Associations of estrogen with modifiable and non-modifiable risk factors for dementia: A narrative review.

Female sex is associated with higher incidence and risk of dementia. Estrogen may represent one important mechanism contributing to the increase in incidence rates. In this review, we synthesize narratively the evidence for associations between estrogen-across the life course from menarche to menopause, estrogen-containing hormonal contraception and hormone replacement therapies, and pregnancy-with potential modifiable risk factors for dementia. These include education, hearing loss, traumatic brain injury, hypertension, alcohol use, obesity, smoking, depression, physical inactivity, diabetes, low-density lipoprotein (LDL) cholesterol, social isolation, air pollution, and untreated visual loss, as well as apolipoprotein E ε4. In addition, evidence is summarized for associations with sleep, diet, and stress. Evidence suggests that estrogen is associated with some of these modifiable risk factors for dementia, particularly LDL cholesterol, smoking, and depression. Research needs to further define these associations and understand whether interventions targeting estrogen levels at key life stages could offer intervention opportunities to reduce future risk of dementia in women. HIGHLIGHTS: Higher dementia risk in women may be associated with estrogen. Estrogen is associated with some of the modifiable risk factors for dementia. However, significant gaps exist in the literature for most risk factors.</p

Movement behaviours in Swedish 3-9-year-olds : prevalence, associations with health markers, and promotion of healthy behaviours

BackgroundMovement behaviours are comprised of physical activity, sedentary behaviour, and sleep and are important for children's physical health. The World Health Organization (WHO) 24-hour movement guidelines integrate these behaviours into three recommendations, recognising their interdependence within the 24- hour day. However, research on the prevalence of Swedish children adhering to these guidelines is limited. Understanding how movement behaviours relate to health markers in children, and how these behaviours can be supported early on, is important for promoting long-term health. In Sweden, nearly all children attend preschool, making it a key arena for health promotion, but limited research has explored how preschools can support healthy movement behaviours.AimThe overall aim of this thesis was to investigate movement behaviours in Swedish children aged 3-9 years, in terms of adherence to movement guidelines and associations with health markers. Another aim was to explore preschool teachers' and principals' perspectives on promoting healthy lifestyle behaviours in the preschool setting, as well as their preferences for digital support.MethodsPaper I: A cross-sectional study in 631 children 3-6 years of age from the Swedish arm of the International Study of Movement Behaviours in the Early Years (SUNRISE). Waist-worn accelerometers were used to measure physical activity and sedentary behaviour, whereas sleep and screen time were assessed through questionnaires. The prevalence of children adhering to the WHO 24- hour movement guidelines together and separately were investigated. Also, associations between reallocating time to either vigorous physical activity (VPA), moderate-to-vigorous physical activity (MVPA), or sedentary behaviour, at the expense of the remaining movement behaviours, with measures of physical fitness, anthropometry, and blood pressure were analysed using compositional data analysis.Paper II: A cross-sectional study in 411 children from Studies of Prospective Health Determinants in Infancy and Childhood (SPINACH) aged 9 years, investigating associations between sleep patterns, based on sleep duration and bedtime, and cardiometabolic risk factors. Sleep duration and bedtime were assessed with wrist-worn accelerometers and cardiometabolic risk factors included blood lipids, glucose, insulin, homeostatic model assessment of insulin resistance (HOMA-IR), a metabolic syndrome score, waist circumference, and blood pressure. Children were categorised into four groups depending on if they adhered to the sleep recommendation or not and if they had an early or late bedtime, based on the median bedtime of the population. Associations between sleep patterns and cardiometabolic risk factors were investigated using analysis of covariance.Paper III: A cross-sectional and longitudinal study in 411 children from SPINACH at 4 and 9 years of age. Movement behaviours were assessed with wrist-worn accelerometers and screen time with questionnaires. Cardiometabolic risk factors included the same measures as in Paper II. Longitudinal associations of increasing sedentary time (at the expense of the remaining movement behaviours) at 4 years of age with cardiometabolic risk factors at 9 years of age were examined, as well cross-sectional associations at 9 years of age, using compositional data analysis. Associations between adherence to the screen time recommendation and cardiometabolic risk factors were also examined.Paper IV: Semi-structured interviews were conducted in 15 preschool teachers and principals. Content analysis was employed to explore perceptions, needs, and prerequisites for promoting healthy lifestyle behaviours in the preschool setting, as well as preferences for potential digital support.ResultsPaper I: Most children met the WHO guidelines for physical activity (95%) and sleep (94%), while fewer met the screen time recommendation (44%). Only 40% adhered to all three guidelines. Increasing VPA or MVPA while evenly reducing the remaining behaviours were associated with a stronger handgrip (both PPaper II: Children adhering to the sleep recommendation combined with having early bedtimes had lower insulin resistance (0.30 versus 0.60, P=0.025), metabolic syndrome score (-0.15 versus 0.42, P=0.029), and insulin levels (6.80 versus 8.87 mIU/L, P=0.034), compared to the group who did not adhere and had late bedtimes. In the model where total sleep time was added as an additional covariate, a significant association was still observed with metabolic syndrome score (-0.19 versus 0.50, respectively, P=0.011).Paper III: Increased sedentary behaviour at the expense of the other movement behaviours at age 9 was associated with higher systolic (P=0.021) and diastolic (P=0.019) blood pressure. In the longitudinal analysis, reallocating time to sedentary behaviour while evenly reducing the remaining behaviours at age 4 was significantly associated with higher diastolic blood pressure (PPaper IV: Teachers and principals considered the preschool environment and themselves as essential in the promotion of healthy lifestyle behaviours in the preschool setting. They highlighted collaboration with parents, a need for clear guidance in policies and curriculum as well as face-to-face support, such as workshops and lectures, complemented by digital support offering a library of easily accessible activities. This was viewed as even more important for teachers less interested in physical activity.ConclusionsThe findings from this thesis highlight the importance of shaping healthy movement behaviours already in the preschool age. High-intensity physical activity, sufficient sleep with early bedtimes, and limited screen time were associated with better markers for physical fitness and cardiometabolic health. Preschools offer a unique setting for supporting movement behaviours and providing teachers with practical guidance and resources, while collaborating with parents may strengthen the promotion of healthy movement behaviours in children.List of scientific papersI. Nilsson E, Tigerstrand H, Delisle Nyström C, Löf M. International study of movement behaviours in the early years (SUNRISE): Results from SUNRISE Sweden. [Manuscript]II. Nilsson E, Delisle Nyström C, Migueles JH, Baurén H, Marín-Jiménez N, Henström M, Torres López L, Löf M. Sleep patterns are associated with cardiometabolic risk factors in nine-year-old Swedish children. Acta Paediatr. 2024;113(8):1891-9.https://doi.org/10.1111/apa.17254III. Nilsson E, Migueles JH, Henriksson P, Delisle Nyström C, Löf M. Higher sedentary behaviour and not following screen time guidelines were associated with unfavourable cardiometabolic outcomes in childhood. Acta Paediatr. 2025.https://doi.org/10.1111/apa.70276IV. Nilsson E, Tigerstrand H, Delisle Nyström C, Söderström E, Alexandrou C, Lof M. Promoting healthy lifestyle behaviours in the preschool setting: perceptions and needs of teachers and principals. BMC Public Health. 2025;25:3042.https://doi.org/10.1186/s12889-025-24379-4</p

