Cold exposure elicits a thermoregulatory response in an organism. If the cold stimulus is sufficient, there is increased heat generation by shivering thermogenesis. Under prolonged action of the cold stimulus, shivering thermogenesis is replaced by non-shivering thermogenesis. Non-shivering thermogenesis in the rat takes place predominantly in brown adipose tissue (BAT), where the uncoupling protein 1 (UCP1) is activated in mitochondria under the control of adrenergic signaling. The activity of UCP1 is crucial for heat production. The coactivator PGC-1α plays an important role in mitochondrial biogenesis and in the energy metabolism of BAT and heart, and its function is associated with cardioprotection. Recently, the cardioprotective effects of mild cold adaptation at 8±1 řC were discovered in our laboratory, which reduced the size of ischemia-reperfusion injury without negative side effects. However, the molecular nature of the events induced by adaptation to mild cold is unknown neither in BAT nor in the heart itself. Therefore, the aims of the thesis were 1) to characterize the development of BAT at the level of expression of selected proteins and mitochondrial markers during acute cold exposure and chronic cold adaptation with subsequent recovery and 2) to determine the role of PGC-1α and...

The PhD thesis is focused on a transcriptomics-based comparison of barley cultivars differing with respect to their low temperature acclimation capacity, with a particular focus on genes transcribed in the leaf and crown. The crown was of interest because of its importance for the winter survival of the plant. To involve both the first and the second phase of hardening, the test plants were exposed first to +3řC for 21 days, followed by - 3řC for one day. Freezing damage was assessed by measuring electrolyte leakage (Papers 2 and 3), using a modified version of a protocol developed by Prášil and Zámečník (1998). The same protocol was adapted to evaluate crown regrowth (Paper 2); for this purpose, the plants were cooled, then replanted and cut above the crown, and their survival rate calculated over the following week. Each RNA sample was queried by hybridization to an Affymetrix 22 K Barley1 GeneChip Genome Array (Close et al. 2004). The data were statistically analysed with the help of the software packages R, MAS 5.0 (Ihaka & Gentleman 1996) (Papers 2 and 3), Gene Spring GX 7.3 (Agilent Technologies, Santa Clara CA) and MapMan (Thimm et al. 2004; Usadel et al. 2005) (Paper 2), the "Self-Organizing Maps" algorithm (Kohonen et al. 1996) (Paper 3) and MIPS FunCat (Ruepp et al. 2004) (Paper 2). Paper...

During cold acclimation the heat production is shifted from shivering to non-shivering thermogenesis, which is mediated by adrenergic signaling. It has also been observed, that cold acclimation may increase the organismal resistence to pathological stimuli and may affect functional parameters of cardiovascular system. However, acute exposure to sever cold is often associated with detrimental effects on the body. We have recently shown that chronic exposure to cold increases the heart's resistance to ischemia-reperfusion injury without negative side effects when mild temperatures are used, however the mechanism of protection is not yet known. The aim of this work was to determine whether: i) if the sensitivity of the heart to ischemia changes already after the first day of cold exposure and does not show any negative effects, ii) if β1-adrenergic signaling plays a role in chronic regimen of cold-induced cardioprotection. The results of this work showed that i) one day of exposure to mild cold did not change the sensitivity of the heart to ischemia and ii) metoprolol treatment reduced the infarct size in the control group, but did not affect the heart of cold-adapted rats. Key words : Heart, rat, beta 1 adrenergic receptors, cold acclimation

