The Role of Circadian Rhythmicity and CLOCK Genes in Psychiatry.

Circadian rhythms are biological oscillations, that perpetuate themselves even in the absence of "zeitgebers" (external time cues), with a period of approximately 24 hours. The master pacemaker is the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN is entrained by environmental factors, particularly light, to the 24-hour light-dark cycle by the Earth's rotation. Peripheral circadian oscillators, located in multiple cell types and tissues, are controlled by signals arising from the SCN and from the environment, particularly food intake, hormonal signals and body-temperature fluctuations. Circadian rhythmicity is observable in almost every cell of living organisms including humans and, for example in cell cultures, these rhythms persist even without the SCN 1 2.

Human Derived Dermal Fibroblasts as in Vitro Research Tool to Study Circadian Rhythmicity in Psychiatric Disorders.

A number of psychiatric disorders are defined by persistent or recurrent sleep-wake disturbances alongside disruptions in circadian rhythm and altered clock gene expression. Circadian rhythms are present not only in the hypothalamic suprachiasmatic nucleus but also in peripheral tissues. In this respect, cultures of human derived dermal fibroblasts may serve as a promising new tool to investigate cellular and molecular mechanisms underlying the pathophysiology of mental illness. In this article, we discuss the advantages of fibroblast cultures to study psychiatric disease. More specifically, we provide an update on recent advances in modeling circadian rhythm disorders using human fibroblasts.

Circadian Clocks in the Regulation of Neurotransmitter Systems.

To anticipate and adapt to daily recurring events defined by the earth's rotation such as light-dark and temperature cycles, most species have developed internal, so-called circadian clocks. These clocks are involved in the regulation of behaviors such as the sleep-wake cycle and the secretion of hormones and neurotransmitters. Disruptions of the circadian system affect cognitive functions and are associated with various diseases that are characterized by altered neurotransmitter signaling. In this review, we summarize the current knowledge about the interplay of the circadian clock and the regulation of psychiatric health and disease.

Synchronization of Fibroblasts Ex Vivo in Psychopharmacology.

The central oscillator for the inner clock is the suprachiasmatic nuclei of the hypothalamus. Furthermore, many peripheral oscillators are present in tissues such as skin. Human derived fibroblasts provide an advantageous model to study circadian rhythmicity as well as the influence of pharmacological drugs on circadian gene expression. Importantly, the synchronization of the circadian system of fibroblasts can be done by different methods. The review presents an overview of the current knowledge of different synchronization methods mostly used in mice or rat fibroblasts. Furthermore, the review sums up and discusses the role of norepinephrine as a possible synchronizer agent.

The impact of COVID-19 on psychiatric and mental health services in Europe: suffering experienced by professionals.

BACKGROUND: The COVID-19 pandemic has not only impacted intensive care units, but all healthcare services generally. This PsyGipo2C project specifically investigates how psychiatry and mental health professionals have been affected by the reorganizations and constraints imposed, which have reshaped their often already difficult working conditions. METHODS: Our research combined quantitative and qualitative methods, surveying and interviewing health professionals of all occupations working in psychiatric and mental health services. A questionnaire was completed by 1241 professionals from 10 European countries, and 13 group interviews were conducted across 5 countries. In addition to this, 31 individual interviews were conducted in Belgium and France. RESULTS: Among the questionnaire respondents, 70.2% felt that their workload had increased, particularly due to their tasks being diversified and due to increased complexity in the provision of care. 48.9% felt that finding a work-life balance had become more difficult, and 59.5% felt their health had been affected by the crisis. The impact of the health crisis nevertheless varied across professions: our data provides insight into how the health measures have had a differential impact on professional tasks and roles across the various categories of occupations, obliging professionals to make various adaptations. The distress incurred has been linked not only to these new constraints in their work, but also to the combination of these with other pressures in their personal lives, which has consequently compromised their well-being and their ability to cope with multiple demands. DISCUSSION: The COVID-19 health crisis has had varying impacts depending on the profession and access to remote work, sometimes leading to conflicts within the teams. The suffering expressed by the professionals was tied to their values and patterns of investment in work. Our research also highlights how these professionals made little use of the psychological supports offered, probably due to a reluctance to acknowledge that their mental health was affected.

