Background: Biological systems adapt to changing environments by reorganizing their cellula r and physiological program with metabolites representing one important response level. Different stresses lead to both conserved and specific responses on the metabolite level which should be reflected in the underl ying metabolic network. Methodology/Principal Findings: Starting from experimental data obtained by a GC-MS based high-throughput metabolic profiling technology we here develop an approach that: (1) extracts network representations from metabolic conditiondependent data by using pairwise correlations, (2) determines the sets of stable and condition-dependent correlations based on a combination of statistical significance and homogeneity tests, and (3) can identify metabolites related to the stress response, which goes beyond simple ob servation s about the changes of metabolic concentrations. The approach was tested with Escherichia colias a model organism observed under four different environmental stress conditions (cold stress, heat stress, oxidative stress, lactose diau xie) and control unperturbed conditions. By constructing the stable network component, which displays a scale free topology and small-world characteristics, we demonstrated that: (1) metabolite hubs in this reconstructed correlation networks are significantly enriched for those contained in biochemical networks such as EcoCyc, (2) particular components of the stable network are enriched for functionally related biochemical path ways, and (3) ind ependently of the response scale, based on their importance in the reorganization of the cor relation network a set of metabolites can be identified which represent hypothetical candidates for adjusting to a stress-specific response. Conclusions/Significance: Network-based tools allowed the identification of stress-dependent and general metabolic correlation networks. This correlation-network-ba sed approach does not rely on major changes in concentration to identify metabolites important for st ress adaptation, but rather on the changes in network properties with respect to metabolites. This should represent a useful complementary technique in addition to more classical approaches.

At the cellular level, the stress response involves the synthesis of a highly conserved family of heat shock proteins (Hsps). These proteins are essential for maintenance of cellular homeostasis, both in times of stress and in normal cell functioning. Some of the most abundant forms of Hsps in the cell are members of the 70 kDa family. Intracellular heat shock protein 70 (Hsp70) expression in response to proteotoxicity is a highly conserved cellular stress response, but little is known about the role of extracellular Hsp70 (eHsp70) in fish. In order to begin characterizing eHsp70 in fish, the hypothesis that an acute stressor will elevate plasma Hsp70 levels in rainbow trout (Oncorhynchus mykiss) was tested. Subsequent in vitro studies examined whether eHsp70 level was modulated by cortisol and if this involved the action of the glucocorticoid receptor (GR), a ligand-activated transcription factor. The effect of cortisol on the eHsp70 response is important to consider because this steroid is elevated as a result of stressor exposure to allow for short-term allocation of energy stores to cope with stress. Cortisol is the primary corticosteroid in fish and exerts its main effects by binding to either GR or mineralocorticoid receptors (MR). Furthermore, eHsp70 has been previously implicated as having important immunoregulatory roles in mammalian models, but nothing has yet been reported in fish. To this end, a hypothesis tested here was that eHsp70 levels will increase after exposure to the bacterial endotoxin lipopolysaccharide (LPS), and that this response is modulated by cortisol. Finally, research on the effects of exogenous Hsp70 has not been reported in lower vertebrates; however, the relevance of this protein in intercellular signaling, especially in regards to immune regulation, is gaining increasing importance in mammalian models. Therefore, an experiment to determine whether Hsp70 would elicit upregulation of key immunoregulatory cytokines was also conducted. To accurately measure the low levels of Hsp70 in the plasma, a competitive antibody-capture enzyme-linked immunosorbent assay (ELISA) was developed. In the in vivo study, fish exposed to an acute heat shock (1h at 10°C above ambient temperature) exhibited a significant elevation in red blood cell Hsp70 levels over a 24 h period. There was also a significant increase in plasma Hsp70 levels at 4 h, but not at 24 h post-heat shock. To more specifically determine how cortisol affected the release of Hsp70, in vitro studies using primary cultures of hepatocytes demonstrated that cortisol significantly decreased eHsp70 levels in the medium at 24 h when compared with untreated controls, and this response was abolished in the presence of a GR antagonist, mifepristone (RU486). This result for the first time established a link between cortisol signaling and eHsp70 release in any animal model. When hepatocytes were exposed to LPS in vitro, eHsp70 levels were significantly lower in the LPS (30 µg/ml) group; however, heat shock abolished this effect at 24 h. Though eHsp70 levels in the heat shocked hepatocytes treated with low-dose LPS (10 µg/ml) was similar to untreated control levels, high-dose LPS treated hepatocytes showed significant elevation of eHsp70 levels above the low dose group. The ability of LPS to modulate eHsp70 release was not observed to be further regulated by cortisol. While this work suggests the modulation of eHsp70 by LPS, the physiological role remains to be elucidated. Finally when hepatocytes were exposed to exogenous Hsp70, there was no effect on key immunoregulatory genes (IL-1β and IL-8) transcript levels; however, the effect of this protein remains to be tested using other cell systems, including immune cells in fish. Overall, eHsp70 concentration was measured in trout plasma using a competitive ELISA and demonstrates for the first time that stressor exposure affects plasma eHsp70 levels in fish. Furthermore, cortisol, the primary corticosteroid in teleosts, modulates eHsp70 release in trout hepatocytes and this is action is mediated by GR signaling. Also, while trout hepatocytes secrete eHsp70 in response to endotoxin shock, a role for eHsp70 in eliciting an immune response is not clear in lower vertebrates. Taken together the results from this study suggest a role for eHsp70 in acute stress adaptation in fish, but the target tissues involved and the physiological responses remain to be elucidated. Further work on the effects of eHsp70 on target tissues effects, and the mechanisms involved, may have important implications in our understanding of the role of this stress protein in cell signaling and stress adaptation in fish.

