The influence of psychosocial stressors, genetic variability, and gender on subjective health complaints: Abusive supervision, genetics, and health complaints

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The influence of psychosocial stressors, genetic variability, and gender on subjective health complaints

Abusive supervision, genetics, and health complaints

By

Ann-Christin Sannes

Thesis submitted for the degree of Philosophia Doctor (PhD)

Faculty of Mathematics and Natural Sciences, University of Oslo, Norway National Institute of Occupational Health, Norway

June 2022

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© Ann-Christin Sannes, 2022

Series of dissertations submitted to the

Faculty of Mathematics and Natural Sciences, University of Oslo No. 2562

ISSN 1501-7710

reproduced or transmitted, in any form or by any means, without permission.

Cover: UiO.

Print production: Graphics Center, University of Oslo.

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Acknowledgements

The work presented in this thesis was based on existing data from the National Institute of

Occupational health (STAMI), the department of work physiology and was funded by the nonprofit organisation Et Liv i Bevegelse (ELIB). I would like to express my sincere gratitude to the following people.

First and foremost, I would like to thank my main supervisor, Johannes Gjerstad. Thank you for giving me this chance when others did not. Thank you for all the interesting and educational discussions both long and short. Not to mention the support, encouragement, opportunities, and new acquaintances I now have because of you – despite the challenges of a pandemic.

Thank you to my co-supervisor Jan Olav Christensen for all the support and encouraging words during those periods with a lot of statistical analyses. Thank you for also providing your knowledge of the psychological aspect to this work. And, for being the only other person with little knowledge on molecular biology during all our meetings. It has been a great comfort not being the only one.

I would also like to extend my greatest appreciation to the engineers Mina Eriksen, Tiril Schjølberg, and Anne-Mari Gjestvang Moe for their time and patients in teaching me the different lab techniques and methods. You have all been essential for many of the joyful moments during this project both professional and otherwise. Thank you for your support, providing me with stacks of books to read and always been available for all my questions. Without you this project would not have been possible.

A special thanks to all the PhD students, postdocs, and other members of the ELIB network which have proven to be a valuable group of people. Not only has the network been inspiring in terms of research but it has also functioned as an impromptu support group. You have all been great!

Last but not least, thank you to all my family and friends for your unconditional support and

encouragement during it all. Thank you for believing in me when I didn’t, despite not understanding what I was working on. And lastly, thank you for letting me vent when times have been hard and for celebrating when they were good. It would not have been possible without you.

Ann-Christin Oslo, June 2022

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Sannes, A., Christensen, J., Matre, D., Nielsen, M. & Gjerstad, J. (2021). Patterns of pain complaints and insomnia symptoms are associated with abusive supervision in the Norwegian working population: a latent class analysis. Scandinavian Journal of Pain, (000010151520210124). doi:10.1515/sjpain-2021- 0124

Paper II

Sannes, A. C., Christensen, J. O., Nielsen, M. B., & Gjerstad, J. (2020). The influence of age, gender and the FKBP5 genotype on subjective health complaints in the Norwegian working population. J Psychosom Res, 139, 110264. doi:10.1016/j.jpsychores.2020.110264

Paper III

Sannes, A.-C., Risøy, A., Christensen, J. O., Nielsen, M. B., & Gjerstad, J. (2021). Spinal pain in employees exposed to abusive supervision: Evidence of a sex and CRHR1 CTC haplotype interaction. Molecular Pain. https://doi.org/10.1177/17448069211042123

Paper IV

Sannes, A.-C., Christensen, J. O., Nielsen, M. B., & Gjerstad, J. Stress-induced headache in the general working population is moderated by the NRCAM rs2300043 genotype. Scandinavian Journal of Pain.

doi: 10.1515/sjpain-2022-0094

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III Summary

Abusive supervision is a strong psychosocial stressor that can influence a variety of subjective health complaints, such as pain and sleep problems. One of the biological processes, related to both types of outcomes, and activated by such an exposure is the HPA axis. Thus, this thesis focuses on the individual susceptibility, such as the influence of genetic factors related to the HPA axis and gender towards such stressors. All studies were based on a probability sample of the general Norwegian working population.

The first paper investigated patterns of subjective health complaints i.e., pain complaints and insomnia symptoms, in those experiencing abusive supervision, in addition to the role of the FKBP5 rs9470080 genotype on these outcomes. The results revealed four groups 1) high pain with insomnia, 2) low pain with insomnia, 3) high pain without insomnia and 4) low pain without insomnia. Significant findings were found for abusive supervision, but not for the FKBP5 genotype. Therefore, further investigation into the effect of this genotype was conducted in paper two.

Paper two aimed to assess the effect of age and the FKBP5 rs9470080 genotype on subjective health complaints. A longitudinal analysis of the relationship between the FKBP5 rs9470080 genotype and subjective health complaints in women showed significant results. An additional cross-sectional analysis showed an effect of both increasing age and the FKBP5 rs9470080 CC genotype on the level of subjective health complaints in women. However, no such findings were seen in men.

The third paper aimed to investigate the relationship between abusive supervision and spinal pain, including the influence of the CRHR1 rs242941/rs242939/rs1876828 haplotype. This analysis was stratified by gender. The findings showed a stronger effect of abusive supervision on the level of spinal pain in females carrying two copies of the CTC allele compared to those with one or no copies. This was not found in men.

The fourth paper investigated the association between abusive supervision and headache, including the influence of NRCAM rs2300043 genotype, stratified by gender. Recent data from our lab show that NRCAM, like FKBP5 and CRHR1, may be involved in the response to stress exposure. This was supported by the findings in this fourth paper where an association was found showing a stronger effect of abusive supervision on headache in male carriers of the rs2300043 C allele. This association was not seen in females.

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1 1 Introduction

The need for us to be part of a collective group and to be accepted have been essential since our early existence. This type of social behaviour has been vital for our survival as it increases safety and ensures a more readily supply of food. Even though society and human interactions have changed since our time as hunter-gatherers, our need for acceptance and belonging have persisted. When such primal needs are not met, they can have consequences for the individual. Situations or events where the threat to e.g., safety, well-being or psychological demands exceeds an individual’s coping resources are called “stressors” [98]. One example of such a stressor is being excluded from a group, which can in turn, initiate an activation of biological systems [7,209]. Such systems are essential for maintaining homeostasis [110], i.e., a balanced state. If the systems are not adequately regulated or if the stressor persists, it can result in physiological and/or psychological manifestations [222].