Lessons Learned from the Covid-19 Crisis: Recommendations for Action to Optimize Occupational Health in Mental Health Professionals in Europe.

Objective: The Covid-19 health crisis has disrupted the organization and functioning of European mental health and psychiatric services, impacting the working conditions - already difficult before the epidemic - of professionals working therein. The Psy-GIPO2C project investigated the impact of the pandemic on these professionals. The Psy-GIPOC2C project has been co-funded by the Agence Nationale de la Recherche française (ANR, French National Research Agency) and the Centre-Val de Loire region (France). Method: This research took the form of a collaborative mixed methods study. The data collected through qualitative and quantitative research were analyzed during a working session held by the research consortium, which resulted in the formulation of recommendations for action to optimize the occupational health of European mental health professionals. Results: This research made it possible to identify and explain the extent to which, and the ways in which, the reorganization of European mental health services has impacted the mental health of the professionals working therein, and, in particular, to highlight the fact that the unprecedented use of digital devices has generated stress, and even tension, within these services. Conclusions: It is important to promote well-being at work among European mental health professionals, by involving them in the development of ad hoc measures (in addition to the existing psychological support measures available) to enable them to recharge their batteries. It is also important to support current and future professionals in their appropriation of digital tools for professional use.

Clock Genes Profiles as Diagnostic Tool in (Childhood) ADHD-A Pilot Study.

Attention deficit hyperactivity disorder (ADHD) is a very common disorder in children and adults. A connection with sleep disorders, and above all, disorders of the circadian rhythm are the subject of research and debate. The circadian system can be represented on different levels. There have been a variety of studies examining 24-h rhythms at the behavioral and endocrine level. At the molecular level, these rhythms are based on a series of feedback loops of core clock genes and proteins. In this paper, we compared the circadian rhythms at the behavioral, endocrine, and molecular levels between children with ADHD and age- and BMI-matched controls, complementing the previous data in adults. In a minimally invasive setting, sleep was assessed via a questionnaire, actigraphy was used to determine the motor activity and light exposure, saliva samples were taken to assess the 24-h profiles of cortisol and melatonin, and buccal mucosa swaps were taken to assess the expression of the clock genes BMAL1 and PER2. We found significant group differences in sleep onset and sleep duration, cortisol secretion profiles, and in the expression of both clock genes. Our data suggest that the analysis of circadian molecular rhythms may provide a new approach for diagnosing ADHD in children and adults.

COVID-19 and Physical Distancing Measures: Experience of Psychiatric Professionals in Europe.

A The COVID-19 pandemic has had a considerable impact on the organization of psychiatric care. The present study examines how care professionals experienced this period and faced these new constraints weighing on their professional practices. Based on a qualitative research methodology, 13 group interviews with healthcare professionals working in psychiatric wards were conducted in five countries in western Europe. To complement this, 31 individual interviews were carried out in Belgium and France. Public health measures hindered certain therapeutic activities, jeopardized communication, and obliged healthcare professionals to modify and adapt their practices. Confronted with a transformation of their usual roles, healthcare professionals feared a deterioration in the quality of care. Impossible to continue in-person care practices, they resorted to online videoconferencing which went against their idea of care in which the encounter holds an essential place. The lockdown contradicted efforts to co-build care pathways toward readaptation, social reintegration, and recovery, thus reviving the perception of psychiatric hospitalization based on isolation.

Molecular Link between Circadian Rhythmicity and Mood Disorders.