Previous studies at the Key Lake uranium mill (Saskatchewan, Canada) suggested the complex effluent discharged alters energetic stores of resident fish species. A second study at the same site demonstrated certain fish from lakes downstream of the mill produce larvae with elevated incidence of developmental deformities. The mechanisms by which energy homeostasis is affected in fish downstream of the Key Lake uranium mill are unknown, and the effects of deformities and altered metabolism on swimming ability have not been explored. Therefore, the overall objective of this thesis was to investigate whether effluent exposed fish exhibited differences in swimming performance and energy homeostasis. To achieve this objective two experiments were conducted. In the first experiment juvenile spottail shiner (Notropis hudsonius) were collected from a lake downstream of the Key Lake uranium mill, and compared to fish collected from a nearby reference lake. In the second experiment larvae were collected from laboratory raised fathead minnow (Pimephales promelas) exposed to 5% diluted uranium mill effluent or control (dechlorinated municipal) water, and reared in the same treatments to 60 days post hatch (dph). No gross deformities were observed in any fish, and only shiner collected from the exposure lake in the field experiment had enlarged heart ventricles relative to body size compared to fish from the reference lake. Swimming performance was similar between shiner from the exposure and reference lakes in the field study, but effluent exposure impaired swimming ability in 60 dph fathead minnow in the laboratory experiment compared to fish from the control water treatment. After swimming performance tests fish were considered fatigued and metabolic endpoints were compared to non-fatigued fish. In both non-fatigued and fatigued shiner, liver glycogen was significantly greater in fish collected from the exposure lake compared to the reference lake. There was no difference in liver triglycerides in non-fatigued shiner between lakes, but liver triglycerides decreased after swimming in the field study reference fish. Muscle energy stores were unaffected by site or swimming in the field experiment. Conversely, whole body triglycerides and glycogen were similar between treatments in non-fatigued fathead minnow in the laboratory experiment. Swimming significantly decreased whole body triglycerides in fathead minnow from both treatments, but whole body glycogen was unaffected. In the field experiment blood endpoints (hematocrit, plasma glucose, lactate) in fatigued and non-fatigued shiner from both lakes further supported the possibility of altered intermediary metabolism or blunted stress response in fish downstream of the Key Lake uranium mill. In the field study, shiner muscle citrate synthase activity (an indicator of tissue aerobic capacity) was similar between lakes, but muscle âhydroxyacyl coenzyme A dehydrogenase activity (an indicator of tissue lipolytic capacity) was elevated. In contrast, laboratory fathead minnow whole body âhydroxyacyl coenzyme A dehydrogenase activity was similar between treatments, but citrate synthase activity was significantly lower in fathead minnow from the 5% effluent treatment. In summary, shiner from the exposure lake in the field experiment had similar swimming endurance and greater energy stores compared to fish from the reference lake, despite metabolic alterations. Fathead minnow from the 5% effluent treatment in the laboratory experiment had reduced swimming endurance that was matched by reduced whole body citrate synthase activity, but no other metabolic alterations were observed. Therefore, effluent exposure caused metabolic alterations in both fathead minnow and shiner, but specific effects between experiments were inconsistent. Overall, the physiological significance of the metabolic and swimming effects of effluent exposure is unclear, but suggests discharged effluent has the potential to negatively affect wild fish survivability.

