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

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.

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Plants have spread to almost everywhere in the world. As they disperse, they meet many different environments to which they may be able to adapt. For a plant species to adapt to a new environment, genetic variation is needed. The individuals differ from each other in their genetic composition, which often means differences in phenotypes. Those individuals that manage to reproduce will form the next generation. With different conditions in different environments, it will not be the same phenotypes that reproduce everywhere. In that way, plant species will form into a mosaic of locally adapted populations varying genetically as the species disperses. After the last ice age plants have started to disperse away from the equators. With increasing latitudes come increasing challenges to migrating plants. As plant species disperse northwards along this gradient of varying conditions individuals are selected for cold adaptive traits like flowering time and freezing tolerance, acquired by cold acclimation. In this way, genetic variation from the original populations for these traits becomes sorted out along a latitudinal cline. The aim of this thesis was to understand how selection along a latitudinal gradient has shaped natural variation in cold adaptive traits in plants dispersing northwards, and specifically, to investigate what variation can be observed in phenotypes for these traits and how these traits correlate with genetic variation in genes known to be involved in cold acclimation. In this study significant variation was found in a sample of the model plan Arabidopsis thaliana accessions in cold adaptive traits flowering time and freezing tolerance. A clear latitudinal cline in the cold adaptive traits freezing tolerance for A. thaliana was observed. Analysis of nucleotide polymorphism for the cold responsive ICE1 (inducer of CBF expression 1) transcription factor revealed a haplotype structure with two allelic clades as well as unusually high levels of synonymous polymorphism. Nucleotide polymorphism analysis for the transcription factors CBF1, CBF2 and CBF3 (C-repeat binding factors) that play a key role in regulating the expression of a group of target genes known as the “CBF regulon” showed a distinct geographical haplotype structure. One haplotype was dominant in southern accessions while in the other northern accessions overrepresented. There was a significant effect of CBF haplotype on both freezing tolerance and flowering time even after correcting for latitude. Significant differences in CBF expression levels were found between the different CBF genes as well as between different accessions. Sequence variation at CBF was shown to have a significant effect on expression levels of CBF2. No clear correlations were found between CBF gene expression and freezing tolerance or temperature sensitivity for any of the accessions used in the study. This highlights the complex relationship between sequence variation in candidate genes and gene expression, and the problems associated with unraveling the genetic basis of ecologically important traits.

Winter rye produces ice-modifying antifreeze proteins upon cold treatment. Two of these antifreeze proteins are members of the large, highly conserved, β-1,3-endoglucanase family. This project was designed to identify glucanase genes that are expressed during cold acclimation, wounding, pathogen infection, drought or treatment with the phytohormones ethylene and MeJa. Additionally, a more detailed proteomic analysis was to be carried out to evaluate the glucanase content of the apoplast of cold-acclimated (CA) winter rye. Results of 2D SDS-PAGE analysis revealed that non-acclimated whole leaf protein extracts contain at least two β-1,3-endoglucanses while CA whole leaf protein extracts contain at least three β-1,3-endoglucanses. Subsequent 2D SDS-PAGE analysis was conducted on the apoplast extracts of NA and CA winter rye plants revealed the limitations of standard 1D SDS-PAGE. The 2-dimensional gel analysis revealed that there is a minimum of 25 proteins within the apoplast of CA winter rye, including at least 5 β-1,3-endoglucanases. Genome walking was used to isolate cold-responsive glucanase genes. The five genes isolated were designated scGlu6, scGlu9, scGlu10, scGlu11 and scGlu12. The cis-element pattern within the promoter of each gene was evaluated using online databases of documented plant cis elements. As expected, all of the promoters contained elements associated with cold, biotic and abiotic stresses, light regulation, and development. The expression patterns predicted by the cis elements in each promoter were compared to the mRNA abundance produced by each gene as detected by semi-quantitative reverse transcriptase PCR. In most cases, the abundance of transcripts arising from each gene loosely corresponded to the expression pattern predicted by the cis elements the corresponding promoter. Transcripts of scGlu9, 10 and 11 were present in cold-treated tissues and are candidates for β-1,3-endoglucanases with antifreeze activity. The results presented in this thesis provide additional insight into the apoplast proteome of CA winter rye plants as well as the complexity of the signals controlling the proteins that reside there. Although there are still a number of unresolved questions, this research opens new directions for future studies in the cold acclimation process in winter rye and specifically for the contribution of β -1,3-endoglucanses.