DIORA family of proteins and functional methods of investigation

This thesis includes projects in basic research that can be divided into two major subparts, one being development of a spatiotemporal genome engineering tool, BLU-VIPR, and the other being the functional investigation into the proteins from the gene family FAM167.To fully understand a biological system more information than the parts involved is needed, how they interact and organize into functional parts is essential for the functional output. Tools that can perturb the genome in a spatiotemporal fashion can help answer questions about how cells behave in space and time. The spatiotemporal tool BLU-VIPR utilizes the light-sensitive protein EL222 to induce production of gRNA upon blue light stimulation, thus allowing the formation of a functional gRNA-Cas complex. Using light to control gRNA production, instead of Cas expression, allows for a flexible system where multiple different CRISPR applications can be performed without cumbersome engineering. Since EL222 induces transcription from the C120 promoter by RNA polymerase II, the gRNA was flanked by self-cleaving ribozymes to allow for the precise excision of the gRNA from the transcript. Using ribozymes also allowed for the production of multiple gRNAs from one transcript as well as production of both gRNA and proteins. The functionality of BLU-VIPR was demonstrated in various CRISPR applications, including: CRISPRa, multiplexed CRISPRa, base editing, and CRISPR knockouts. BLU-VIPR enabled simultaneous induction of gRNA and protein, with very low leakiness observed in dark conditions. Additionally, the functionality of the system was demonstrated in vivo by CRISPR mediated knockouts in mouse T lymphocytes. This positions BLU-VIPR as a tool that can be used for investigating biological systems in a spatiotemporal fashion with high resolution, allowing for novel questions to be answered about the role of proteins in different contexts.Despite large efforts into sequencing the human genome, and by proteomics and transcriptomics proving that many genes are expressed, many proteins still have little or no functional description. One such poorly described gene family is FAM167, which contain two protein coding genes, FAM167A and FAM167B, with the proteins being denoted DIORA1 and DIORA2, respectively. To identify interactors for the DIORA proteins, we used BioID-based proximity proteomics and identified MRCK proteins as interactors to both DIORA1 and DIORA2. This was further validated in co-immunoprecipitation studies. For the DIORA1 protein, this interaction was further characterized by expressing mutants of the DIORA1 and MRCK proteins in co-immunoprecipitation experiments, resulting in three individual domains that allowed for interaction between the proteins. To determine the cellular function of DIORA proteins, we used CRISPRi to stably knockdown DIORA expression in neuroblastoma cell lines (DIORA1) and a melanoma cell line (DIORA2). Both DIORA1 and DIORA2 knockdown led to significant changes in transcriptional programs in the respective cell lines, with genes involved in epithelial to mesenchymal transition upregulated after DIORA1 knockdown and downregulated after DIORA2 knockdown. This was verified on a protein level in DIORA1 knockdown cells. Functional investigation of cellular motility was performed, and both DIORA1 and DIORA2 knockdown cells displayed altered invasion. DIORA2 knockdown cells also displayed altered expression of genes involved in oxidative phosphorylation, and had overall lower oxygen consumption, reduced mitochondrial RNA and DNA levels, and altered ADP/ATP ratio. These studies demonstrate an interest in further investigation of the role the DIORA proteins have in cellular motility, especially in the context of cancer cells with a potential mechanistic link between the MRCK kinases and DIORA proteins.List of scientific papersI. Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo Diego Velasquez Pulgarin, Nathalie Pelo, Lin Ferrandiz, Tilen Tršelič, William A. Nyberg, Gary Bowlin, Alexander Espinosa Nucleic Acids Research, Volume 53, Issue 6, 11 April 2025, gkaf213. https://doi.org/10.1093/nar/gkaf213II. Autoimmunity-associated DIORA1 binds the MRCK family of serine/threonine kinases and controls cell motility Tilen Tršelič, Nathalie Pelo, Gregoire Martin de Fremont, Vaishnavi S. Iyer, Elina Richardsdotter Andersson, Vijole Ottosson, David Alexander Frei, Elisa Baas, William A. Nyberg, Guðný Ella Thorlacius, Lara Mentlein, Sanjaykumar V. Boddul, Ioana Sandu, Diego Velasquez Pulgarin, Ákos Végvári, Carmen Gerlach, Fredrik Wermeling, Maria Sunnerhagen, Björn Wallner, Alexander Espinosa, and Marie Wahren-Herlenius PNAS, October 3 2025, 122 (40) e2426917122. https://doi.org/10.1073/pnas.2426917122III. Identification of DIORA2 as a novel regulator of melanoma cell invasion and mitochondrial function Nathalie Pelo, Diego Velasquez Pulgarin, Lara Mentlein, William A. Nyberg, Vijole Ottosson, Ekaterina Zhuravleva, Gregoire Martin de Fremont, Tabassom Mohajershojai, Christina Gerstner, Liv Eidsmo, Elina Richardsdotter Andersson, Tilen Tršelič, Alexander Espinosa, Marie Wahren-Herlenius [Manuscript]</p