The term stress has been cross-utilised in the literature referring to different elements such as stressful stimulus, stress response and stress effect (for review see [157]). The term stress is a concept that has been used interchangeably in biological, physiological, psychological, and social research. In this thesis the term stressor refers to a stressful stimulus e.g., environmental events or situations that can be a challenge to an organism’s homeostasis. This type of stressful stimulus can elicit both psychological coping mechanisms and biological processes [13].

The biological process in terms of how a stressor is handled to maintain homeostasis is called allostasis [268]. This is an active process that through change maintains stability [173]. One example of how this is regulated is the activation of the hypothalamic-pituitary-adrenal (HPA) axis and its role in the stress response. In certain situations, the activity levels of such a response might be prolonged or altered, which can lead to allostatic load or overload [268]. These can be situations relating to the environment such as social interactions, that can lead to e.g., chronic pain [173]. How an individual cope and respond to such stress is highly individual and multifactorial. One example of a stressor that relates to e.g., social interactions is psychosocial stress.

1.1 Psychosocial stress

The feeling of acceptance and belonging can be threatened in certain environments or situations by e.g., psychosocial stress. Such situations include those that may threaten one’s social value, esteem, status and competence based on other people’s perception [67]. The response to such exposure is

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dependent on several structures. The process starts in the brain centres responsible for integration and evaluation of the sensory input, including appraising its meaning or significance [67]. This initial step occurs in the thalamus and the prefrontal cortex (PFC), before the signals are sent to the limbic system (e.g., amygdala and hippocampus) for generation of potential emotional responses. In a normal setting, where the stressor is removed within a reasonable amount of time, biological responses activate to restore homeostasis, which promotes restoration to baseline functioning [36]. However, in a situation where such stress is experienced frequently or persistently, it may result in a dysregulation of these responses [288]. It is in such instances that these events may result in both psychological and somatic symptoms through affecting the physiology of the individual. Previous research has found a neurophysiological change in connections between brain areas such as the amygdala and the PFC in response to stress [222]. In addition, being exposed chronic stress has also shown to reduce the hippocampal size [158]. In turn such changes may be of great importance for the individual.

Research on the impact of experiencing psychosocial stress, in regard to health outcomes has been increasing during the past decades. Evidence show that experiencing this type of stress is associated with e.g., changes in social perception and behaviour [274], biological aging [289], obesity [85] and even a reduction in survival rate [214]. Furthermore, several reviews have shown associations between psychosocial stress and negative outcomes, such as depressive symptoms, anxiety, alcohol use disorders, worsening of autoimmune diseases, cardiovascular disease, sleep quality and musculoskeletal complaints [34,50,120,229,245,253]. Also, musculoskeletal complaints and headache have been shown to be associated with factors such as low perceived fairness and leadership style [50,253]. This and more has been uncovered as an increasing base of epidemiological research has consistently documented the impact of psychosocial work characteristics [50,253]. However, comparatively few studies have investigated the underlying biological mechanisms and the role of genetic factors in shaping the response to sub-optimal working conditions. Not only are such consequences detrimental for the individual.

Psychosocial stress may reduce physical ability and social contact with other, but it may also increase demand on health care services and socioeconomic cost in terms of sick-leave and absenteeism from work. Based on the wide psychological and physiological impact that psychosocial stress might have, further assessing the consequences of such exposure is of importance. Settings in which these situations might occur can be many and varied. It can be amongst family and friends or colleagues at work. This thesis will focus psychosocial stress in the workplace.

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3 1.1.1 Abusive supervision

One example of psychosocial stress is abusive supervision [165]. This type of leadership style entails persistent hostile verbal and nonverbal behaviours, excluding physical contact [118,259]. Exposure to such leadership style can be highly damaging to those affected. It has been shown that it may result in both somatic complaints, including sleep [51,77,91,99,123,205], psychological symptomatology [176,223], such as emotional exhaustion (for review see [165]) and outcomes resembling post- traumatic stress disorder [262]. A study from the US estimated that 10-16% of workers are exposed to such behaviour from their superiors [260]. These numbers are closely related to the data set used in this thesis based on a Norwegian probability sample, with a prevalence of 9-10%. There may, however, be a difference regarding the definition of abusive supervision. There currently does not exist a consensus regarding the cut-off value that represents the threshold of abusive supervision. Hence, these numbers must be interpreted with some reservations.

One important factor as to why abusive supervision can induce negative effects has been proposed to be related to the power difference between the employer and the employee (for review see [198]). If a mismanaged power difference is allowed to persist over time the employee may process this behaviour similar to threat detection, which can lead to an increased likelihood of continued perception of the behaviour as abusive [161]. This in turn can lead to a negative spiral which may result in an unnecessary duration of the stressor.

Like other social stressors, abusive supervision activates biological stress responses such as the sympathetic nervous system (SNS) and the HPA axis. In this thesis the focus will be on the HPA axis.

1.2 HPA axis

The HPA axis is a neuroendocrine system heavily involved in the stress response in humans. This system is highly important in maintaining homeostasis in response to e.g., external stressors.

The HPA axis is initiated by input from cortical and limbic brain regions to the paraventricular nucleus of the hypothalamus [110,290] in response to a stressor. This in turn releases corticotropin-releasing hormone (CRH), which leads to the pituitary release of adrenocorticotropic hormone (ACTH), from the anterior pituitary gland. Further, ACTH promotes adrenal secretion of glucocorticoids e.g., cortisol.

These circulating glucocorticoids (CORTs) interact with essentially every organ and tissue [188] through

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two main receptors; mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). Both these receptors are intracellular which can activate or repress several glucocorticoid responsive genes [188].

Under normal conditions the HPA axis is self-regulatory (see Figure 1). This is regulated by the free circulating CORTs which activates a negative feedback loop that regulates further secretion of CRH.

This self-regulatory function is important in maintaining an appropriate level of activity by adjusting the secretion of CRH and thus the regulating the cascade of events back to baseline levels.

Figure 1. Schematic overview of the HPA axis. Created with BioRender.com

In addition to the HPA axis’ role in the biological stress response it is also involved in regulating both the circadian rhythm [184,188,254] and the ultradian rhythm [276]. The circadian rhythm shows a cyclic activation with its peak in the morning, just before waking [276], and its most known function is the 24-hour sleep-wake cycle [254]. The ultradian rhythm refers to discrete pulses of CORTs released from the adrenal cortex [276]. This pulsatile secretion is important in maintaining general tissue responsiveness which is important in response to e.g., stressful stimuli.

Moreover, psychosocial stress, and a potential consecutive change in cortisol levels, has previously been linked to several negative outcomes such as pain disorders [212] (for reviews see [73,98]).