In the Caribbean, Acropora cervicornis (staghorn coral) exemplifies the worldwide anthropogenic decline of reef-building corals. From the mid-Pleistocene through the mid-1900s, A. cervicornis was a dominant framework builder, providing complex habitat for reef organisms. Since the 1980s, populations of A. cervicornis have declined by as much as 98%. Despite the overall decline, scattered remnants persist, and some appear to be thriving. As in recent studies on other acroporids, if we can identify variation in traits related to resilience in the remaining A. cervicornis, and understand the genetic basis of such variation, we could better forecast the species’ future response to climate change, and inform ongoing restoration efforts. Here, I compare phenotypic and transcriptomic indicators of resilience in A. cervicornis from two nearby but environmentally-disparate habitats on Turneffe Atoll, Belize: Calabash Caye forereef and Blackbird Caye backreef. Blackbird exhibits significantly higher flow, light, average temperature, and temperature variation. Over four years, I conducted a longitudinal study of 122 tagged coral colonies. Corals from Blackbird and Calabash, which I confirmed to be genetically distinct based upon single nucleotide polymorphisms, exhibited pronounced differences in traits related to resilience including the proportion of healthy tissue, chlorophyll, growth, and wound-healing. By most measures, Blackbird corals displayed superior indicators of resilience. Through a two-year reciprocal transplant study involving 120 corals, I identified substantial environmental plasticity in these traits, e.g., Blackbird corals transplanted to Calabash exhibited higher chlorophyll levels and more rapid wound healing than when grown in Blackbird, exceeding the native Calabash corals. RNA sequencing and assembly of site-specific transcriptomes revealed greater diversity of transcripts and genes from photosynthetic symbionts at Blackbird but greater diversity of bacterial associates at Calabash. Single nucleotide polymorphism (SNP) analyses using RNAseq data determined that corals from the two sites were separate putative populations. Principal components analysis of gene expression in natives and transplants revealed a clear distinction based on site of origin, but also a clear effect of environment. Thousands of differentially expressed genes distinguished the sites, including many genes implicated in heat stress, oxidative stress and UV-light stress. This genetic and phenotypic diversity of remnant staghorn populations on Turneffe represents a potential basis for future re-expansion of this important framework builder through natural or assisted shifts toward resilient populations. / 2023-02-03T00:00:00Z

Daphnia is a widespread freshwater zooplankton species, which is both a classic and emerging new model for research in ecological physiology, ecotoxicology and evolutionary biology of adaptation to novel environments. Heat tolerance in Daphnia is known to depend both upon evolutionary history of a genotype and on individuals' acclimation to elevated temperature and to correlate with the level of haemoglobin expression. We demonstrate the existence of north-south gradient of heat tolerance in North American D. pulex, which is not associated with any parallel changes in haemoglobin expression. Geographically distinct clones differ in the way their haemoglobin expression changes due to acclimation to a sub-stressful (28°C) temperature, but these changes are not correlated with the latitude of clones' origin. Likewise, the effect of acclimation to sub-stressful temperature is independent from, and cannot be fully explained by, haemoglobin expression changes during acclimation. The degree of oxidative damage to haemoglobin, measured as the ratio of absorbance at 540:576 nm at the acclimation temperature, is a strong predictor of 28°C-acclimated Daphnia survival during an acute heat exposure. The comparison of haemoglobin expression in resistant and tolerant clones acclimated to different temperatures indicates that tolerant clones exhibit canalization of haemoglobin expression, possessing a high level of haemoglobin even at non-stressful temperatures. We discuss the evolutionary biology of adaptation and acclimation to elevated temperatures in an ecologically important component of freshwater ecosystems in the context of global climate change.

Examples of phenotypic plasticity—the ability of organisms of identical genotypes to produce different phenotypes in response to the environment—are abundant, but often lack data on the causative physiology and biochemistry. Phenotypes associated with increased protection against or reduced damage from harmful environments may, in fact, be downstream effects of hidden adaptive responses that remain elusive to experimental measurement or be obscured by homeostatic or over-compensatory effects. The freshwater zooplankton crustacean Daphnia drastically increases its heat tolerance as the result of acclimation to high temperatures, an effect often assumed to be based on plastic responses allowing better protection against oxidative stress. Using several geographically distant Daphnia magna genotypes, we demonstrate that the more heat tolerant individuals have a higher total antioxidant capacity (TAC) both in the comparison of heat-acclimated vs. non heat-acclimated females and in the comparison of females to age- and body size-matched males, which show lower heat tolerance than females. However, experimental manipulations of hypothesized antioxidant pathways by either glutathione addition or glutathione synthesis inhibition had no effect on heat tolerance. Lipid peroxidation (LPO), contrary to expectations, did not appear to be a predictive measure of susceptibility to thermal damage: LPO was higher, not lower, in more heat tolerant heat-acclimated individuals after exposure to a lethally high temperature. We hypothesize that LPO may be maintained in Daphnia at a constant level in the absence of acute exposure to elevated temperature and increase as a by-product of a possible protective antioxidant mechanism during such exposure. This conclusion is corroborated by the observed short-term and long-term changes in phospholipid composition that included an increase in fatty acid saturation at 28 °C and up-regulation of certain long-chain polyunsaturated fatty acids. Phospholipid composition was more strongly affected by recently experienced temperature (4-day transfer) than by long-term (2 generations) temperature acclimation. This is consistent with partial loss of thermal tolerance after a short-term switch to a reciprocal temperature. As predicted under the homeoviscous adaptation hypothesis, the more heat tolerant Daphnia showed lower membrane fluidity than their less heat tolerant counterparts, in comparison both between acclimation temperatures and among different genotypes. We conclude that thermal tolerance in Daphnia is influenced by total antioxidant capacity and membrane fluidity at high temperatures, with both effects possibly reflecting changes in phospholipid composition.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20099号 / 生博第359号 / 新制||生||47(附属図書館) / 33215 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 福澤 秀哉, 教授 佐藤 文彦, 教授 河内 孝之 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The study aimed to assess whether a short-term, high-intensity cold acclimation protocol would modulate finger vasomotor [i.e., finger temperature (TF), cold induced vasodilation (CIVD)] responses and regional thermo-perception to localized cooling. Six men performed a hand cold provocation (consisting of a 30-min immersion in 8°C water), while being whole-body immersed, once, in 21°C water (i.e., cold trial; HYPO), and, the following day, in 35.5°C water (i.e., normothermic trial; NORM). The local cold provocations were repeated, in the same order, after 10 days. In the intervening period, the subjects undertook a whole-body cold acclimation pro-tocol, consisting of daily whole-body 14°C-water immersions for 5 consecutive days, for a maximum of 2 h, while the skin temperature of the right hand was maintained at 35.6 (0.1)°C. Thermal (rectal temperature, skin temperature, finger temperature) cardiorespiratory (mean arterial pressure (MAP), heart rate and oxygen uptake), and perceptual responses (thermal sensation and comfort, pain, affective valence) were monitored throughout the trials. The acclimation protocol resulted in hypothermic adaptation (i.e., habituation), which was characterized by a modest reduction in shivering and an attenuation of whole-body thermal discomfort. The main finding of the study was that, regardless of subjects’ thermal status, the 5-day whole-body cold acclimation protocol did not alter TF (P > 0.1) and CIVD responses (P > 0.2) during local cold stress. Yet, after the acclimation, the cold-induced increase in MAP was reduced and tended to be reduced during the HYPO (P = 0.05) and NORM (P = 0.14) local cold provocation trials, respectively. Furthermore, the perceived thermal discomfort and pain in the immersed hand appeared to be alleviated in all post-acclimation trials.