Metastatic colorectal cancer from an epidemiological and immunological perspective

Colorectal cancer (CRC) is the third most common type of cancer and around 8000 persons in Sweden get the diagnosis each year. One in five will have metastases at diagnosis, i.e., synchronous, and almost as many will develop metastases during follow up, i.e., metachronous. Median survival after diagnosis of metastatic CRC (mCRC) is 1-1.5 years. The most common metastatic locations are the liver, lungs, and peritoneum. When the extent of tumour spread in these organs is limited, surgical resection of the metastases may be feasible. Only a fraction of patients is treated with metastatic site surgery. The treatments are imperfect and puts the patient's body under major strain and risk. Despite this, prognosis of mCRC is largely dependent on if the primary tumour and metastases can be surgically removed or not.There is a need to increase our understanding of who benefits from the surgical treatments and who does not. Furthermore, previous research has shown that access to metastatic site surgery may be unequal. Lastly, a deeper understanding of the immune system's role in CRC could be crucial, as it influences disease progression and may be central to the discovery of novel treatment options. The overarching aim of this thesis is to improve the prognosis of patients with mCRC by deepening our understanding of the disease and its treatment through epidemiological and immunological approaches.Study I investigated overall survival of 131 patients with CRC and peritoneal metastases (PM) undergoing direct (without neoadjuvant chemotherapy) cytoreductive surgery and hyperthermic intraperitoneal chemotherapy with complete cytoreduction (CCO-1), between 2012 and 2019. The patients' PM were confirmed by histopathological examination, and potential extraperitoneal metastases treated before end of follow-up. The median overall survival was 40.3 months. In a multivariable model including potential prognostic factors, peritoneal carcinomatosis index > 16 was the only factor studied associated with decreased survival. The study showed that overall survival, from an institution with direct surgery as the standard, was similar to reported median overall survival from other recent cohorts where neoadjuvant chemotherapy was standard.Study II examined the association between hospital volume and metastatic site surgery for 9,968 adult patients diagnosed with synchronous mCRC in 2009- 2016, registered in the Swedish CRC database linkage CRCBaSe. Patients at high-volume hospitals were more often treated with metastatic site surgery. When adjusting for that high-volume hospitals were more often university hospitals, in addition to adjustment of identified confounders, only hospital level remained associated with chance of metastatic site surgery. In summary, being cared for at a hospital with proximity to metastatic site surgery, i.e., a university hospital increased the odds of receiving metastatic site surgery.In study III the association between sex and metastatic site surgery was investigated for 12,201 patients with synchronous mCRC diagnosed in 2007- 2016 and registered in CRCBaSe. Women received 23% less metastatic site surgery for mCRC despite adjusting for factors affecting patient selection, e.g., age, comorbidities, and primary tumour location. Furthermore, the effect of sex on receival of metastatic site surgery was modulated by hospital level and year of diagnosis. This indicates that non-biological reasons contribute to the sex- based differences observed in metastatic site surgery rates.In study IV the impact of patient income on cancer-specific survival after diagnosis of synchronous or metachronous mCRC was evaluated. In total, 33,498 patients diagnosed with CRC in 2007-2021 were identified using CRCBaSe, including follow-up throughout 2022. Patient income was associated with relative survival and the effect was time-varying with the largest impact in the first years after metastasis diagnosis. Furthermore, we found no support for a temporal trend affecting the impact of income on mCRC survival. Thus, patient income continues to be a factor associated with inferior survival after mCRC diagnosis.In study V we explored innate lymphoid cells (ILCs)' transcriptional patterns and differentiation capacities in tissue samples of primary colon cancer, PM from CRC, and macroscopically unaffected colon. Patients undergoing direct surgery for primary colon cancer and/or PM from CRC at Karolinska University Hospitals from 2020 were recruited. Single cell RNA sequencing of tissue samples from 11 patients revealed that primary colon cancer and CRC-PM are infiltrated by 14 transcriptionally different clusters of ILCs. In all three tissue types, two clusters of immature ILCs were present, annotated early NK cells and naïve ILCs. The naïve ILCs in tumours had more transcriptional similarities to ILCs type 1 (ILC1s) and tissue-resident natural killer cells, than ILCs from unaffected colon. We used a flow cytometry panel, based on the gene expression and surface protein signatures of the immature cells. Thereby, we could confirm the existence of the immature cell clusters in another patient population of eight patients contributing with seven primary tumours and seven colon tissue samples. The differentiation capacity of the immature cells was investigated using in vitro differentiation assays of an additional 14 patients contributing with paired primary tumour and unaffected colon samples. The experiments showed that naïve ILCs from primary colon tumours had an increased capacity to generate cells with a ILC1 or tissue-resident natural killer cell phenotype. Our findings contribute to the understanding of ILCs role in CRC and provides insights that could potentially be used in future therapies aiming to increase the innate immune response in CRC.List of scientific papersI. Direct surgery with Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy for patients with Colorectal Peritoneal Metastases. Ljunggren M, Nordenvall C, Palmer G. Eur J Surg Oncol. 2021 Nov;47(11):2865-2872. https://doi.org/10.1016/j.ejso.2021.05.046II. Hospital factors and metastatic surgery in colorectal cancer patients, a population-based cohort study. Ljunggren M, Weibull CE, Rosander E, Palmer G, Glimelius B, Martling A, Nordenvall C. BMC Cancer. 2022 Aug 19;22(1):907. https://doi.org/10.1186/s12885-022-10005-8III. Sex differences in metastatic surgery following diagnosis of synchronous metastatic colorectal cancer. Ljunggren M, Weibull CE, Palmer G, Osterlund E, Glimelius B, Martling A, Nordenvall C. Int J Cancer. 2023 Feb 1;152(3):363-373. https://doi.org/10.1002/ijc.34255IV. Low income has a negative effect on survival following diagnosis of metastatic colorectal cancer – a population-based cohort study Ljunggren M, Dietrich CE, Merk C, Palmer G, Martling A, Nordenvall C. Cancer Med. [Accepted]V. Tumor-infiltrating immature innate lymphoid cells in colorectal cancer and peritoneal metastases are dysregulated and biased towards tissue-resident NK cell differentiation. Marchalot A, Ljunggren M, Weigel W, Stamper C, Tibbitt C, Meninger I, Vinay Pandey R, Franklin M, Ahlberg M, Lindforss U, Jansson-Palmer G, Nordenvall C, Mjösberg J. [Manuscript]</p