BACKGROUND: The internal clock is driven by circadian genes [e.g., Clock, Bmal1, Per1-3, Cry1-2], hormones [e.g., melatonin, cortisol], as well as zeitgeber ['synchronisers']. Chronic disturbances in the circadian rhythm in Objectives: The aim of this review is to summarise the current knowledge and literature regarding circadian rhythms in the context of mood disorders, focussing on the role of circadian genes, hormones, and neurotransmitters. METHODS: The review presents the current knowledge and literature regarding circadian rhythms in mood disorders using the Pubmed database. Articles with a focus on circadian rhythms and mood disorders [n=123], particularly from 1973 to 2020, were included. RESULTS: The article suggests a molecular link between disruptions in the circadian rhythm and mood disorders. Circadian disturbances, caused by the dysregulation of circadian genes, hormones, and neurotransmitters, often result in a clinical picture resembling depression. CONCLUSION: The article suggests a molecular link between disruptions in the circadian rhythm and mood disorders. Circadian disturbances, caused by the dysregulation of circadian genes, hormones, and neurotransmitters, often result in a clinical picture resembling depression.

Opportunities and Challenges for Professionals in Psychiatry and Mental Health Care Using Digital Technologies During the COVID-19 Pandemic: Systematic Review.

BACKGROUND: The COVID-19 pandemic has required psychiatric and mental health professionals to change their practices to reduce the risk of transmission of SARS-CoV-2, in particular by favoring remote monitoring and assessment via digital technologies. OBJECTIVE: As part of a research project that was cofunded by the French National Research Agency (ARN) and the Centre-Val de Loire Region, the aim of this systematic literature review was to investigate how such uses of digital technologies have been developing. METHODS: This systematic review was conducted following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. The search was carried out in the MEDLINE (ie, PubMed) and Cairn databases, as well as in a platform specializing in mental health, Ascodocpsy. The search yielded 558 results for the year 2020. After applying inclusion and exclusion criteria, first on titles and abstracts and then on full texts, 61 articles were included. RESULTS: The analysis of the literature revealed a heterogeneous integration of digital technologies, not only depending on countries, contexts, and local regulations, but also depending on the modalities of care. Notwithstanding these variations, the use of videoconferencing has developed significantly, affecting working conditions and therapeutic relationships. For many psychiatric and mental health professionals, the pandemic has been an opportunity to build up their experience of remote care and, thus, better identify the possibilities and limits of these digital technologies. CONCLUSIONS: New uses of such technologies essentially consist of a transition from the classic consultation model toward teleconsultation and make less use of the specific potential of artificial intelligence. As professionals were not prepared for these uses, they were confronted with practical difficulties and ethical questions, such as the place of digital technology in care, confidentiality and protection of personal data, and equity in access to care. The COVID-19 health crisis questions how the organization of health care integrates the possibilities offered by digital technology, in particular to promote the autonomy and empowerment of mental health service users.

International professional practices in mental health, organization of psychiatric care, and COVID-19: A survey protocol.

OBJECTIVE: Our project aims to provide: an overview of the impact of the COVID-19 pandemic on the field of mental health professionals in 23 countries;a model of recommendations for good practice and proposals for methods and digital tools to improve the well-being at work of mental health professionals and the quality of services offered during crisis and post-crisis periods;an in-depth ethics review of the assessment of the use of numerical tools for psychiatry professionals and patient support, including teleconsulting. METHODS: This is a large international survey conducted among 2,000 mental health professionals in 23 countries over a 12-month period. This survey will be based on 30 individual interviews and 20 focus group sessions, and a digital questionnaire will be sent online to 2,000 professionals based on the criteria of gender, age, professional experience, psychiatric specialty, context of work in psychiatry, and geographical location. Regarding the development of telepsychiatry during the COVID-19 pandemic, a pilot study on the use of digital tools will be carried out on 100 clients of psychiatry professionals in France and Belgium. DISCUSSION-CONCLUSION: This study will contribute to the co-construction of an international organization and monitoring system that takes into account psychiatric health professionals as major resources to fight against the COVID-19 pandemic and to develop efficient processes for preparing and anticipating crises by reducing psychosocial risks as much as possible. This project also aims to design tools for remote medicine and to develop the use of numerical tools for monitoring and supporting professionals and helping professionals to build the conditions for satisfactory operational work during crises and post-crisis situations, using adapted organizational methods. Our ongoing research should support professionals in the search for existing concrete solutions to cope with emergency work situations while maintaining an optimal quality of life.

