||N5045(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 阪井 康能, 教授 矢﨑 一史, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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The debilitating impact of climate change on crop production remains an issue of global concern. Climate change has over the years been a driving force of extreme weather conditions that can adversely impact the growth, development, and yield of crops. Plants are able to combat the effects of stress conditions using complicated signaling networks and pathways. Several chemicals and phytohormones have been identified as part of these signaling pathways. However, the role of phytohormones in facilitating the transmission of extracellular stress stimuli into intracellular responses in plants remains unclear. This challenge is a consequence of the difficulty in studying real-time plant trafficking mechanisms in live plants and understanding how phytohormones facilitate this process. The objective of this study is to explore the mechanism of phytosulfokine trafficking in Arabidopsis thaliana with the aid of a fiber-optic fluoresce microscope that was built specifically for this purpose. Phytosulfokine (PSK) is a peptide hormone that is involved in both plant growth and stress response signaling. PSK and tetramethyl-rhodamine labelled PSK (TAMRA-PSK) were delivered to the roots and leaves of different Arabidopsis thaliana genotypes (wild type, PSK receptor deficient, overly expressed PSK receptor). With the aid fluorescence micrographs captured by the fiber-optic microscope PSK was observed to be transported form the abaxial surface of the leaf to the adaxial surface, the movement of PSK from the roots to the leaves was also observed with significant difference in mobility in the different plant genotypes. The more effective phytosulfokine mobility observed in the genotype with overly expressed PSK receptors suggests that PSK mobility is receptor dependent. Further quantitative analysis via different extraction methods and HPLC analysis will test the amount free PSK and receptor bound PSK in root and leaf tissues to better understand the mechanism of phytosulfokine in plant signaling.

Eukaryotic translation initiation factor 2 (eIF2) is a heterotrimeric G-protein that plays a critical role in protein synthesis regulation. eIF2-GTP binds Met-tRNAi to form the eIF2-GTP:Met-tRNAi ternary complex (TC), that is recruited to the 40S ribosomal subunit. Following GTP hydrolysis, eIF2-GDP is recycled back to TC by its guanine nucleotide exchange factor (GEF), eIF2B. Phosphorylation of the eIF2α subunit in response to various cellular stresses converts eIF2 into a competitive inhibitor of eIF2B, triggering the integrated stress response. Dysregulation of eIF2B activity is associated with a number of pathologies, including neurodegenerative diseases, metabolic disorders, and cancer. However, despite decades of research, the underlying molecular mechanisms remain unknown. This is due in large part to the absence of a structural understanding of the eIF2B assembly and of the eIF2B:eIF2 interaction. Common methods, such as yeast genetics, have been unable to unambiguously determine these mechanisms. Meanwhile, expanded interest in the integrated stress response has uncovered a diverse array of pathologies for which therapeutic modulation of the eIF2B:eIF2 interaction may ameliorate or overcome disease states. In this dissertation, a combination of structural and biochemical techniques is employed to elucidate the mechanisms of eIF2B action and its regulation by eIF2α phosphorylation. The aim is to provide a direct, unambiguous, structural understanding of eIF2B assembly and of eIF2B’s interactions with phosphorylated and unphosphorylated eIF2α. The work described here was among the first to challenge the widely held notion of a pentameric eIF2B assembly, as eventually confirmed by the recent publication of eIF2B’s crystal structure. The work further aims to overturn another long-standing assumption regarding the nature of inhibition of eIF2B activity: that competitive inhibition is mediated by a “direct effect” of the negatively charged phosphate group on the eIF2α:eIF2B interaction. Instead, we present evidence for an “indirect effect,” whereby phosphorylation disrupts a novel intramolecular interface within eIF2α, exposing an eIF2α surface that binds eIF2B and is responsible for inhibition of eIF2B. In the end, we combine a structural model of the eIF2B:eIF2 complex with our novel mechanism of inhibition, placing them within the larger thermodynamic context of eIF2-GDP recycling by eIF2B. / 2017-09-08T00:00:00Z

My Ph.D. research project in the Bhagwati Gupta lab focuses on understanding the mechanism by which the Axin family of scaffolding proteins functions to regulate biological processes in multicellular eukaryotes. Towards this, I am using the nematode (worm) Caenorhabditis elegans as an animal model to investigate the role of one of the Axin homologs, PRY-1. Studies in various model systems and humans have shown that the Axin family of proteins plays crucial roles during cell proliferation, cell differentiation, and organ formation. Such a role of Axin depends on the negative regulation of the WNT signaling cascade. Consistent with these, alterations in Axin function are associated with developmental abnormalities and age-associated diseases such as axis duplication, neuroectodermal defect, and muscle degeneration. As a scaffolding protein, Axin family members bind to and recruit multiple protein partners that are both WNT dependent and independent. However, how Axin interacts with these factors to regulate molecular events is not well understood. While some Axin-interacting factors have been identified, many more remain to be discovered. My project deals with the identification and functional characterization of pry-1/Axin interactors in C. elegans. The key findings of my Ph.D. research are published in five peer-reviewed papers. Collectively, the results demonstrate that PRY-1 is necessary to regulate lipid metabolism, stress response, muscle health, and aging. I have shown that PRY-1 utilizes multiple pathways to control these diverse processes. Specifically, PRY-1 functions via the SREBP transcription factor homolog SBP-1 to regulate yolk lipoprotein expression to promote lipid synthesis. The analysis of pry-1’s role in aging and muscle health has revealed its interactions with the energy sensor AMPK homolog AAK-2, thereby affecting the function of the Insulin/IGF1 signaling (IIS) transcriptional regulator DAF-16/FOXO. Moreover, I have identified several mRNA genes and microRNAs that function downstream of PRY-1/Axin signaling to either suppress or enhance pry-1 mutant defects. All these novel interactors have mammalian homologs. Altogether, these findings form the basis to pursue future work to investigate the conserved mechanism of Axin signaling and hold the potential for effective intervention to delay aging and age-associated muscle deterioration. / Thesis / Doctor of Philosophy (PhD)