Promoting change in secure settings : adaptation and evaluation of the adolescent community reinforcement

BackgroundAdolescents in secure care constitute one of the most vulnerable groups in society, commonly contending with intersecting adversities, substance use, and involvement in severe crime. Also, they are in a setting where personal freedom is restricted. Despite the complexity of their needs and the high risk of continuing a destructive pathway into adulthood, few interventions have been rigorously evaluated or tailored for delivery in closed secure settings in Sweden. The Adolescent Community Reinforcement Approach (A-CRA), originally developed for outpatient services, has demonstrated effectiveness in promoting abstinence and prosocial behaviour in community contexts, but had not previously been tested within secure care.AimsThe main objective of this thesis was to examine A-CRA in secure settings from multiple perspectives: adolescents' and therapists' experiences, feasibility in the secure environment, potential processes of change it engages, and what outcomes it yields in comparison with treatment-as-usual. More specifically, the effectiveness of A-CRA on social, emotional, and behavioural problems, substance use, and prosocial behaviour.MethodsFour interlinked studies were conducted:I. Therapists' experiences of conducting A-CRA in secure care were explored and specifically how treatments were perceived, how A-CRA was adapted to the secure setting, and potential facilitators and barriers in delivery. Interviews were analysed using qualitative content analysis with an inductive approach (N = 7).II. A potential novel change process for A-CRA in secure care was investigated through a mixed-methods study of adolescents' goal-talk. First, content analysis with a deductive approach was used to develop a coding manual using a Relational Frame Theory framework, and second, the association between the coded goal talk and change in A-CRA was investigated (N = 12).III. Feasibility, acceptability and preliminary treatment effects was investigated through a mixed methods randomised trial comparing A-CRA as an addition to treatment-as-usual (TAU) with TAU alone and investigating adolescents' experiences of undergoing A-CRA in secure care through interviews (N = 42).IV. Randomised controlled trial evaluating the effectiveness of A-CRA in secure care by comparing A-CRA + treatment-as-usual (TAU) with TAU alone (N = 84).ResultsI. Therapists reported that A-CRA was useful in secure care with only minor adaptations. The programme’s flexibility and scope for individual tailoring were viewed as enhancing engagement, and a strong therapeutic alliance was considered essential to success.II. Among adolescents receiving A-CRA, goal-related verbal behaviour (goal-talk) was feasible to identify, code, and analyse. Appetitive goal-talk showed strong associations with reduced substance use and with movement towards personally endorsed values.III. A-CRA proved feasible and acceptable in secure care, with a 77% completion rate. Adolescents generally described positive treatment experiences. Registry indicators suggested lower relapse to substance use in A-CRA + TAU (54%) than in TAU (92%), whereas rates of serious norm-violating behaviour were similar across conditions. Recruitment and randomisation procedures were workable, however, administering paper self-reports led to substantial missing data.IV. In the definitive trial, both A-CRA + TAU and TAU yielded improvements in emotional, social, and behavioural problems, alongside gains in prosocial behaviour. Differences between groups were not clearly distinguishable in the full sample. In the completer sub-sample, adolescents receiving A-CRA + TAU showed greater improvement in emotional symptoms and conduct problems than those receiving TAU alone.ConclusionsA-CRA can be delivered in secure care with minor adaptations and is perceived as acceptable and helpful to adolescents and therapists. Notably, the flexibility of A-CRA, allowing therapists to tailor sessions to adolescents' specific goals and needs, was seen by therapists and adolescents to increase engagement, which is often difficult to maintain in compulsory secure care. Measuring goal-talk is a promising approach to capturing proximal change processes in this context. Across studies, A-CRA achieved effects comparable to those typically observed in outpatient delivery, however, the evidence does not indicate a clear advantage over other CBT-based treatments currently provided in secure care.ImplicationsPolicy and service implications include that A-CRA is perceived as helpful by therapists and adolescents and that treatment can be delivered as intended with smaller adaptations. Furthermore, A-CRA decreases problem behaviours in secure settings. Lastly, that flexibility and focus on individual goals as part of treatment seems to foster engagement and motivation for change towards prosocial pathways for adolescents within compulsory, secure care, where personal freedom is already very restricted.List of scientific papersI. Mälarstig, I., Tyrberg, M. J., Lundgren, T., & Alfonsson, S. (2023). Experiences of conducting a substance use disorder treatment, A-CRA, in compulsory institutional care for youth-The challenge of promoting openness in a closed, temporary setting. Children and youth services review, 148, 106850. https://doi.org/10.1016/j.childyouth.2023.106850II. Mälarstig, I., Törneke, N., Lundgren, T., Alfonsson, S., & Tyrberg, M. J. (2025). "Finding your values is important. You only die once."- Analysing goal-talk in incarcerated adolescents struggling with substance use from a Relational Frame Theory perspective. Journal of Contextual Behavioral Science, 100947. https://doi.org/10.1016/j.jcbs.2025.100947III. Mälarstig, I., Spännargård, Å., Garke, M. Å., Tyrberg, M., Lundgren, T., & Alfonsson, S. (2025). Adolescent Community Reinforcement Approach in Secure care for Adolescents with Substance Use and Severe Norm-Violating behavior: A Randomised Feasibility Trial. [Submitted]IV. Mälarstig, I., Spännargård, Å., Garke, M. Å., Sahlin, H., Lundgren, T., & Alfonsson, S. (2025). The effectiveness of the Adolescent Community Reinforcement Approach in secure care for adolescents with substance use and severe norm-violating behavior: A randomised controlled trial. [Manuscript]</p

From drugs to climate : associations between external factors and profound hyponatremia