Previous studies at the Key Lake uranium mill (Saskatchewan, Canada) suggested the complex effluent discharged alters energetic stores of resident fish species. A second study at the same site demonstrated certain fish from lakes downstream of the mill produce larvae with elevated incidence of developmental deformities. The mechanisms by which energy homeostasis is affected in fish downstream of the Key Lake uranium mill are unknown, and the effects of deformities and altered metabolism on swimming ability have not been explored. Therefore, the overall objective of this thesis was to investigate whether effluent exposed fish exhibited differences in swimming performance and energy homeostasis. To achieve this objective two experiments were conducted. In the first experiment juvenile spottail shiner (Notropis hudsonius) were collected from a lake downstream of the Key Lake uranium mill, and compared to fish collected from a nearby reference lake. In the second experiment larvae were collected from laboratory raised fathead minnow (Pimephales promelas) exposed to 5% diluted uranium mill effluent or control (dechlorinated municipal) water, and reared in the same treatments to 60 days post hatch (dph). No gross deformities were observed in any fish, and only shiner collected from the exposure lake in the field experiment had enlarged heart ventricles relative to body size compared to fish from the reference lake. Swimming performance was similar between shiner from the exposure and reference lakes in the field study, but effluent exposure impaired swimming ability in 60 dph fathead minnow in the laboratory experiment compared to fish from the control water treatment. After swimming performance tests fish were considered fatigued and metabolic endpoints were compared to non-fatigued fish. In both non-fatigued and fatigued shiner, liver glycogen was significantly greater in fish collected from the exposure lake compared to the reference lake. There was no difference in liver triglycerides in non-fatigued shiner between lakes, but liver triglycerides decreased after swimming in the field study reference fish. Muscle energy stores were unaffected by site or swimming in the field experiment. Conversely, whole body triglycerides and glycogen were similar between treatments in non-fatigued fathead minnow in the laboratory experiment. Swimming significantly decreased whole body triglycerides in fathead minnow from both treatments, but whole body glycogen was unaffected. In the field experiment blood endpoints (hematocrit, plasma glucose, lactate) in fatigued and non-fatigued shiner from both lakes further supported the possibility of altered intermediary metabolism or blunted stress response in fish downstream of the Key Lake uranium mill. In the field study, shiner muscle citrate synthase activity (an indicator of tissue aerobic capacity) was similar between lakes, but muscle âhydroxyacyl coenzyme A dehydrogenase activity (an indicator of tissue lipolytic capacity) was elevated. In contrast, laboratory fathead minnow whole body âhydroxyacyl coenzyme A dehydrogenase activity was similar between treatments, but citrate synthase activity was significantly lower in fathead minnow from the 5% effluent treatment. In summary, shiner from the exposure lake in the field experiment had similar swimming endurance and greater energy stores compared to fish from the reference lake, despite metabolic alterations. Fathead minnow from the 5% effluent treatment in the laboratory experiment had reduced swimming endurance that was matched by reduced whole body citrate synthase activity, but no other metabolic alterations were observed. Therefore, effluent exposure caused metabolic alterations in both fathead minnow and shiner, but specific effects between experiments were inconsistent. Overall, the physiological significance of the metabolic and swimming effects of effluent exposure is unclear, but suggests discharged effluent has the potential to negatively affect wild fish survivability.

The present study investigates the effects of cognitive inhibitory mechanisms and tendency to suppress thoughts on the frequency and intensity of traumatic intrusions within the trauma film paradigm. Non clinical participants&rsquo / response inhibition and proactive inhibition levels and tendency to suppress thoughts were measured prior to exposure to a trauma film. One week after seeing the trauma film, participants reported the frequency and intensity of trauma film related intrusions with an intrusion diary and Impact of Events Scale. No significant effect of response inhibition, proactive inhibition and thought suppression tendency was found on the frequency and intensity of trauma film related intrusions. Findings of the study are discussed.