Thermal adaptation in freshwater planktonic crustacean Daphnia pulex has been investigated by means of heatshock (35°C) experiments using individuals acclimated to different water temperatures (18, 24, and 28°C). Retention of mobility and survival during the heatshock experiments differed among clones and strongly depended on acclimation temperature: clones acclimated to substressful temperature (28°C) survived significantly longer than clones acclimated to lower temperatures. Both retention of mobility and survival correlated with geographic latitude of the clones' origin. There was, on the other hand, no significant correlation between clones' performance in heatshock experiments and hemoglobin content. Clones do not differ in the degree of the effect of acclimation on survival during heatshock but differ with respect to the effect of acclimation on mobility retention.

The thermotolerance, an adaptive phenomenon that is accompanied by the phenotypic plasticity which is the adjustment of physiology, biochemistry and metabolism of every cellular function by the hidden mechanism. Mitochondrion, the powerhouse of the cell that determines the functional integrity of every cellular homeostasis and functional phycological processes should provide its association in regulating the thermotolerance as well. This study assessed the mitochondrial function in regulating and determining the limit of thermo tolerance in the Daphnia magna of different geographical regions of the world, mainly sub grouped as temperature tolerant clones (IL) and temperature sensitive clones (GB). The acclimation effects or the adjustment of the preexisting biological properties help the organism adjust its biological processes to the changing habitat to maintain the cellular functional integrity. The clonal divergence as well as the acclimation show a clear pattern in limiting the thermotolerance and the prediction is the temperature tolerant clones should show higher adjustment of the mitochondrial function than temperature sensitive ones. We hypothesize that the damage in the mitochondrial membrane integrity by different mito-toxins should decrease the heat tolerance by decreasing the membrane potential and fluidity. The integrated mitochondrial function was assessed in acclimated clones by using the molecular studies as well as observation of behavioral and phenotypic plasticity. Due to the specific effects of each mito-toxins (CCCP, NaN3 and DNP) on different complexes (I-IV and ATP synthase) in ETC, we determined the mitochondrial membrane integrity by the Rhodamine 123 alongside with the lactate assay for measuring the mitochondrial integrity. Among all these three mito-toxins, CCCP show significant effect on limiting the heat tolerance. The lower lactate accumulation was observed in the temperature-tolerant clones acclimated in cold temperatures (18°C) which indicates the higher mitochondrial adjustment than the temperature sensitive clones. The concluding remark is that thermal tolerance is determined by the adjustment of mitochondrial function which accompanied with the adjustment to the mitochondrial respiration as well as the adjustment to membrane potential and fluidity.