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Considering the wide affinity of cortisol receptors throughout the body it might have an impact on several processes and therefore also lead to a variety of physiological consequences such as pain.

1.3 Pain

The experience of pain is possibly one of the most relatable experiences and is defined as:

“An unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage” [204].

Further, this definition has been expanded by several key points [204]. Three of the points important for this thesis are; 1) pain is always a personal experience that is influenced to a varying degree by biological, psychological, and social factors, 2) although pain usually serves an adaptive role, it may have adverse effects on function, social and psychological well-being, and 3) pain and nociception are different phenomena.

The advantage of pain has had an evolutionary value. Pain draws attention to an area that might be damaged or potentially close to damage. Further, it serves as a reminder that actions are needed to reduce further harm to the area, to seek protection or safety. One example can be that an injured animal hides, avoiding being an easy meal [277], removing itself from predators that may have taken advantage of its vulnerability. This protective response increases the chances of healing, which is pertinent to survival. Moreover, social modulation of pain has also had an evolutionary advantage.

Deciding when to express pain and when not to depends on the situation. It has been proposed that one of the factors affecting this decision is the relationship between those in proximity and what type of information it may reveal [133]. Pain that increases wanted attention or further helps the situation is more likely to be expressed than pain that might lead to negative consequences, e.g., being left out or left behind. This information might be important in studies involving self-reported pain outcomes, e.g., reporting increased levels of pain might lead to an advantageous outcome for the individual.

Pain is multidimensional and has been categorised in several ways. It can be divided into dimensions such as 1) sensory; the location and intensity of the pain, 2) emotional; how unpleasant the experience is and 3) cognitive; the interpretation of pain based on past experiences and the response it elicits [59].

Moreover, pain can also be divided into acute and chronic. Acute pain is pain lasting 3 months or less and is normally, but not always, associated with injury [48]. Chronic pain, on the other hand, lasts longer than 3 months [266]. It can start as acute pain, with actual tissue damage but may persist even after tissue healing. This type of pain experience can be caused by dysfunctional sensory processes

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due to an altered relationship between the site of injury and higher level processing, and can lead to a failure to return to the prior state or “reset” [48]. Chronic pain has been shown to be relatively common with an estimated prevalence of 24 - 31% in Norway [145,217].

Further, chronic pain can lead to neurophysiological changes and activity in several brain areas. One study observed a decrease in parietal activity suggesting an attenuation of attentional networks [18].

In addition, the same study found that a frontal increase in activity was seen, possibly as a result of cognitive and emotional enhanced processes [18]. Also, a shift in brain activity from sensory regions to emotional/limbic regions such as mPFC, anterior cingulate cortex (ACC), and amygdala, has been found in chronic pain [107]. These areas are highly involved in the social modulation of pain which is an important aspect. Moreover, it has been proposed that there is a shift in the efferent channels from periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) from inhibitory to facilitation that also contributes to chronic pain [271].

Additionally, it has also been shown that both brain morphology [16] and activity [22] in those with chronic pain is not equivalent to those who are not in pain. Reduced grey matter tissue in the PFC and the thalamus of chronic pain patients have been reported [16]. Further, a decreased level of excitatory neurotransmitters in the PFC consistent with PFC dysfunction [93] has been found in similar patient groups. Hence, suggesting a neurobiological consequence of pain.

Some of the manifestations of these changes have been seen in relation to cognitive abilities. One example is that patients with chronic back pain seems to demonstrate poorer decision-making [15,187]. This may be important for decision making in all aspects of daily life such as work and otherwise. However, the silver lining is that these changes are not permanent. This is good news considering the relatively high prevalence of chronic pain in Norway [145,217].

To understand the complexity of pain it is also important to keep in mind the intricacy of the system responsible for relaying the messages i.e., the nervous system. The experience of pain is the end result of a chain of events that starts with smaller structures and somewhat less complex processes. As mentioned as a key point earlier there is a difference between pain and nociception.

1.3.1 Nociception

To better understand the complexity of pain a brief introduction to the process of nociception is necessary. This is best achieved by understanding the structures involved, both peripheral and central, their role in the process and how it all connects.

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Nociception, from the Latin word nocere meaning “to harm/hurt”, is the process by which organisms detect potential or actual damaging stimuli [237] e.g., leads you to change your position when you’ve sat in one position for too long. Thus, protecting your body from potential harm and therefore pain.

A dedicated class of sensory afferents called nociceptors transmits this information. Nociceptors include different fibre types which have different qualities, such as Aδ- and C-fibres [48]. These can be found in a range of different tissues, such as skin, muscle, bone and connective tissue [48]. A nociceptor is defined as “a high-threshold sensory receptor of the peripheral somatosensory nervous system that is capable of transducing and encoding noxious stimuli” [196].

The route these signals take starts with the activation of e.g. Aδ- or C-fibres. In turn, these signals travels to the dorsal horn (in the spinal cord), and ascends through e.g. the spinothalamic or spinohypothalamic tracts [265] before reaching the higher processing centres of the brain, such as the limbic and cortical areas previously mentioned. However, an important point is that activation of nociceptors does not necessarily lead to pain. Pain is a conscious experience caused by the individuals’

interpretation of the nociceptive information. The final experience is an amalgamation of memories, emotions, pathological and cognitive factors [265]. In other words, nociceptive signals can arise without passing the threshold for pain perception [21]. Activation of these can have several different consequences. One example is that continuous nociceptive input to supraspinal targets such as within the limbic system 1) reorganizes memories and their associations within the cortex; and 2) reorganizes motivational and memory consolidation properties [18]. Nociceptors have a great potential for plasticity (the ability to adapt [94] in response to demand), which is important in the process of sensitization [237]. This process includes stimulating non-responsive neurons to become responsive, or neurons responding at a reduced threshold and/or produces a response of greater magnitude. In turn, the pathways involved in nociceptive signalling are activated more extensively and/or strongly.

One example of this can be dorsal horn facilitation from intense or prolonged nociceptive input. This in turn lowers pain threshold, amplifies nociceptive responses and expands the receptive fields of nociceptive higher order neurons to incorporate non-injured areas near the wound and normally non- nociceptive sensory signals [129].

Due to the complexity and number of structures involved in both nociception and the experience of pain a brief overview of some of the related areas will be presented.

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8 1.3.2 The neuroanatomy of pain

The complex processing of nociceptive input, leading to the experience of pain, involves a vast number of brain areas. For the purpose of this thesis a short description of some of these areas will be given.