Background:Hyponatremia is the most common electrolyte disturbance and associated with substantial morbidity and mortality. Despite its clinical relevance, many aspects of hyponatremia's epidemiology, including pharmacological and environmental influences remain challenging for clinicians. This doctoral thesis addresses key knowledge gaps using population-based register data to investigate drug- induced hyponatremia, climate-related risk factors, and the value of an integrated cohort with sodium concentrations in focus.Aims:The overarching goal of this thesis is to improve understanding of hyponatremia by examining its pharmacological and environmental determinants. Specifically, the aims were to:1- Investigate the time-dependent risk of hyponatremia associated with proton pump inhibitors (PPIs).2- Establish and describe the Stockholm Sodium Cohort (SSC), a population- based database for sodium research.3- Assess the association between outdoor temperature and severe hyponatremia.4- Evaluate the risk of profound hyponatremia linked to selective serotonin reuptake inhibitors (SSRIs) and venlafaxine.Methods:All studies used population-based registers. Study 1 obtained data from the National Patient Register on hospitalized patients due to hyponatremia between 2006 and 2014. Studies 2-4 utilized the SSC, a cohort of 1.6 million residents of Stockholm County with at least one recorded serum sodium measurement between 2005 and 2018. Individual-level sodium test results were linked to national registers covering demographics, socioeconomic status, diagnoses, health care contacts, and dispensed medications. Study 1 used a case-control design with over 56,000 individuals to analyze the time-course of hyponatremia after omeprazole/esomeprazole initiation. Study 2 describes the creation and representativeness of the SSC. Study 3 was a retrospective cohort analysis assessing temperature-hyponatremia links using daily temperature data matched to residential area. Study 4 applied a within-individual design including 234,217 new users of SSRIs/venlafaxine to analyze the temporal risk of profound hyponatremia.Results:In Study 1, the adjusted odds ratio (aOR) for hospitalization due to hyponatremia was highest within the first week of PPI treatment (aOR 6.87), declining gradually over five weeks. Ongoing use was associated with a modest risk (aOR 1.10). Study 2 showed that the SSC covered 64% of the regional population and over 90% of patients with major chronic diseases, demonstrating its strong representativeness for clinical and pharmacoepidemiologic research. In Study 3, 51,143 episodes of severe hyponatremia (serum sodium Conclusion:This thesis demonstrates that hyponatremia is strongly associated with both pharmacologic exposures and environmental factors. The findings emphasize that PPIs and serotonergic antidepressants pose the greatest risk shortly after initiation, supporting early and targeted sodium monitoring in high-risk populations, particularly elderly women. Moreover, outdoor temperature is an important factor in severe hyponatremia risk, which is projected to rise substantially with future climate warming. The establishment of the SSC provides a robust and generalizable platform for studying sodium-related conditions on a population level. Together, these studies provide new, clinically actionable insights that may inform drug safety monitoring, geriatric care, and public health responses to climate change.List of scientific papersI. Issa I, Skov J, Falhammar H, Calissendorff J, Lindh JD, Mannheimer B. Time- dependent association between omeprazole and esomeprazole and hospitalization due to hyponatremia. Eur J Clin Pharmacol. 2023 Jan;79(1):71-77. https://doi.org/10.1007/s00228-022-03423-xII. Issa I, Skov J, Falhammar H, Franko MA, Lindh JD, Mannheimer B. Establishment and representativeness of the Stockholm Sodium Cohort: A laboratorial and pharmacoepidemiologic database covering 1.6 million individuals in the Stockholm County. Ann Epidemiol. 2024 Mar;91:1-7. https://doi.org/10.1016/j.annepidem.2024.01.005III. Issa I, Skov J, Falhammar H, Lindh JD, Mannheimer B. The Association of Outdoor Temperature with Severe Hyponatremia. J Am Soc Nephrol. 2025 Mar 1;36(3):435-440. doi: 10.1681/ASN.0000000519.https://doi.org/10.1681/ASN.0000000519IV. Issa I, Skov J, Falhammar H, Roos M, Lindh JD, Mannheimer B. The association of selective serotonin reuptake inhibitors and venlafaxine with profound hyponatremia. Eur J Endocrinol. 2025 Jun 30;193(1):179-187. https://doi.org/10.1093/ejendo/lvaf140</p

Visual motion processing in visual motion hypersensitivity patients ensuing from mild traumatic brain injury