Apoptosis is a conserved program of cell death that promotes organism homeostasis in all stages of life. Two main pathways activate caspases, which are cysteinyl-aspartate proteases that execute apoptosis. The extrinsic pathway is initiated by cell surface death receptors, while the intrinsic pathway is initiated by intracellular signals that cause permeabilization of the outer mitochondrial membrane (MOMP). The Bcl-2 protein family regulates MOMP, which causes the release of several pro-apoptotic proteins (such as cytochrome c, Smac) into the cytosol. Bcl-2 proteins share homology in up to four "BH" domains and are subdivided into three subgroups. Pro-apoptotic Bax and Bak catalyze pore formation in the mitochondria, while anti-apoptotic members (Bcl-2, Mcl-1) inhibit MOMP. The third subgroup, termed BH3-only, promotes MOMP by either antagonizing Bcl-2 proteins or by directly activating Bax/Bak, and initiate apoptosis in response to various stressors, including heat shock (HS). Hyperthermia or acute HS reportedly induces apoptosis through caspase-2-mediated cleavage of BID, engaging the intrinsic pathway. However, additional evidence suggests that this pathway could represent an amplification loop. Thus we hypothesized that during HS, another BH3-only protein such as BIM, that does not require cleavage, could engage MOMP. Herein, we report that BIM mediates an alternative HS-induced apoptosis pathway. Cells lacking BIM are resistant to HS and exhibit better short and long-term survival than either Bid[superscript -/-] or Bax[superscript -/-]Bak[superscript -/-]. Moreover, caspase-2 induces apoptosis in Bim[superscript -/-] but not Bid[superscript -/-] cells, implying that caspase-2 kills exclusively through BID. Interestingly, Bim[superscript -/-] and Bax[superscript -/-]Bak[superscript -/-] cells are entirely resistant to MOMP, but the Bax[superscript -/-]Bak[superscript -/-] cells still undergo caspase-3 activation and remain partially sensitive to HS, indicating that BIM triggers caspase-3 activation upstream of mitochondria. Thus, BIM plays an important role in HS-induced apoptosis. Hyperthermia has clinical applications for the treatment of solid tumors. Unfortunately, a practical limitation is the development of thermotolerance, which confers resistance not only to subsequent HS but also to radiotherapy and chemotherapy. Therefore, a better understanding of the molecular mechanisms involved both in heat-induced apoptosis and thermotolerance could lead to new therapeutic interventions. Here we also show evidence for a putative role for the stress kinase JNK signaling pathway in the regulation of thermotolerance. / text

Hybrid plants and animals, like corn and the domestic dog, grow larger and more vigorously than their parents, a common phenomenon known as hybrid vigor or heterosis. In hybrids between Arabidopsis ecotypes or species (in allotetraploids), altered expression of circadian clock genes leads to increased starch and chlorophyll content and greater biomass. In plants and animals, circadian clock regulation plays a key role in optimizing metabolic pathways, increasing fitness, and controlling responses to biotic and abiotic stresses. In the allotetraploids, the increased level of heterosis is likely caused by interspecific hybridization as well as genome doubling. However, it is unknown how genome dosage and allelic effects influence heterosis, and whether additional clock output traits, such as stress responses, are altered in hybrids. In three related projects, the effects of genomic hybridization (including parent-of-origin effects) and genome dosage on heterosis were elucidated. In my first project, I found that although ploidy influenced many traits, including seed and cell size, biomass and circadian clock gene expression were most strongly influenced by hybridization. Additionally, parent-of-origin effects between reciprocal hybrids were frequently observed for many traits. In my second project, I described a unique role for RNA-directed DNA methylation (mainly CHH methylation) in mediating the parent-of-origin effect on expression of the circadian clock gene CCA1 in reciprocal hybrids. Altered CCA1 expression peaks were associated with heterosis of biomass accumulation in the reciprocal hybrids. Lastly, I used transcriptome sequencing in hybrids at different times of day to examine changes in downstream clock-regulated pathways. In the hybrids, many genes in photosynthetic pathways were upregulated, while many genes involved in biotic and abiotic stresses were repressed during the morning and afternoon, respectively. Additionally, natural variation between parents in stress-responsive gene expression was found to be crucial for producing vigorous hybrids. These conceptual advances increase the mechanistic understanding of heterosis, and may guide selection of parents for making better hybrids. / text

