When proteins are unable to fold properly in the endoplasmic reticulum (ER), the resultant formation of misfolded proteins causes stress of the ER. Cells with ER stress often have a higher abundance of reactive oxygen species (ROS). Previous studies suggest that ROS could aggravate ER stress by further disrupting the ER protein folding process. More recent studies suggest that the unfolded protein response signaling pathways activated by ER stress could lead to the production of ROS. Such studies lead to the hypothesis that ER stress could be promoted by ROS, and vice versa. The aim of the present study is to test the above hypothesis by studying how ROS could be generated in ER-stressed cells. This is followed by investigating if ROS could increase or decrease the level of ER stress in cells. Finally, the extent of ER stress induced cell death in the presence and absence of ROS is assessed. The treatment of HeLa cells with tunicamycin (Tm), a common ER-stress inducing agent, resulted in the elevation of intracellular ROS that could be detected with the ROS-reactive probe dichlorodihydrofluorescein (DCF), but not dihydroethidium which is relatively specific towards superoxide anion. The Tm-induced elevation of ROS could be prevented by co-incubation of cells with thiol reductants such as dithiothreitol and N-acetylcysteine but not with the free radical scavenger ascorbate. The tunicamycin-induced elevation of ROS level could also be prevented by the over-expression of catalase in HeLa. These data is consistent with the idea that hydrogen peroxide is a major form of ROS produced in Tm-treated cells. In addition to elevation of ROS level, HeLa cells treated with tunicamycin also resulted in the phosphorylation of PERK and eIF2α, and the splicing of XBP-1. In the presence of cycloheximide to inhibit protein synthesis so as to deplete protein substrates for folding in the ER, tunicamycin-induced ER stress was greatly minimized as was evident by the absence of both the phosphorylation of PERK and splicing of XBP-1. However, the phosphorylation of eIF2α and elevation of DCF-detectable ROS remained unaffected. The cycloheximde-resistant phosphorylation of eIF2α could be prevented when cells were co-treated with thiol reductants, or upon the over-expression of catalase. These data suggest that the production of ROS in Tm-treated cells does not require the presence of ER stress as a prerequisite. Furthermore, the ROS so produced could induce phosphorylation of eIF2α without the need to cause ER stress in the first place. The quenching of ROS through the use of thiol reductants, or the over-expression of catalase, had no effect on inhibition of protein synthesis in cells treated with tunicamycin. However, the extent of cell death was significantly increased. The data obtained in this study is not consistent with the idea that ROS is a downstream product of ER stress, capable of inducing more ER-stress by a feedback mechanism. Therefore, a mutually enhancing effect between ER stress and ROS may not exist. The ROS found in stressed cells may serve to extend cellular survival under the condition of continuous stress. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

This thesis examines human performance under sub-optimal working conditions during work with complex and highly-automated process control systems. The operational context focuses on applications in extended spaceflight but the generic approach allows for generalisations beyond this target work environment The methodological approach is based on the use of a computerised multiple-task environment to carry out generic simulations of real work environments (micro-worlds) with a high level of ecological validity. For that purpose, a PC-based task. environment was developed to simulate the operation of a life support system in a spacecraft. This task environment has been used in lab-based experiments with trained participants from the student population and with real space crews during large-scale mission simulations. A series of six experiments was carried out (3lab and 3 field studies) to investigate the impact of different configurations of sub-optimal working conditions and unfavourable operator states, using the following independent variables: sleep deprivation, dialogue control, social isolation and confinement, training, noise, extended lay-off period and different types of system faults (corresponding to variations in workload). The task environment comprised up to five tasks, allowing for the observation of differential effects of the independent variables on different levels of cognitive activity. Dependent variables included primary task performance, secondary task performance, system control behaviour, information sampling behaviour, and subjective state measures. The findings suggested that primary performance was rarely affected, whereas certain secondary task measures and, notably, information sampling strategies appeared to be good indicators of changes in demand under the unfavourable conditions. The isolation and confinement experiments revealed no serious breakdown of performance among the crew but some indications of strain were observed. The use of two different training approaches displayed a very complex picture, with no method showing clear superiority over the other concerning performance, though there were differences in knowledge structure and system management behaviour. An important implication of the experimental work is that a broad methodological approach is needed in order to investigate the complex adjustment patterns displayed by individuals during the management of task demands under unfavourable conditions.