Mild traumatic brain injury (mTBI) represents a pervasive public health issue, precipitating a range of persistent post-concussion symptoms (PCS) in up to 82% of individuals. Among the most challenging of these is visual motion hypersensitivity (VMH), where patients experience profound dizziness and disorientation in visually complex environments, a phenomenon often described as "supermarket syndrome." This condition creates a significant diagnostic conundrum, as patients report severe, life-altering symptoms despite the absence of overt structural damage on standard clinical neuroimaging, which has historically led to the misattribution of symptoms to psychosomatic origins. The prevailing theory to explain VMH has been one of "visual dependence," a model that posits the brain over relies on visual cues to compensate for a primary deficit in the vestibular system. While applicable in some cases, this framework is critically insufficient for the substantial and poorly understood cohort of patients who suffer from severe VMH while presenting with a functionally intact vestibular system. This discrepancy highlights a fundamental gap in our understanding and necessitates a new conceptual model of VMH as a primary disorder of visual processing. This thesis was therefore designed to systematically investigate the neurosensory underpinnings of VMH in this specific non-vestibular PCS population, employing a multi-level approach from low-level sensory processing to integrated sensorimotor control.The research involved recruiting and comparing patients with chronic PCS-VMH against age-matched healthy controls across three complementary studies. The methodology was sequential and multi-modal. Study I utilized precisely controlled visual, vestibular and visuo-vestibular stimulation while recording three-dimensional gaze-stabilizing eye movements, specifically ocular torsion and vertical vergence. This allowed for the dissociation of the optokinetic reflex (OKR) and the vestibulo-ocular reflex (VOR), providing a quantitative measure of how visual motion signals modulate subcortical sensorimotor pathways and influence the velocity storage mechanism (VSM). Study II employed complex optokinetic paradigms with varying stimulation patterns (coherent vs. incoherent motion) presented to different visual field locations (central vs. peripheral) to probe the functional consequences of VMH on oculomotor control under challenging conditions that mimic real-world visual stimuli. The objective oculomotor data were then correlated with subjective symptom severity, as measured by the Visual Vertigo Analog Scale (VVAS) and the Dizziness Handicap Inventory (DHI). Study III shifted focus to fundamental perceptual mechanisms, using a custom-developed psychophysical apparatus to measure Critical Flicker Frequency (CFF) thresholds via a staircase procedure at multiple retinal eccentricities. This approach was designed to isolate the integrity of low-level visual processing, using CFF variability as a proxy for internal perceptual noise. Data from all studies were analyzed using advanced statistical models, including generalized linear mixed models (GLMM), to account for repeated measures, covariates and variability between subjects.Study I demonstrated a clear sensory imbalance: patients exhibited pathologically enhanced optokinetic responses, with significantly increased oculomotor gain and slow-phase velocities during visual and visuo-vestibular stimulations, alongside an expedited onset of optokinetic nystagmus during visual stimulations. Crucially, their vestibular-only responses were normal, indicating that the VSM's intrinsic properties were preserved but were being hijacked by a disinhibited, pathologically potent visual feedback loop. Study II confirmed that this hypersensitivity translates into maladaptive motor control, with patients demonstrating exaggerated oculomotor responses with a trend towards complex peripheral motion and a significantly weaker correlation between their torsional and vergence eye movements, suggesting poorer motor coordination. Critically, the magnitude of these objective oculomotor biomarkers was strongly and significantly correlated with the patients' subjective symptom scores on both the VVAS and DHI. Study III traced the origin of this dysfunction to an elemental level, revealing that patients' CFF thresholds were pathologically influenced by perceptual noise. The modulating effect of CFF variability on elevating the final perceptual threshold was five times greater in the PCS group than in controls. Furthermore, a negative correlation was found between CFF variability and time since injury, suggesting slow, partial neurological recalibration or compensatory processes over time.In conclusion, this thesis provides further insight into the ways abnormal visual- motion processing influences visuo-vestibular control and explores plausible pathophysiological mechanisms. The collective findings demonstrate that this debilitating condition is not arising from compensation for vestibular loss but is driven by a primary dysfunction within the visual system itself. The evidence points to a foundational deficit in low-level sensory processing characterized by an abnormal sensitivity to internal neural noise. This sensory instability appears to necessitate a compensatory upregulation of gain across visual pathways, which in turn manifests as the exaggerated and disinhibited oculomotor responses that are directly linked to the patient's clinical symptoms. These results establish specific oculomotors and perceptual measures as potent objective biomarkers that can aid in the diagnosis, stratification, and monitoring of rehabilitation progress in patients with PCS. By re-framing visual motion hypersensitivity as a disorder of pathological gain control rooted in sensory noise, this work provides a new, evidence-based framework to guide the development of more targeted and effective therapeutic strategies for this challenging patient population.List of scientific papersI. Frattini, D., Rosen, N., & Wibble, T. (2024). A Proposed Mechanism for Visual Vertigo: Post-Concussion Patients Have Higher Gain From Visual Input Into Subcortical Gaze Stabilization. Investigative ophthalmology & visual science, 65(4), 26. https://doi.org/10.1167/iovs.65.4.26II. Wibble, T., Frattini, D., Benassi, M., Bolzani, R., & Pansell, T. (2023). Concussed patients with visually induced dizziness exhibit increased ocular torsion and vertical vergence during optokinetic gaze- stabilization. Scientific reports, 13(1), 3690. https://doi.org/10.1038/s41598-023-30668-yIII. Frattini, D., Benassi, M., Wibble, T., Nilsson, M., Bolzani, R. & Pansell, T. (2025). Temporal Visual Processing Deficits in Post-Concussion Syndrome. [Submitted]</p