1.3.2.1 The limbic system

The limbic system’s main role is the integration of experiences from the external world with the biological processes. In other words, it coordinates reflexes and behaviours needed to maintain a state of homeostasis. It integrates endocrine function and autonomic activity with social behaviours that combined are important physical and cognitive responses. There are several brain areas relating to the limbic system (see Figure 2). Even though they all are important in their own way, only a few of the areas will be presented for the purpose of this thesis.

The thalamus, which consists of multiple nuclei, has several essential roles and functional components.

These nuclei can be divided into three categories: 1) sensorimotor, 2) limbic and 3) a combination of 1

& 2 [273]. Once the input is received, it is then processed and passed onto e.g., cortical areas [14], hippocampus [243] and amygdala [227]. The functional components of the thalamus have been

The limbic system

Figure 2. Schematic overview of some of the structures within the limbic system. Created with BioRender.com

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divided into five major groupings [227]; 1) arousal and pain regulation, 2) all sensory domains except olfaction, 3) motor language function, 4) cognitive functions, and 5) mood and motivation.

The hippocampus is highly important in creating memories and the ability to learn new information.

The hippocampus builds cognitive maps through which humans e.g., recognise their relation to objects and events, both present and past. It is due to these functions that the hippocampus is important in the processing of both psychosocial stressors and pain. Moreover, this area is highly integrated with other limbic structures such as the amygdala.

The amygdala has an important role in coordinating autonomic and emotional responses. This area is important in linking emotional and motivational responses to external stimuli, such as being exposed to a psychosocial stressor. It also contributes to learning and memory, which are key functions for the stress response and pain processing. Like the hippocampus it is highly connected to other brain areas, such as the PFC [187], which regulates e.g., behaviour and reasoning [84]. Moreover, it is also connected to the hypothalamus thus its importance in the homeostatic regulation of autonomic, endocrine, and immune responses.

In addition to its previously mentioned neuroendocrine functions relating to the HPA axis, the hypothalamus has an important role in the integration of inputs from areas such as the amygdala and the hippocampus with ascending signals from the spinal cord and brain stem. Through its regulation of autonomic nerve impulses, it evokes the physical manifestations of emotion, such as sweating, increased heart rate and disturbances in the gastrointestinal tract. This connection to the response of emotional and cognitive reactions is due to its connection to e.g., the PFC.

1.3.2.2 Other related brain areas

The PFC has several functions that are highly important in the processing and experience of pain. These can be categorised into e.g.; [233] 1) executive functions, such as initiating and applying behaviour, attention, information processing and emotional regulation, 2) encoding and retrieval of memory, and 3) intelligence i.e. verbal expression, abstraction and formulating behavioural plans. All of which play a role when exposed to a psychosocial stressor. Another important area that is highly interconnected with the aforementioned areas is the insula. This area is involved in e.g., emotion, attention, memory, somatosensation, and pain [143,267]. Additionally, the ACC has been called one of the main brain regions that signalise pain, or emotional pain [18]. It is involved in the mediation of the emotional

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response to pain [233] and the emotional/affective properties of pain as well as the anticipatory response to pain [18].

1.3.3 Psychosocial stress and pain

The mechanisms behind the relationship between psychosocial stress and pain is still not fully understood. However, evidence show an observed increase in activation of the pain matrix in the brain e.g., PFC, ACC, and insula [75,125]. Additionally, studies have discovered an increased dopamine release in limbic areas following psychological stress [201], though this association needs further investigation.

Despite the need for more research, one proposed model attempts to describe the chain of events from chronic stress exposure to increased pain signalling. Persistent exposure increases activation of areas such as the ACC and the insula, which may lead to adaptive changes, which are still unclear. This influences the amygdala resulting in an alteration of opioid receptor binding, which in turn e.g., activate opioid receptors in the RVM. In the spinal cord a transient increase in pro-inflammatory mediators, such as tumour necrosis factor alpha, is also seen which results in enhanced pain signalling [125].

Moreover, the mechanisms are likely multifaceted. The relationship between psychosocial stress and pain can be influenced by behavioural changes. Examples of this can be a change in eating, drinking and exercise habits. These changes, alone or combined, could induce e.g., weight changes which is unfavourable in relation to pain conditions. In addition, being subjected to long-term hormonal dysregulation of hormones, such as cortisol, can lead to an increase in negative health outcomes [8].

The mechanisms behind this could be cortisol’s important role in the regulation of inflammation which in turn is a possible explanation for the increase in the unfavourable outcomes [54,235].

1.4 Subjective health complaints

Subjective health complaints, such as pain, comorbid pain and insomnia can include several different afflictions which can have an effect on both the individual and the society. The consequences are therefore varied, ranging from a change in quality of life on an individual level to a socioeconomic burden on the society. A model that explains the interactions between biological, psychological, and social aspects on pain is the biopsychosocial model (see Figure 3). This model has been said to be the

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most used model in research relating to e.g., chronic pain [275]. By including all three aspects the multidimensionality and reciprocal influence of e.g., chronic pain [175] is explained. Based on the variables of interest in this thesis this model will be referred to in the coming sections.

Figure 3. Biopsychosocial model of pain with examples of the different aspects. Created in Biorender.

Moreover, the included health complaints are all relatively prevalent and show a natural connection to the biological stress response, which can be a consequence of or influenced by exposure to a psychosocial stressor.

1.4.1 Musculoskeletal pain

The musculoskeletal (MSK) system is an intricate and dynamic system which consists of several structures such as bone, cartilage, intervertebral discs, tendons, ligaments, and muscles. All these structures are highly interconnected and therefore highly dependent on one another for general function [213]. Considering the complexity of this system it might not be surprising that it is also prone to a variety of disorders and afflictions. MSK pain is an umbrella term that includes afflictions such as, but not limited to, arthritis, carpal tunnel syndrome, ligament strains/sprains, herniated and/or degenerative disc disease and low back and/or neck pain. The cause of MSK problems can be many and multifactorial. Factors can be both external e.g., physical strain (work, trauma etc), and internal, such as aging, genetics, minerals, vitamins, and hormones. Some of the hormones known to have an impact are, e.g., relaxin [66], oestrogen [234] and cortisol [202]. As previously mentioned, cortisol is the end product of the HPA axis. Hence, it is plausible that being exposed to a psychosocial stressor may have an impact on MSK pain.