The Early Impacts of the COVID-19 Pandemic on Mental Health Facilities and Psychiatric Professionals.

(1) Background: While in many countries, the psychiatric and mental health sectors had been in crisis for years, the onset of a novel coronavirus pandemic impacted their structures, organizations, and professionals worldwide. (2) Methods: To document the early impacts of the COVID-19 health crisis on psychiatry and mental health sectors, a systematic review of the international literature published in 2020 was conducted in PubMed (MEDLINE), Cairn.info, and SantéPsy (Ascodocpsy) databases. (3) Results: After applying inclusion and exclusion criteria, 72 articles from scientific journals were selected, including papers documenting the early impact of the COVID-19 pandemic on the organization of psychiatric care delivery, work processes in psychiatry and mental health units, and personal experiences of mental health professionals. This review identified the contributions aimed at preventing the onset of mental disorders in the early stages of the health crisis. It lists the organizational changes that have been implemented in the first place to ensure continuity of psychiatric care while reducing the risk of SARS-CoV-2 transmission. It questions the evolution of the rights and duties of mental health professionals in the first months of the pandemic. (4) Discussion and conclusions: Although this literature review exclusively documented the early impacts of the COVID-19 health crisis, it is of significant interest, as it pictures the unprecedent situation in which psychiatry and mental health care professionals found themselves in the first stages of the pandemic. This work is a preliminary step of a study to be conducted with mental health professionals on an international scale-the Psy-GIPO2C project-based on more than 15 group interviews, 30 individual interviews, and 2000 questionnaires. The final aim of this study is to formulate concrete recommendations for decision-makers to improve work in psychiatry and mental health.

Dopamine adjusts the circadian gene expression of Per2 and Per3 in human dermal fibroblasts from ADHD patients.

A link between dopamine levels, circadian gene expression, and attention deficit hyperactivity disorder (ADHD) has already been demonstrated. The aim of this study was to investigate the extent of these relationships by measuring circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after dopamine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different dopamine concentrations in human dermal fibroblast (HDF) cultures, the rhythmicity of circadian gene expression (Clock, Bmal1, Per1-3, Cry1) was analyzed via qRT-PCR. We found no statistical significant effect in the actigraphy of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, wake after sleep onset, and total number of wake bouts. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. Dopamine has no effect on Per3 expression in healthy controls, but produces a significant difference in the ADHD group at ZT24 and ZT28. In the ADHD group, incubation with dopamine, either 1 µM or 10 µM, resulted in an adjustment of Per3 expression to control levels. A similar effect also was found in the expression of Per2. Statistical significant differences in the expression of Per2 (ZT4) in the control group compared to the ADHD group were found, following incubation with dopamine. The present study illustrates that dopamine impacts on circadian function. The results lead to the suggestion that dopamine may improve the sleep quality as well as ADHD symptoms by adjustment of the circadian gene expression, especially for Per2 and Per3.

Norepinephrine influences the circadian clock in human dermal fibroblasts from study participants with a diagnosis of attention-deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) is characterized by changes to the circadian process. Many medications used to treat the condition, influence norepinephrine levels. Several studies have, in addition, reported that norepinephrine itself has an effect on circadian function. The aim of this study was to investigate the circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after norepinephrine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with an ADHD diagnosis. Circadian preference was evaluated with German Morningness-Eveningness Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different norepinephrine concentrations in HDF cultures, the rhythmicity of circadian gene expression was analyzed via qRT-PCR. The exposure of 1 µM norepinephrine to confluent cultures of human dermal fibroblasts from participants with a diagnosis of ADHD, was shown to dampen Per1 rhythmicity. The expression of Bmal1, Per1 and Per3 in control subjects was also influenced by incubation with 1 µM norepinephrine. Cultures from the ADHD group revealed no statistically significant overall differences in circadian gene expression, between cultures with and without norepinephrine incubation. Per3 expression showed a significant ZT × group interaction via mixed ANOVA. Per3 expression at ZT4 was significant higher in the group of control samples incubated with 1 µM norepinephrine, compared to the control group without norepinephrine. This effect was also shown in the control samples incubated with 1 µM norepinephrine and cultures from subjects with ADHD without norepinephrine incubation. Per3 expression differed between the healthy control group and the ADHD group without norepinephrine incubation at ZT28. The results of the present study illustrate that norepinephrine impacts on circadian function. In both groups, control group and cultures taken from subjects with ADHD, the expression of the periodic genes (Per1-3) was significantly influenced by incubation with norepinephrine.