Non-invasive predictors of prognosis in chronic liver disease

If the liver is exposed to continuous damage over time, accumulation of liver fibrosis occurs in all chronic liver diseases. The amount of fibrosis is traditionally evaluated via liver biopsy and is most often staged as fibrosis stage 0-4, where stage 4 is termed liver cirrhosis, and is the end-stage of chronic liver diseases, regardless of the etiology. This is considered an irreversible condition and is associated with an increased morbidity and mortality, including an increased risk of hepatocellular carcinoma (HCC), and biannual screening with ultrasound scans is recommended to identify smaller tumors eligible for curative treatment.Fibrosis stage by liver biopsy is the marker that best correlates with progression to cirrhosis, decompensated cirrhosis, and hepatocellular carcinoma. However, a liver biopsy is invasive and not an optimal method since it is resource-intensive, comes with a risk of complications, carries a risk of sampling error and interobserver variability, and is not suitable to screen large populations with, and the prevalence of the most common chronic liver disease, metabolic dysfunction- associated steatotic liver disease (MASLD), is 38% globally. Non-invasive tests have the potential to be used instead of a liver biopsy. In this thesis, we aimed to investigate different non-invasive tests and their performance in chronic liver disease, with a special emphasis on MASLD. We investigated non-invasive tests both as screening tools to identify cases with a higher risk of progression, and their performance to predict important outcomes such as progression to cirrhosis, decompensated cirrhosis, HCC, liver transplantation, and liver-related death.In study I, a case-control study, cases were patients with cirrhosis who had died from HCC between 2004 and 2020 in Stockholm County. They were matched to controls, patients with cirrhosis who had not died from HCC, based on age, sex, etiology of cirrhosis, year of cirrhosis diagnosis, hospital of cirrhosis-diagnosis, MELD score, and Child-Pugh Score. Using an adjusted logistic regression model, we investigated the exposure to HCC-surveillance between cases and controls. 72 cases and 72 controls were matched, and we found that HCC surveillance was associated with decreased HCC-related mortality, with an odds ratio (OR) of 0.37 (95%CI=0.16-0.88). The analysis showed that controls who did not die from HCC were more exposed to screening ultrasounds compared to cases, suggesting a protective effect.In study II, we collected liver stiffness measurements (LSM) by vibration- controlled transient elastography (VCTE) from 16 hospitals in Sweden. After linkage to nationwide healthcare registers, we created a cohort of 14,000 patients where disease etiology, comorbidities and outcomes were based on data from the registers. We used a Cox regression model to investigate if LSM by VCTE could be used to predict progression to an outcome associated with portal hypertension or hepatocellular carcinoma. A total of 402 (2.7%) patients developed an outcome associated with portal hypertension, and the risk of an outcome was 48-fold higher in patients with an LSM >25 kPa compared to patients with an LSM 25 kPa had a 34-fold increased rate of hepatocellular carcinoma compared to patients with an LSM In study III, a cross-sectional study with MASLD-patients, we compared the ability of the non-invasive tests FIB-4 (a score based on ALT, AST, platelet count, and age) and ADAPT (a score based on age, diabetes, the collagen formation biomarker PRO-C3, and platelet count) to detect patients with an LSM >8 kPa or >12 kPa. We showed that ADAPT had modestly better discriminative ability than FIB-4 in detecting an LSM >8 kPa in patients with MASLD, and a higher net benefit among across the probability threshold range of 15% - 40%, which means that ADAPT has a higher net benefit as a first-line screening test of fibrosis compared to FIB-4, if we are willing to do 100 LSM by VCTE to find 15-40 patients with an LSM >8 kPa.In study IV, we used longitudinal data from patients with MASLD to investigate how the non-invasive tests LSM by VCTE, FIB-4 and CAP changed over three years. We compared changes in mean values over time, both in the full population and between subgroups where the exposure to disease modifiers such as weight loss and alcohol consumption differed. We also described fluctuations between established categories of LSM and FIB-4, aimed to describe the risk of advanced fibrosis, over time. We could demonstrate that the mean change over time in LSM by VCTE (-0.13 kPa/year, 95%CI =- 0.39 to +0.13) and FIB-4 (0.04/year, 95%CI=0.01-0.06) was negligible, but there were considerable intraindividual fluctuations over time where 55 patients (29%) changed LSM category (12 kPa) between baseline and one year, and 51 patients (27%) changed LSM category between one and three years of follow-up.In conclusion, this thesis found that HCC surveillance in patients with cirrhosis is associated with a decreased HCC-related mortality, which suggests that patients with cirrhosis eligible for curative treatment should continue to be included in HCC-surveillance programs. We also showed that LSM by VCTE is a non-invasive biomarker suitable for risk stratification in patients with chronic liver disease. ADAPT performs modestly better than FIB-4 when used as a first-line test to screen for patients with an LSM >8 kPa, however the added value of ADAPT as a first-line test is unknown and warrants further evaluation. Mean LSM by VCTE and FIB-4 does not change much over three years on a group level, but disease monitoring with non-invasive tests remains challenging with intraindividual fluctuations over time. Further research is needed to find better ways of disease monitoring, where true progressors can be separated from non-progressors with fluctuations due to measurement errors of non-invasive tests.List of scientific papersI. Hegmar H, Bengtsson B, Ullman Nilsson S, Rowe I, Stål P, Hagström H. HCC-surveillance is associated with improved HCC-related mortality in patients with cirrhosis - a case-control study. Scand J Gastroenterol. 2025 Jun 17:1-11. doi: 10.1080/00365521.2025.2517208. Online ahead of print.https://doi.org/10.1080/00365521.2025.2517208II. Hegmar H, Wester A, Aleman S, Backman J, Degerman E, Ekvall H, Lund K, Lundgren Å, Nasr P, Shahnavaz A, Vessby J, Westin J, Önnerhag K, Hagström H. Liver stiffness predicts progression to liver-related events in patients with chronic liver disease - A cohort study of 14 414 patients. Liver Int. 2024 Jul;44(7):1689-1699. https://doi.org/10.1111/liv.15919III. Hegmar H, Wiggers T, Nasr P, Vessby J, Kechagias S, Nyhlin N, Marschall HU, Danielsson Borssen Å, Strandberg R, Karsdal M, Julie Leeming D, Ekstedt M, Hagström H. Performance of novel collagen turnover biomarkers to detect increased liver stiffness in MASLD. J Intern Med. 2024 Aug;296(2):177-186. https://doi.org/10.1111/joim.13813IV. Hegmar H, Strandberg R, Shang Y, Vessby J, Danielsson Borssén Å, Nyhlin N, Kechagias S, Stål P, SWEHEP study group, Ekstedt M, Hagström H. Dynamics of non-invasive tests in metabolic dysfunction-associated steatotic liver disease. [Manuscript]</p

Exploring the human oligodendrocyte lineage during development and adulthood through the lens of single-cell methods