The physiological process of cold acclimation protects plants native to the temperate regions of the world from the deleterious effects of low and freezing temperatures. This is achieved by a series of transcriptional, regulatory, and metabolic changes that enable continued growth and survival. Within minutes of exposure to temperatures below ca. 10°C, a complex cascade of transcriptional events is initiated to accomplish these changes. The initial alarm phase favors the rapid induction of a library of stress proteins with protective functions (e.g. COR proteins). This is followed by a cold hardened phase, characterized by maximal freezing tolerance, which continues until either the stress is removed, or the plant's metabolic and/or developmental state can no longer support maximal resistance. We have studied some of the important transcription factors and transcriptional changes associated with the initial alarm and later hardened phases of cold acclimation in the herbaceous annual Arabidopsis thaliana and the woody perennial Populus spp. We confirmed the functionality of the CBF-mediated signaling cascade in Poplar overexpressing AtCBF1, but noted that regulon composition and endogenous poplar CBF ortholog induction appeared to be tissue-specific. The lack of statistically significant DRE enrichment in the Poplar AtCBF1 regulons led us to investige cis-element abundance in the cold-associated transcription factor regulons of publicly available microarray data from Arabidopsis, leading to the development of a gene voting method of microarray analysis that we used to test for regulatory associations between transcription factors and their downstream cis-elements and gene targets. This analysis resulted in a new transcriptional model of the ICE1-mediated signaling cascade and implicated a role for phytochrome A. Application of this same method to microarray data from arabidopsis leaves developed at low temperature allowed us to identify a new cis-element, called DDT, which possessed enhancer-blocking function during the alarm stage of cold stress, but was enriched in the promoters of genes upregulated during the later cold hardened stages. As leaf growth and development at low temperature correlated with the enhancement freeze tolerance in Arabidopsis, we compared the transcriptomes of rapidly growing and fully grown poplar leaves at night (when both low temperatures and PhyA status might play important roles in nature), in the hopes of comparing this data with that of cold-treated leaves in the future. We identified the nocturnal mode of leaf growth in Populus deltoides as predominantly proliferative as opposed to expansive, and potentially linked to cellular carbohydrate status.

Abstract Natural seasonal variation and the effects of cold treatment and nitrogen fertilization on protein expression with special emphasis on dehydrin proteins, were studied using different aged Scots pine (Pinus sylvestris L.). Several different dehydrins were found and their expression depended on the tissue type, tree age or specific treatment. Their concentrations fluctuated seasonally and in response to nitrogen fertilization, but no effects of low temperature on the dehydrins of seedlings were observed. A 60-kDa dehydrin was associated with cold acclimation in the bud and bark tissues of mature trees and in the needles of seedlings. In the needles of mature trees, this dehydrin was associated with springtime desiccation, which was detected as a significant decrease in the osmotic potential of needles. The quantity and quality of soluble proteins altered seasonally in Scots pine tissues, but low temperature treatment alone did not have any effect on the proteins. Soluble protein concentration increased during autumn and decreased in spring in buds and bark, but not in the needles of mature trees. In needles of seedlings, however, protein concentrations altered seasonally. Several proteins, of varying molecular weights, were more abundant in winter in all the tissues studied and some increased in concentration in the nitrogen-fertilized seedlings. The role of these proteins as a storage reserve in Scots pine is discussed. The osmotic potential of needles showed seasonal fluctuation, being high in the summer and low during the winter. Low temperature treatment decreased the osmotic and water potential of needles and increased the concentrations of soluble sugars in seedlings. Based on carbohydrate analyses, the metabolism of seedlings acclimated to low temperature in less than ten days. Nitrogen fertilization increased the content of total nitrogen and the soluble protein concentrations in the needles of seedlings and the growth both in the mature trees and seedlings. Although the frost resistance showed no response to nitrogen-fertilization, the soluble proteins and dehydrins were affected in a manner that suggested an earlier growth resumption of spring in the fertilized trees.