Atomoxetine and circadian gene expression in human dermal fibroblasts from study participants with a diagnosis of attention-deficit hyperactivity disorder.

Atomoxetine (ATO) is a second line medication for attention-deficit hyperactivity disorder (ADHD). We proposed that part of the therapeutic profile of ATO may be through circadian rhythm modulation. Thus, the aim of this study was to investigate the circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after ATO exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with a diagnosis of ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different ATO concentrations in HDF cultures, the rhythmicity of circadian gene expression was analyzed via qRT-PCR. No statistical significant effect of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, sleep WASO and total number of wake bouts was observed. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. ATO induced the rhythmicity of Clock in the ADHD group. This effect, however, was not observed in HDF cultures of healthy controls. Bmal1 and Per2 expression showed a significant ZT × group interaction via mixed ANOVA. Strong positive correlations for chronotype and circadian genes were observed for Bmal1, Cry1 and Per3 among the study participants. Statistical significant different Clock, Bmal1 and Per3 expressions were observed in HDFs exposed to ATO collected from ADHD participants exhibiting neutral and moderate evening preference, as well as healthy participants with morning preferences. The results of the present study illustrate that ATO impacts on circadian function, particularly on Clock, Bmal1 and Per2 gene expression.

Remdesivir shifts circadian rhythmicity to eveningness; similar to the most prevalent chronotype in ADHD.

Circadian clocks control immunity and virus replication, as well as pharmacokinetics and efficacy therapeutics. The aim of this study was to investigate the extent of these relationships by measuring circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after remdesivir exposure. In the current study, we analysed circadian gene expression in a cohort of participants without a neuropsychiatric diagnosis. After ex vivo exposure to remdesivir to human dermal fibroblast (HDF) cultures and dexamethasone synchronization, the rhythmicity of circadian gene expression (Clock, Bmal1, Per1-3, Cry1) was analysed via qRT-PCR. In this study, D-MEQ scores indicated that participants without a neuropsychiatric diagnosis had no evening preference. Remdesivir leads to a slight phase-shift in Clock, Per1 and Per2. Significant different expressions of Bmal1 and Per3 were detected after remdesivir exposure: Bmal1 at ZT8 (t(22) = 3.26, p = 0.004), ZT24 (t(22) = - 2.66, p = 0.015), ZT28 (t(20) = - 2.14, p = 0.045) and Per3 at ZT8 (t(22) = - 4.27, p < 0.001) and ZT12 (t(22) = - 2.61, p = 0.016). A significant difference between chronotype and circadian gene expression for Bmal1, Cry1 and Per3 was observed. The present study shows that remdesivir has an impact on circadian function. It is well known that the circadian rhythm effects sleep and, moreover, sleep quality. The results suggest that remdesivir medication may alter sleep quality in participants without a neuropsychiatric diagnosis and shifts chronotype to eveningness; similar as prevalent in ADHD.

Evolutionary conservations, changes of circadian rhythms and their effect on circadian disturbances and therapeutic approaches.