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MSK pain is one of the most prevalent reasons for visiting healthcare providers and reason for sick leave. It has a great impact not only on the individual’s quality of life and health, but also on society in terms of socioeconomic burden. As many as approximately 80% of the Norwegian adult population experience pain originating from the MSK system at least once [121,140]. The most commonly reported areas of MSK complaints are low back [121] and neck [294]. A rapport from 2019 showed that the socioeconomic cost, in Norway alone, was as much as 165 billion NOK annually [71].

Additionally, the leading cause of disability globally has been found to be low back and neck pain [68].

For both men and women, low back pain (LBP) was the number one leading cause for years lived with disability, whereas neck pain has previously been ranked as number eleven and nine [3]. Some of the known risk factors for MSK pain are genetic factors [59,113], sex [68,80], smoking, obesity and occupational factors [68,104]. Additionally, LBP can also be influenced by other comorbidities such as sleep problems and widespread pain [251].

In LBP patients, as well as in those with other chronic pain conditions, the prevalence of pain in more than one location has been reported to be anywhere between 10-89% [47,53,62,132], for review see [106,291]. The mechanism behind these common occurrences is not yet fully understood. However, suggestions have been made that generalised central processes and reduced descending inhibitory control mechanisms are involved in patients with pain in more than one site [88]. Further explained by an increased facilitation, pain in more than one area is associated with several negative consequences such as risk of chronicity [263], reduced quality of life [47], poorer general health [186,292], decreased probability of recovery from LBP [291], and sleep problems [186].

Thus, due to the increased probability for co-complaints in other areas of the spine if the primary complaint is already in on area [103], and the high prevalence of low back- and neck pain, the focus in this thesis will be MSK pain relating to the spine.

LBP is a multidimensional complaint, which includes and affects all parts of the biopsychosocial model [104]. Pain has an impact on our biology by activating systems, both endocrine such as the HPA axis and neurophysiological. Regarding neurophysiological consequences, changes within the descending pain modulatory network have been implicated in chronic pain [265]. In addition to evidence indicating that patients with chronic LBP show a 2-11% reduction in neocortical grey matter volume, which has been related to pain duration [17]. However, the consequences do not only have an impact biologically. The psychological aspect of pain has also been investigated. Studies have shown that there is an impact on the mental health of those affected with MSK pain [251] (for review see [156]).

Moreover, pain also impacts the social aspect of the biopsychosocial model. It can have an impact on the person’s quality of life, including e.g., social interaction, physical function, and financial

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consequences due to absenteeism from work. This in turn might result in loosing social interactions normally encountered in the workplace. The combination of these three aspects underlines the complexity of the consequences as a result of low back pain.

As previously mentioned, LBP is one of the most common reasons of disability worldwide which includes the working population [68]. This can lead to absenteeism from work and sick leave, which in turn might result in both a loss of productivity and a societal cost through benefit payments. However, LBP can have a great impact on both an individual and societal level and have a wide range of presentations [43,49]. Importantly, LBP is often short-lasting and has little to no serious consequences [49]. Additionally, it is important to underline that LBP is a symptom [122], and may vary widely in terms of duration (acute/chronic) and origin. The most common form of LBP is non-specific LBP [104]

and has no apparent structural origin. It is defined as discomfort and pain below the costal margin and above the gluteal folds [117], which may or may not be accompanied by referred leg pain [9]. As a contrast, specific LBP is pain originating from structures such as the intervertebral disks, the surrounding tissues, or the spine itself [216]. This type of LBP accounts for less than 15% of all back pain [9]. Examples of this can be disk herniation, spondylolisthesis, muscle strain, fractures, or tumours.

Like LBP, neck pain can also be divided into non-specific and specific, acute, and chronic. The non- specific variant is defined as discomfort or pain in the neck that may or may not involve the shoulder girdle with or without referred pain down the arms [80]. The lifetime prevalence of neck pain has been estimated to be between 14 – 71 % in the adult population (for review see [80]), and is a common cause of disability globally [68]. The natural history of non-specific neck pain is normally between days to weeks, but it can also become recurrent or chronic [27].

As with any type of chronic pain, MSK afflictions may influence other aspects of daily life. One aspect that seems to be influenced directly is sleep. Studies show that 40 - 60% struggle with sleep due to chronic pain complaints [38,134]. Moreover, this relationship seems to be interrelated, meaning that chronic pain increases sleep difficulties and vice versa (for reviews see [29,238]). It seems that neither the number of pain sites nor the duration of pain is as important as the intensity and the affective dimensions of pain, such as fear [136], in affecting sleep. Due to the, seemingly undeniable, interrelationship between pain and sleep it seems important to include it when investigating pain conditions.

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14 1.4.2 Insomnia symptoms

The second component within the investigated subjective health complaints in this thesis relate to sleep. Sleep has been defined as “a natural and reversable state of reduced responsiveness to external stimuli and relative inactivity, accompanied by a loss of consciousness” [206]. Neuroanatomically, sleep involves several brain areas, one of which is the suprachiasmatic nucleus (SCN) in the hypothalamus [33,180]. Within the SCN is the molecular oscillators, also called the biological clock [52,230], that is responsible for generating a 24-hour cycle [33,124]. This cyclical rhythm is regulated by a change in input of light going from day to night [224]. However, there also seems to be a close link to a more homeostatically regulated aspect of sleep (for review see [11,239]), based in the hypothalamus. It is due to this link to the hypothalamus, the initial site of the HPA axis, that sleep is of interest in this thesis. In addition, previous evidence have shown a link between psychosocial stress (for review see [293]), the effect on sleep and its potential consequences. Even though all the aspects of sleep are yet to be determined [224], lack of sleep has an impact on several bodily functions, e.g.

digestion and cardiovascular problems (for review see [293]). Moreover, it is essential for biological processes such as brain development and maintaining normal brain function [10], including memory processes (for review see [206]).

Difficulty sleeping is commonly called insomnia. Insomnia is defined as “persistent difficulty with sleep initiating, duration, consolidation or quality that occurs despite adequate opportunity and circumstance for sleep, and results in some form of daytime impairment” [193]. Insomnia is one of the most prevalent sleep disorders, with about ⅓ of adults experiencing insomnia symptoms [28].

However, it seems that this estimate may contain some variations. A study assessing the prevalence of insomnia experienced by patients through the general practitioners (GP) office found that over 50%

reported insomnia [28]. Another study reported a 13% prevalence using the same diagnostic criteria and insomnia scale [192], this study was however conducted on a population based scale. A third study showed that 30% of adults reported one or more symptoms of insomnia [215]. Even though these numbers vary, they give an indication of the prevalence despite the different populations investigated.