Biological processes in living organisms consist of a vast number of different molecular networks and interactions, which are complex and often hidden from our understanding. This work is focused on recovery of such details for two quite distant examples: acclimation to extreme freezing tolerance in Siberian spruce (Picea obovata) and detection of proteins associated with prostate cancer. The first biological system in the study, upon P. obovata, is interesting by this species ability to adapt and sustain extremely low temperatures, such as -60⁰C or below. Despite decades of investigations, the essential features and mechanisms of the amazing ability of this species still remains unclear. To enhance knowledge about extreme freezing tolerance, the metabolome and proteome of P. obovata’s needles were collected during the tree’s acclimation period, ranging from mid August to January, and have been analyzed. The second system within this study is the plasma proteome analysis of high risk prostate cancer (PCa) patients, with and without bone metastases. PCa is one of the most common cancers among Swedish men, which can abruptly develop into an aggressive, lethal disease. The diagnostic tools, including PSA-tests, are insufficient in predicting the disease’s aggressiveness and novel prognostic markers are urgently required. Both biological systems have been analyzed following similar steps: by two-dimensional difference gel electrophoresis (2D-DIGE) techniques, followed by protein identification using mass spectrometry (MS) analysis and multivariate methods. Data processing has been utilized for searching for proteins that serve as unique indicators for characterizing the status of the systems. In addition, the gas chromatography-mass spectrometry (GC-MS) study of the metabolic content of P.obovata’s needles, from the extended observation period, has been performed. The studies of both systems, combined with thorough statistical analysis of experimental outcomes, have resulted in novel insights and features for both P. obovata and prostate cancer. In particular, it has been shown that dehydrins, Hsp70s, AAA+ ATPases, lipocalin and several proteins involved in cellular metabolism etc., can be uniquely associated with acclimation to extreme freezing in conifers. Metabolomic analysis of P. obovata needles has revealed systematic metabolic changes in carbohydrate and lipid metabolism. Substantial increase of raffinose, accumulation of desaturated fatty acids, sugar acids, sugar alcohols, amino acids and polyamines that may act as compatible solutes or cryoprotectants have all been observed during the acclimation process. Relevant proteins for prostate cancer progression and aggressiveness have been identified in the plasma proteome study, for patients with and without bone metastasis. Proteins associated with lipid transport, coagulation, inflammation and immune response have been found among them.

The constitutively obsereved up-regulation of Hsp70 expression often led to premature conclusions about its critical role as a repair mechanism of cold injury that is, besides, expressed by protein misfolding/denaturation. In this study, we analyze the cold tolerance and the expression of 24 different mRNA transcripts of Hsp complex and other genes, that are associated with the repair of injury caused by cold. We use two strains of D. melanogaster: the wild type and the mutant type Hsp70- null, that lacks all 6 copies of the gene hsp70. We found out, that the larvae of two strains do not differ in their patterns of target genes expression during long term acclimation nor during recovery from chronic cold exposure and acute cold shock, therefore there is no transcriptional compensation of any other Hsp gene for the missing hsp70 in Hsp70- strain. The cold tolerance of Hsp70- strain larvae was impaired only, when they were exposed to strong acute cold shock by temperatures below -8°C. No difference in cold tolerance was observed, when the larvae were exposed to chronic cold exposure in 0°C or to mild acute cold shock by temperatures up to -4°C. Based on our results we assess, that the cold injury caused by strong acute cold schock is of another nature than caused by mild cold conditions and only in the first case Hsp70 expression is critical for the repair of cold injury in Drosophila melanogaster larvae.