The circadian rhythm is essential for the interaction of all living organisms with their environments. Several processes, such as thermoregulation, metabolism, cognition and memory, are regulated by the internal clock. Disturbances in the circadian rhythm have been shown to lead to the development of neuropsychiatric disorders, including attention-deficit hyperactivity disorder (ADHD). Interestingly, the mechanism of the circadian rhythms has been conserved in many different species, and misalignment between circadian rhythms and the environment results in evolutionary regression and lifespan reduction. This review summarises the conserved mechanism of the internal clock and its major interspecies differences. In addition, it focuses on effects the circadian rhythm disturbances, especially in cases of ADHD, and describes the possibility of recombinant proteins generated by eukaryotic expression systems as therapeutic agents as well as CRISPR/Cas9 technology as a potential tool for research and therapy. The aim is to give an overview about the evolutionary conserved mechanism as well as the changes of the circadian clock. Furthermore, current knowledge about circadian rhythm disturbances and therapeutic approaches is discussed.

The Collateral Damage of the COVID-19 Outbreak on Mental Health and Psychiatry.

The potential consequences of the COVID-19 outbreak are multifarious and remain largely unknown. Deaths as a direct result of the condition are already in the millions, and the number of indirect deaths is likely to be even higher. Pre-existing historical inequalities are compounded by the virus, driving increased rates of infection and deaths amongst people who use drugs and alcohol, those belonging to racial-ethnic minority groups, poorer communities, LBGTQ+ populations, healthcare workers, and other members of the care economy; all of whom are already at increased risk of adverse mental health effects. In this paper we suggest that a central role of mental health practitioners is advocacy: both for people who use psychiatric services and for those who, due to the effects of the pandemic, are at an increased risk of needing to do so.

EEG Data Quality: Determinants and Impact in a Multicenter Study of Children, Adolescents, and Adults with Attention-Deficit/Hyperactivity Disorder (ADHD).

Electroencephalography (EEG) represents a widely established method for assessing altered and typically developing brain function. However, systematic studies on EEG data quality, its correlates, and consequences are scarce. To address this research gap, the current study focused on the percentage of artifact-free segments after standard EEG pre-processing as a data quality index. We analyzed participant-related and methodological influences, and validity by replicating landmark EEG effects. Further, effects of data quality on spectral power analyses beyond participant-related characteristics were explored. EEG data from a multicenter ADHD-cohort (age range 6 to 45 years), and a non-ADHD school-age control group were analyzed (ntotal = 305). Resting-state data during eyes open, and eyes closed conditions, and task-related data during a cued Continuous Performance Task (CPT) were collected. After pre-processing, general linear models, and stepwise regression models were fitted to the data. We found that EEG data quality was strongly related to demographic characteristics, but not to methodological factors. We were able to replicate maturational, task, and ADHD effects reported in the EEG literature, establishing a link with EEG-landmark effects. Furthermore, we showed that poor data quality significantly increases spectral power beyond effects of maturation and symptom severity. Taken together, the current results indicate that with a careful design and systematic quality control, informative large-scale multicenter trials characterizing neurophysiological mechanisms in neurodevelopmental disorders across the lifespan are feasible. Nevertheless, results are restricted to the limitations reported. Future work will clarify predictive value.

Feigning ADHD and stimulant misuse among Dutch university students.

The increasing number of university students seeking diagnosis of attention-deficit/hyperactivity disorder (ADHD), and findings of an increased stimulant misuse among university students, has raised concerns regarding the credibility of the symptoms of those students. However, most of our current knowledge refers to university students in North America and less is known about this issue on European campuses. The present survey aimed to collect opinions on feigning ADHD and to estimate the prevalence of stimulant misuse among 1071 university students in the Netherlands. The majority of students expressed liberal attitudes towards feigning ADHD. Also, a substantial number of respondents considered feigning ADHD themselves or know someone who feigns ADHD. Furthermore, 68% of students assumed benefits of taking stimulants without prescription and 16% have indeed already taken stimulants without prescription. Feigning ADHD and misuse of prescription medication are prevalent issues among Dutch students. The results underline the need for a careful diagnostic evaluation of individuals for ADHD. Furthermore, efforts are required in order to prevent stimulant drug trafficking and misuse among university students.