How insomnia presents might be slightly different between those affected, but some of the main complaints are trouble falling asleep, trouble with coherent sleep and/or early awakening [97,215].

The causes of sleep problems might be many, varied and multifactorial, and can like other afflictions, cover all aspects of the biopsychosocial model. Some of the psychosocial stressors that might cause acute insomnia are interpersonal conflicts or stress at work [211]. This type of insomnia often ceases after removal of the stressor. However, previous evidence show that just over 20% of those affected

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by acute insomnia can transition into chronic insomnia [74]. Hence, understanding influencing factors might be important in preventing chronification of insomnia.

1.4.3 Abdominal pain

Abdominal pain is another common symptom that can present throughout life [119,135] and may have several causative factors. Some might be somatic, such as appendicitis, while others might be a result of environmental factors such as social stress, e.g., functional gastrointestinal disorders (FGIDs). For the purpose of this thesis the latter will be the focus. For complaints without a purely structural origin, the biopsychosocial model offers some insight when exploring causative or influencing factors.

One group of disorders that are influenced by several components of the biopsychosocial model are FGIDs [269]. Such conditions are complex and multifactorial including genetic and environmental factors. One proposed explanatory model for the interaction of psychosocial and biological factors is the gut-brain axis which is influenced by the e.g., the HPA axis (for review see [167]). Due to this proposed link, it seems important to include abdominal pain when investigating associations between stressful stimuli and health complaints in the general population.

1.4.4 Headache

The last health complaint included in this thesis is headache. As many as 50-90% of adults have experienced headache within the last year [244,248]. This high prevalence consists of several different types of headaches. The two most common headache types, tension-type headache (TTH), and migraine, alone, affects almost three billion people globally [55]. These are both classified as primary headaches, meaning they have no clear aetiology [20], i.e., not due to an underlying condition. The most common primary headache is TTH, with a lifetime prevalence of 30-78% of the European adult population [1,126,244,248]. The mechanism of this type of headache is not fully understood but is has been divided into subcategories such as episodic or chronic [90]. However, for the episodic variant, peripheral pain mechanisms is suggested to be involved, whereas central pain mechanisms may have an influence on the chronic variant [1].

Migraine headache has an estimated prevalence of 15-35% in Europe [244,248]. The pathophysiology of migraine is yet to be fully understood. It has long been proposed that migraine has a neurological or vascular origin. Neither of these have been accepted as conclusive, and other proposed mechanisms have been presented. This includes suggestions such as involvement of trigeminovascular pathways,

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genetic predisposition and a brain state of altered excitability (for review see [92]). Moreover, there is evidence showing that TTH and migraine can also co-occur [160].

As with abdominal pain headache complaints are also influenced by several components of the biopsychosocial model. More specifically, an association between psychological stress, the HPA axis and headaches has previously been shown (for review see [185]). Based on this association and the high prevalence it is natural to also include headache as part of the chosen subjective health complaints investigated in this thesis.

1.5 Genetic factors

As previously mentioned there seems to be a genetic component in relation to all the chosen health complaints in this thesis [97,108,151]. Due to the connection between these afflictions and the structures and functions of the HPA axis, genes and genetic variations related to the stress response was chosen as part of this thesis. However, there are several different methods and approaches when investigating genetic influences. Hence, some of which will be briefly mentioned.

1.5.1 Different methods and approaches

There are several methods available to assess genetic factors and their influences. All methods have strengths and limitations, which makes the choice of method dependant on the research question.

One of the most well-known methods are twin studies. Twin studies has been utilised to study the influence of genetics for decades. This method can consist of several different designs that provides different information [255]. One benefit is the possibility of estimating the proportion of variance within a trait that can be attributed to genetic variation versus the proportion that is due to shared/unshared environment [219]. This method is useful in investigating the relative importance of the interaction between genes and the environment [105,255]. However, some limitations to this method can be e.g., that the results cannot be generalised to a larger population, due to lack of randomisation. Nor can it provide any information on the genes responsible.

Another method is Genome Wide Association Studies (GWAS) [220], which is a method that investigates a wide range of genetic variability (for review see [100]). One of the benefits of utilising this method is that there is no need for a priori hypothesis. This open-minded approach presents findings that are not susceptible to any form of preconception. However, a GWAS study only provides

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data relating to the alleles with large enough effects. Moreover, it finds loci (the location of the gene), not genes, which can complicate identifying any pathogenic changes. In addition, to ensure a reliable result a high sample size, thousands of subjects, is required, which is costly.

The candidate gene approach, on the other hand, is more affordable. This method offers information on the association between a specific genetic variant and selected outcome. The genetic variants can include, e.g., a genotype, based on a single nucleotide polymorphism (SNPs), or a haplotype (combination of alleles inherited together [199]). These studies focus on a priori hypothesis about the aetiology of the outcome, and are often conducted on a population-based sample [252]. The result can e.g., give an indication on whether a risk allele is more prevalent in the subjects with the outcome of interest [144,199], or if it has an influence on the relationship between an exposure and an outcome.

This in turn can be useful as a first step in further investigations of potential causal pathways and mechanisms. Proposed guidelines for these type of studies are: biological plausibility, strength of association and dose-response relationship and consistency [252]. However, like for the other methods this also suffers from some limitations. One limitation is that it does not consider the complex interplay between multiple genes and mechanisms. Moreover, even though one genetic variant might not have the penetrance needed to show statistical significance it doesn’t mean that it doesn’t play a role in the overall mechanism. Additionally, this approach also suffers from the “streetlight effect”, a type of observational bias [96] which is based on looking where it is easiest to look. It has been described as losing e.g., your keys somewhere along a road and only choosing to look where the streetlight lights up the road, even though the keys can be lost anywhere along the road.

1.5.2 Variations, interactions, and factors

One of the potential factors influencing the response to stressful situations is the individual’s genomic architecture such as genetic variations. This variation is caused by the genes present and order of the base pairs constituting a certain DNA sequence. One such variation is SNPs which are distributed throughout the genome, occurring at specific locations and are highly abundant [78]. Further, there are variations within each SNP. These variations are associated with susceptibility and vulnerability to disease [232], in addition to the regulation of biological processes relating to e.g., a stressor, such as the HPA axis [79], and brain processing [56]. Interestingly, accumulation of stressful events has shown to alter brain activity based on genetic variance [45]. Moreover, life experiences have also been shown to alter the epigenome, which may have a lasting influence upon the expression of some genes [87].

These are changes that modifies the genome without altering the DNA sequences. Importantly, these changes can be either beneficial or increase the risk of negative outcomes.