Do person by situation effects influence physiological stress response? Despite being relatively uncontested since being theorized nearly 80 years ago, the fight-or-flight model of stress response has suffered criticism for its one-size-fits-all approach in light of the historical gender bias in the literature. In contrast, the tend-and-befriend model of stress response argues that females are driven to care for their offspring (tend) and band together with others (befriend) in response to stress. Despite evidence suggesting the importance individual differences in the effect of affiliation and social support on stress, past research has generally overlooked markers of individual difference and personality. The major aim of this dissertation was to identify and examine potential person by situation effects on stress physiology, illuminating under what conditions and for whom affiliation and social support may buffer against the stress response and aid recovery in the wake of a stressor. Two studies were conducted to evaluate the role of individual difference factors of gender and personality (i.e., person effects) and the availability of affiliation with a similar other (study 1) or a trusted friend (study 2) compared with no available affiliation (i.e., social situation effects) on stress response and recovery. Study 1 revealed that availability of affiliation with a similar other did not have a protective influence on stress physiology in general. However, personality (openness to experience) and gender together, along with availability of affiliation, were important predictors of stress physiology over time. Study 2 indicated that the availability of affiliation with a trusted friend had a protective effect on stress physiology across each time point. Further, extraversion, conscientiousness, and openness to experience each appear to be important predictors of the influence of availability of affiliation with a close friend on stress physiology over time. In sum, this dissertation found evidence of person by situation effects on stress physiology across two studies. In both studies, the effect of the availability of affiliation differed based on individual difference factors of personality, not on gender. As such, tend-and-befriend may be better conceptualized not as a uniquely female response, but rather a stress response that depends on individual difference characteristics of personality. Further, future stress response models and research should consider personality as an important marker of individual difference in physiological stress response. / text

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

Analgesias induced by an interaction between restraint and morphine, an interaction between exposure to a novel environment and morphine, and by restraint alone were all shown to be dependent upon an increase in brain tryptophan uptake. Further investigation of the analgesia induced by an interaction between retraint and morphine revealed that the increase in brain tryptophan uptake was induced by sympathetic activity and that the nucleus raphe magnus, the nucleus raphe dorsalis, and the periaqueductal gray were critical to the analgesia. Examination of the endogenous opiod activity critical to analgesia induced by restraint alone revealed that the opioid activity was in the central nervous system and independent of tryptophan uptake. The findings reported in this thesis may be delineating a general mechanism for analgesia that involves stress, serotonin, and opioids.

The peripheral and central mechanisms mediating the modulatory effects of glucose and acute stress in rats were investigated using two versions of an appetitive win-stay task. Post-training injections of glucose, but not fructose, enhanced retention on the closed maze task. Acquisition of this task was found to be impaired by lesions of the fimbria-fornix (FF). Further experiments showed that while the celiac ganglion and the FF normally participate in suppressing the memory-enhancing effect of an acute stressor, neither structure is involved in mediating the effect of glucose on memory. Post-training injections of glucose, but not fructose, enhanced retention on the open maze task. Although acquisition of this task was not affected by FF lesions, both the celiac ganglion and the FF participate in mediating the memory-enhancing effect of glucose. Together, the results suggest that the peripheral and central mechanisms studied here are both substance- and task-specific. The modulatory effects of different types of stress, and the issue of whether task acquisition and memory modulation are anatomically distinct are discussed.

To assess the changes in glycolysis of skeletal muscles within metabolic stress and to test whether metabolic stress-induced oxidative stress and malnutrition were associated with these changes, slow- (soleus) and fast-twitch extensor digitorum longus (EDL) muscles were studied in zymosan-induced critically ill, pair-fed and control rats for 7 days. Thiobarbituric acid reactive species (TBARS) concentrations were increased in both stressed and pair-fed rats. In slow-twitch muscle, the fructose-1,6-bisphosphate (F-1,6-P2)/fructose-6-phosphate (F-6-P) ratio was decreased in stressed rats and was not altered with increased food intake. F-1,6-P2/F-6-P ratio in soleus was correlated with both TBARS and muscle dry weight. In EDL, the F-1,6-P2/F-6-P was unaffected and neither oxidative stress nor muscle weight correlated with the ratio. In conclusion, metabolic stress-induced oxidative stress and malnutrition influenced glycolytic slowdown only in slow-twitch muscle.

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