Even though much is known about oligodendroglia based on animal models, human oligodendroglia biology knowledge is still lacking. In this thesis, we attempt to characterize human oligodendroglia molecular profiles using single-cell technologies. In papers I and II, we explored human oligodendroglia in the context of development. We identified oligodendrogenesis already occurring as early as 8 postconceptional weeks and revealed the molecular changes in OPC specification with single-cell RNA-seq. We also inspected the epigenetic landscape using single-cell ATAC-seq, revealing which transcription factors are active in OPC specification. Using spatial transcriptomics, we managed to map the OPC to ventral regions, suggesting that the first wave of OPCs also arises from ventral regions as in the mouse. Following the same principles, we investigated the second trimester of gestation in paper II. We characterized the molecular profile of the second-trimester oligodendroglia using single-nuclei RNA-seq and single-nuclei ATAC-seq. We investigated the molecular changes in OPC specification and compared it with the first trimester, showing some overlap, but several unique genes, indicating that in the second trimester OPC specification follows a different pathway. Next, we investigated the human oligodendroglia in adulthood in papers III and IV. Using single-nuclei RNA-seq we characterized the oligodendroglia from different regions-cortex, cerebellum, and spinal cord-and identified unique oligodendroglia populations. We also investigated the changes in oligodendroglia transcriptomic by age and sex. While we observed that aged individuals have reduced expression of genes related to myelination-indicating lower myelination capacity-, sex differences were more subtle. Finally, we investigated the epigenomic landscape of adult human oligodendroglia in paper IV. We used a combination of single-nuclei ATAC-seq and single-nuclei CUT&Tag to investigate the regulatory mechanism in human oligodendroglia. We identified a new candidate SOX10 enhancer in oligodendrocytes and observed the epigenetic priming of the HOX gene family in spinal cord oligodendrocytes. Together, we provided a deep molecular characterization of human oligodendroglia both in development and adulthood.List of scientific papersI. Developmental landscape of human forebrain at a single-cell level identifies early waves of oligodendrogenesis. David van Bruggen, Fabio Pohl, Christoffer Mattsson Langseth, Petra Kukanja, Hower Lee, Alejandro Mossi Albiach, Mukund Kabbe, Mandy Meijer, Sten Linnarsson, Markus M. Hilscher, Mats Nilsson, Erik Sundström, Gonçalo Castelo-Branco. Developmental Cell 2022. https://doi.org/10.1016/j.devcel.2022.04.016II. Specification of oligodendrocyte progenitor cells and their precursors during diferent stages of human neural development. Fabio Pohl, Luise A. Seeker, Mukund Kabbe, Eneritz Agirre, Neemat Mahmud, Carmen Abaurre, David Van Bruggen, Nadine Bestard- Cuche, Zahra Moslehi, Gioele La Manno, Steve Goldman, Anna Williams, Gonçalo Castelo-Branco. [Manuscript]III. Brain matters: unveiling the distinct contributions of region, age, and sex to glia diversity and CNS function. Luise A. Seeker, Nadine Bestard-Cuche, Sarah Jäkel, Nina-Lydia Kazakou, Sunniva M. K. Bøstrand, Laura J. Wagstaff, Justyna Cholewa-Waclaw, Alastair M. Kilpatrick, David Van Bruggen, Mukund Kabbe, Fabio Baldivia Pohl, Zahra Moslehi, Neil C. Henderson, Catalina A. Vallejos, Gioele La Manno, Goncalo Castelo-Branco, Anna Williams. Acta Neuropathologica Communications 2023. https://doi.org/10.1186/s40478-023-01568-zIV. Single-nuclei histone modification profiling of the adult human central nervous system unveils epigenetic memory of developmental programs. Mukund Kabbe, Eneritz Agirre, Karl E. Carlström, Fabio Baldivia Pohl, Nicolas Ruffin, David van Bruggen, Mandy Meijer, Luise A. Seeker, Nadine Bestard-Cuche, Alex R. Lederer, Jilin Zhang, Virpi Ahola, Steven A. Goldman, Marek Bartosovic, Maja Jagodic, Anna Williams, Gonçalo Castelo-Branco. BioRxiv 2024. https://doi.org/10.1101/2024.04.15.589512 [Manuscript Preprint]</p

Molecular mechanisms of WNT signaling and receptor-transducer coupling

Wingless and int-1 (WNT) signaling is a group of evolutionarily conserved signal transduction pathways that are vital for fundamental cellular processes. The signals are transduced via receptors at the cell membrane, Frizzleds (FZDs), which belong to class F of GPCRs. In this thesis, the focus is on WNT signaling, the binding of WNT-3A to FZDs, and their coupling to their primary intracellular transducer, Dishevelled (DVL), constitutively, i.e. in the absence of ligands.WNT-3A has been extensively studied for its role in beta-catenin dependent signaling. In this work, eGFP-WNT-3A was used in combination with HiBiT-tagged FZD1-10 to determine the binding kinetics in a live cell nanoBRET assay. The kinetic parameters were determined from both kinetic and saturation binding curves. Among all FZDs, except for FZD3 and FZD9 for which binding could not be detected, the determined affinities range from single to double digit nanomolar concentrations. Additionally, the utility of this assay for other experimental modes was shown, including competition binding with commercial preparations of WNT- 3A, WNT-5A, WNT-5B, WNT-10B, WNT-11 and WNT-16B.In addition to ligand induced signaling, constitutive activity, which is defined as coupling of the receptor to its transducer in the absence of stimulation by a ligand can have important functional consequences. To this end, G protein tri-cistronic activity sensors (G-CASE) were designed and in addition to measuring ligand induced G protein coupling, were validated specifically for determining constitutive coupling in GPCRs.FZDs couple constitutively to DVL. To this end, a FZD6-DVL3 complex was formed and stabilized for cryogenic electron microscopy (cryoEM) experiments, which enabled the determination of the structure at an overall resolution of 3.6 Å. The structure consists of FZD6 and residues 409-495 of DVL3 (DEP domain). The receptor exhibits a typical GPCR-like organization with seven transmembrane helices. The interaction of FZD6 with DEP is characterized by two sites: 1) a lipophilic cavity formed by residues L3935.69, V3965.72, V3995.75 and I4005.76 on FZD6 and residues L434 and I436 on DVL3 and 2) a polar site formed by residues R22612.49, K4126.28, R4166.32 and K4988.49 on FZD6 and K435 on DVL3, surrounding an unassigned density. Single, alanine point mutations in R22612.49, R4166.32, K4988.49, as well as the molecular switch W4937.55 in the vicinity, reduce the relative affinity of FZD6 to DVL3 (amino acids 243-496). The moiety in this unassigned density may play a central role in coordinating the polar residues surrounding this site.Overall, the work presented in this thesis adds to our knowledge on WNT signaling and receptor-transducer coupling methodologically with new approaches to study WNT-FZD kinetics and constitutive GPCR coupling, as well as mechanistically with novel information on the molecular interaction of FZD6-DVL3, with the first structure of a FZD-DVL complex reconstituted in a lipid environment.List of scientific papersI. Quantitative Profiling of WNT-3A Binding to All Human Frizzled Paralogues in HEK293 Cells by NanoBIT/BRET Assessments. Paweł Kozielewicz, Rawan Shekhani, Stefanie Moser, Carl-Fredrik Bowin, Janine Wesslowski, Gary Davidson, Gunnar Schulte. ACS Pharmacol Transl Sci. 2021 May 11;4(3):1235-1245.https://doi.org/10.1021/acsptsci.1c00084II. Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors. Hannes Schihada, Rawan Shekhani, Gunnar Schulte. Sci Signal. 2021 Sep 7;14(699):eabf1653. https://doi.org/10.1126/scisignal.abf1653III. Structural insight into the functional interaction between Frizzled 6 and the transducer protein Dishevelled. Rawan Shekhani, Magdalena M Scharf, Jan Hendrik Voss, Jia Yu Ho, Fanny Peysson, Shi Min Tan, Allan H. Pang, Jitender Kumar, Vítězslav Bryja, Konstantinos Tripsianes, Sébastien Granier, Rémy Sounier, Yong Zi Tan, Gunnar Schulte. [Manuscript]</p