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The events that might induce changes in gene expression based on surroundings and experiences is called, gene x environment (GE) interactions. GE interactions can present the individuals risk for a given outcome, such as pain sensitivity, if exposed to sustained or severe stress [48]. Events like these might have an impact on epigenetic mechanisms [170]. Epigenetic mechanisms are changes in the gene expression that does not alter the DNA sequence and can be a result of external factors [115,172].

These changes allow rapid adaptation and are highly dynamic in relation to the environment [87], which might lead to positive and appropriate changes. Epigenetic changes has also been shown to occur at an allele-specific level [174], indicating that genetic architecture is important to stress response.

Moreover, several genetic factors, such as FKBP5 and CRHR1, are known to be associated in the biological response to stress [79] and thus in its regulation. Some of these acts as ligands (a molecule that transmits signals between and/or within cells) through GR activity and other cellular processes [79,290] once a stressor is perceived. These genes are found expressed in several central brain areas relating to stress, pain and sleep [236]. Another genetic factor that is expressed in similar areas, which is also found to be influenced by exposure to stress, is the gene encoding NRCAM. This gene has previously been investigated in relation to negative affect [191], neuropsychiatric conditions [40,183]

and neural development [101].

As these different genetic factors have previously shown to influence different outcomes of stress exposure, and their affinity in pain related structures and brain matrix, they will be further discussed with the intent to examine the possible associations between psychosocial stress and health outcomes.

1.5.2.1 FKBP5

The gene encoding the FK506-binding protein 5 (FKBP5) molecule belongs to the FK506-binding protein family [290] and is located on the short arm of chromosome 6 (6p21.31). Previous studies suggest that this molecule has an inhibitory effect on the GR signalling [290] and its sensitivity to cortisol [35,42].

This inhibitory effect occurs through ligand binding, receptor activation and intracellular transcriptional processes [225]. The FKBP5 molecule acts as a co-chaperone that changes folding and activity of other proteins [87,290].

Once exposure to a stressor occurs an increase in FKBP5 expression is seen in several brain areas [188].

The largest changes have been observed in the amygdala and the PVN. Moreover, the amygdala has shown structural and functional changes if exposed to stress [116]. Further, expression of this gene seems to be age dependent. With increasing age, an increase in FKBP5 expression has been observed

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in several brain areas [30,130]. These changes, in combination with FKBP5 might be of importance for the mechanism responsible for negative outcomes on an individual level.

Previous studies have found associations between FKBP5 and depression [250], bipolar disorder [286], PTSD [285], Alzheimer’s disease [30] and worse cognitive performance [83]. This indicates that the FKBP5 gene may play a role neuroendocrine dysregulation, especially if exposed to stress. It has also been suggested that FKBP5 alter pain sensitivity, which supports the idea that it may drive persistent pain states [162].

1.5.2.2 CRHR1

The gene encoding corticotropin-releasing hormone type 1 receptor (CRHR1). Located on the long arm of chromosome 17 (17q21.31), this G-protein coupled receptor is responsible for binding neuropeptides of the CRH family [142]. This happens by enabling the G-protein-coupled peptide receptor activity as well as corticotrophin-releasing factor activity [4]. It is through these functions that this gene maintains normal hormonal responses to stress.

The CRHR1 receptors are, like FKBP5, expressed in several brain areas, such as the prefrontal cortex, amygdala, and the pituitary gland [112,236]. These are areas important for cognitive function and supraspinal nociceptive processing [236]. Moreover, it is one of the important mediators of the HPA axis [208]. Due to its connection with the regulation of cortisol it has been a factor of interest in regard to several negative outcomes, such as depression [76,150,153,154], alcoholism [142,208] and suicide [280].

1.5.2.3 NRCAM

Another interesting gene is the gene encoding Neuronal Cell Adhesion Molecule (NRCAM). Located on the long arm of chromosome 7 (7q31.1), this gene is involved in a wide variety of functions in neuronal development [149], such as axon guidance, neuron to neuron adhesion as well as cell to cell communication [5]. NRCAM belongs to the immunoglobulin superfamily where it can act as both a ligand and a receptor [5,159]. It is expressed in neurons and glial cells [159], nodes of Ranvier [61] and a wide variety of brain areas such as the cerebral cortex, hippocampus and the amygdala [236].

Furthermore, it has also been found to be expressed in structures important to the HPA axis such as the hypothalamus and the adrenal glands [236]. Additionally, it has been found to be important for

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neural circuit formation during development [246,247]. Interestingly, it has previously been associated with several disorders such as schizophrenia [138], autism [166], and neuropathic pain [149].

1.6 Sex and gender differences

Research exploring the differences between men and women in relation to a variety of outcomes has been of interest for some time. However, historically, it was perceived as too challenging including women as participants due to their biological complexity [203], e.g., hormones, thus they were excluded. In later years this has changed and new knowledge on influencing factors have been uncovered in both men and women. The differences are believed to be influenced by a combination of biological, psychological and socio-cultural factors [200].

In the literature, the terms sex and gender are often used interchangeably despite there being a distinct difference between the two terms. Sex has been described as characteristics that are biologically defined, whereas gender is based on features constructed by society [194] which also relates to identity. Depending on the research question this may or may not be important. If the scope of the study is to assess societal expectations depending on gender, then assigned sex at birth might be less important. If the purpose is to investigate biological processes that are related to sex differences, based on genetics or hormonal components, knowing the sex assigned at birth might be more important. In the questionnaire used in this thesis the participants were asked whether they were male or female. As this might not necessarily be their assigned sex at birth, the results in this thesis ought to be considered as gender dependent.

However, between men and women there are differences in biological factors such as genetic, hormonal, and neural components. Starting with the genetic component, most humans have 23 pairs of chromosomes whereof the 23^rd pair are the sex chromosomes. This is the chromosome pair that differs between men and women, which gives rise to the biological differences between the sexes.

Men have XY, and women have XX. This means that genetic predisposition could present with a sex dependent influence on several outcomes, e.g., pain and sleep. As a consequence of the different chromosomal difference, men and women have different levels of various hormones, e.g., sex hormones. Sex hormones may influence several parts of e.g., the central nervous system, including direct and indirect effects in pain processing [81]. An example of how hormonal changes might impact pain has been shown in relation to migraine before and after puberty. Before puberty girls and boys were affected approximately equally [81]. However, post puberty, the lifetime prevalence increased in women compared to men. Another example of the hormonal impact in relation to pain has been

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observed depending on which phase of the menstrual cycle women are in [109,240], reporting higher sensitivity to pain during certain parts of the cycle.