Levels of macromolecules fluctuate both spatially and temporally in individual cells. Such heterogeneity could be exploited for bet hedging in uncertain environments, or be suppressed by negative feedback if perturbations are deleterious. For the master stress-response regulator in Escherichia coli, RpoS, both of these scenarios have been suggested. RpoS levels are also exceedingly low and controlled by the ClpXP protease, which reportedly displays extreme spatial heterogeneity. However, little is known quantitatively about RpoS dynamics. This is partly because no functional protein fusions exist, but also because the quantitative tools for studying fluctuations and localizations are limited, particularly ones that can be independently validated. Here I develop such methods and begin applying them to RpoS. Protein localization measurements increasingly rely on fluorescent protein fusions and are difficult to verify independently. I designed a non-intrusive method for validating localization patterns in live bacterial cells by exploiting post-division heterogeneity in downstream processes. Applying this assay to the ClpXP protease, widely reported to form biologically relevant foci, revealed in fact that the protease molecules are not specifically localized inside cells, as confirmed by four independent methods. I further evaluated 20+ commonly used fluorescent reporters and found that many cause severe mislocalization when fused to homo-oligomers, likely due to avidity effects. Further reinvestigating other foci-forming proteins strongly suggests that the previously reported foci were all caused by the fluorescent proteins used. For mRNAs – which are often present in low numbers per cell and major sources of non-genetic heterogeneity – existing single-cell assays have unknown accuracy: the experimental counting errors could completely over-shadow the natural variation. I therefore optimized and cross-evaluated two single-molecule mRNA detection methods. Several problems were identified and solutions discussed. I succeeded in building a functional RpoS protein fusion, and used bulk methods to show that the RpoS feedback loop is effectively not operating during exponential- phase growth. Mathematical analyses and initial experiments in a microfluidic device further suggest that the RpoS system has several unusual properties contributing towards extremely fast stress response. A stochastic analysis further suggests that the RpoS feedback loop cannot suppress spontaneous fluctuations, and preliminary experiments indicate that large deviations might indeed play important roles.

Bacteria use quorum sensing, a cell to cell signaling mechanism mediated by small molecules that are produced by specific signal molecule synthases, to regulate gene expression in response to population density. In Vibrio anguillarum, the quorum-sensing phosphorelay channels information from three hybrid sensor kinases VanN, VanQ, CqsS that sense signal molecules produced by the synthases VanM, VanS and CqsA, onto the phosphotransferase VanU, to regulate activity of the response regulator VanO. VanO activates transcription of quorum-sensing regulatory RNAs (Qrr), which work together with the RNA chaperone Hfq to repress expression of the transcriptional regulator VanT. The work presented in this thesis characterizes quorum-sensing independent and quorum-sensing dependent mechanisms that regulate VanT expression. Moreover, an in vivo imaging system was established, as a means to study V. anguillarum infections in the rainbow trout infection model. Two quorum-sensing independent mechanisms regulating VanT expression were identified. First, the sigma factor RpoS indirectly activates VanT expression during transition into stationary growth phase by inhibiting hfq expression. Both, RpoS and VanT are crucial for stress response. Second, a type VI secretion system (T6SS) has a novel function as a signal sensing mechanism to regulate rpoS and vanT expression. Consequently, RpoS, quorum sensing and T6SS form a global network that senses stress and modulates stress response to ensure survival of the bacteria. Further analysis of the quorum-sensing dependent regulation of VanT expression by the phosphorelay system revealed that four qrr genes are expressed continuously during growth. The phosphotransferase VanU is suggested to activate two response regulators, VanO and a predicted second response regulator. Activated VanO induces expression of the Qrr sRNAs, whereas, the predicted response regulator represses expression of the Qrr sRNAs. Thus, VanU has a pivotal role in the regulation of VanT expression. The signal synthase VanM and VanT form a regulatory loop, in which VanM represses VanT by inducing expression of the Qrr sRNAs and VanT directly activates vanM expression to repress its own expression. Moreover, Hfq destabilizes vanM mRNA, repressing vanM expression. VanT forms another regulatory loop with the transcriptional regulator LuxT, in which LuxT activates vanT expression and VanT directly represses luxT expression. V. anguillarum is an opportunistic pathogen that causes vibriosis, a terminal hemorrhagic septicemia. The spatial and temporal progression of the infection was analyzed using the whole animal with an in vivo bioluminescent imaging method. Initial studies showed that colonization of the fish skin requires the siderophore, the RNA chaperone Hfq and the exopolysaccharide transport system, which protects against the innate immunity on the skin. Colonization of the fish skin is crucial for disease.

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