Moreover, studies have found a difference in activation of several brain areas (e.g., insula, amygdala) in response to pain between sex/gender [26,81,137,181]. This evidence might be due to the neuroplasticity which can be a result of both nature and nurture. As these areas are highly involved in e.g., memory, the response might be dependent on past experiences and coping strategies. So far, there is evidence showing that women report pain at higher intensities, frequencies and in more locations than men for some conditions [189,200,203]. These conditions might include e.g., sleep problems [151,215], abdominal pain, headache and musculoskeletal pain [81]. Further, several studies have been conducted regarding gender differences investigating several different types of pain (for review see [81]). Despite investigating different types of pain, men seem to be more resilient than women [68].

Other than the proposed biological difference, other reasons might be psychological and socio-cultural differences that can have a great impact on the expression and reporting of pain [200]. This means that any gender difference observed in one demographic might not be representable on a global scale.

Most epidemiological research looking into this topic i.e., psychosocial stress in the workplace, has been conducted in Europe and especially Scandinavia [81], meaning that it might not be representable for other demographic areas. It seems probable that the observed reported pain levels and pain experiences might reflect a combination of factors some of which have been mentioned in this section.

Importantly, this topic still suffers from knowledge gaps that needs further investigation to uncovered mechanisms and processes that can explain the differences in full.

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22 2 Aims

The overall aim of this thesis was to examine the effect of psychosocial stress, in the form of abusive supervision moderated by genetic variants and investigate gender differences in the working population. The moderating influence of genetic variants and gender may be important for resilience and adaptability in response to certain exposures and might also influence different outcomes. Hence, we aimed to:

1. Examine subgroups within the working population based on the FKBP5 rs9470080 genotype, reported abusive supervision and subjective health complaints. Thus, uncovering epidemiological patterns that previously have not been investigated. This will be done in three steps:

a. Uncover patterns of pain complaints and sleep problems, i.e., subjective health complaints, in the general Norwegian working population

b. Determine whether abusive supervision or the FKBP5 rs9470080 genotype is associated with these patterns

c. Examine the interaction between abusive supervision and the FKBP5 rs9470080 genotype regarding subjective health complaints

2. Investigate the association between age and the FKBP5 rs9470080 genotype on subjective health complaints, and the differences between men and women. Thereby, further exploring any potential associations and implications of the FKBP5 rs9470080 genotype.

3. Assess for any association between abusive supervision and musculoskeletal pain relating to the spine, moderated by the CRHR1 rs242941/rs242939/rs1876828 haplotype. Thus, looking into other genotypes relating to cortisol and their potential effects.

4. Examine the reported level of headache in those exposed to abusive supervision, moderated by the NRCAM rs2300043 genotype. By looking into a genotype not previously associated with pain conditions, new and interesting findings might be revealed.

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23 3 Methods

For this thesis, the collected data was based on a probability sample of the general Norwegian working population. A total of 5000 subjects, between 18 and 60 years of age, were randomly selected from The Norwegian Central Employee Register with help from Statistics Norway. The questionnaires were distributed by the Norwegian Postal Service. Of the total number of subjects (1608 individuals, 32%) who gave consent a saliva collection kit was also sent. As a result, a total of 1226 contributed with saliva samples. The data was collected at three time points over a period of 18-20 months. In the first paper the analysed data was extracted from time point one, resulting in a total of 1073 included subjects. In paper II, data from all three time points was used; the cross-sectional analyses were based on data from time point 1 which contained 1060 subjects, whereas the longitudinal data, which included all three time points, resulted in 745 subjects. For paper III and IV data from the first time point was analysed, providing 745 and 1185 subjects, respectively.

3.1 Instruments

3.1.1 Abusive supervision

Participants were asked to provide information regarding their experience of their employer’s leadership style by stating how often certain proposed situations occurred. This consisted of a 5-item version of the Tepper’s “Abusive Supervision Scale” [6,259,261] reflecting an abusive leadership style.

These items were “my closest leader…”; 1) critiques me in front of others, 2) tells me my thoughts and feelings are stupid, 3) says I am useless, 4) negative remarks about me in front of others and 5) ridicule me. The response categories ranged from “never”, “rarely”, “once in a while”, “quite often” and “very often or always”, which were coded from 0 – 4. For the purpose of this thesis whether or not one classified as experiencing abusive supervision was defined as a mean average of 0.8 or more. This cut off was decided based on the probability that this score might reflect experiencing that type of behaviour more than once.

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24 3.1.2 Subjective health complaints

The participants were asked to indicate level of experienced subjective health complaints during the last 12 months. The questions comprised of 8 items representing pain complaints and insomnia symptoms. The 5 first items reflecting pain were 1) headache, 2) neck pain, 3) upper back pain, 4) lower back pain and 5) abdominal pain. The 3 items reflecting insomnia symptoms were 1) problems with sleep onset, 2) problems with sleep maintenance and 3) early wakening. The response categories for all items ranged from “not bothered”, “a little bothered”, “considerably bothered” and “seriously bothered”, coded 0 – 3. For paper I and II all eight items were investigated, for paper III items 2-4 were assessed and named spinal pain, whereas item 1 was examined in paper IV.

3.2 Genotyping

Genomic DNA was extracted from the received saliva samples utilising OrageneRNA sample collection kit (DNA Genotech Inc. Kanata, Ontario, Canada). SNP genotyping, of FKBP5 rs9470080 (paper I & II), CRHR1 rs242941, rs242939 and rs1876282 (paper III) and NRCAM rs2300043 (paper IV), was carried out using predesigned TaqMan SNP genotyping assays for the respective SNPs. Approximately 10 ng genomic DNA was amplified in a 5 µl reaction mixture in a 384-well plate containing 1x TaqMan genotyping master mix (Applied Biosystems) and 1x assay mix, the latter containing the respective primers and probes. The probes were labelled with the reporter dye FAM or VIC to distinguish between the two alleles. In accordance with the procedure in our earlier studies [123,205], an ABI 79000HT sequence detection system was used. Negative controls were included in every run.

3.3 Haplotyping

Phase v.2.1.1 was used to define the CRHR1 haplotypes (paper III). The haplotyping based on the SNPs rs242941/rs242939/rs1876828 was then categorised into individuals with two copies of the CTC allelic combination, individuals with one copy of CTC and individuals with no copies. Approximately 10% of the samples were re-genotyped and the concordance rate was 100%.

The influence of psychosocial stressors, genetic variability, and gender on subjective health complaints: Abusive supervision, genetics, and health complaints