Advancing assessments of endocrine disruptors using adverse outcome pathways and novel methodologies

Protecting human and environmental health from adverse effects of endocrine disruptors (EDs) is a high priority in the EU. These substances cause a range of adverse effects, such as reproductive toxicity, neurotoxicity, metabolic disruption, and cancer. If an organism is exposed during a developmental life stage, the effects may also be permanent. Assessing if a substance has endocrine disrupting properties is a lengthy and complex process that requires considerable amounts of animal data. There is a demand to phase-out animal testing in the EU and, simultaneously, there is growing use of alternative methods, like in silico, in chemico, and in vitro methods. Hence, there is a need, and an opportunity, to move toward ED assessments based on mechanistic data from alternative methods. However, there is a lack of methodology to connect these types of novel and complex data to adverse outcomes for use in ED assessments.The aim of my doctoral project was to develop and explore methodologies for the assessment of endocrine disruptors based on mechanistic data. The Adverse Outcome Pathway (AOP) framework was crucial to achieve this and was used across all subprojects of the thesis. I explored many complex aspects of the AOP framework, including development and application of AOP network and quantitative AOPs (qAOPs). Literature-based, computational, and experimental approaches were utilized, as well as combinations of these.In Study I, I developed a data-driven approach to generate AOP networks. This approach combines a systematic approach to identifying relevant AOPs in the AOP-Wiki with a computational approach to process and filter AOP-Wiki data. The output could then be imported into a software for visualization and analysis of networks.In Study II, I conducted and compared a standard ED assessment according to current guidance, to a mechanism-based assessment without in vivo data. A thyroid-related AOP network was utilized for identifying relevant search terms and for mapping collected data to identify a potential Mode of Action. This case study was performed using perfluorooctane sulfonic acid (PFOS) as a model substance, investigating its potential for thyroid disruption and developmental neurotoxicity.In Study III, RNA-sequencing of zebrafish embryos exposed to either cadmium (Cd) or 3,3',4,4',5-pentachlorobiphenyl (PCB126) was performed to investigate how transcriptomics data can be coupled to an AOP network. Multiple different data analysis approaches were explored, and their potential connection to an estrogen, androgen, thyroid, and steroidogenesis (EATS) related AOP network was evaluated. Ultimately, I developed and compared a data-driven and an expert-driven approach to connect Gene Ontology (GO) terms from enrichment analysis to key events (KEs) in the network.In Study IV, I developed an approach to quantify a Key Event Relationship (KER) based on data from published literature, and applied this approach on a KER linking decreased circulating testosterone levels to decreased sperm count. The approach consists of two parts, (1) A structured search strategy, study reliability assessment, and standardized data extraction to collect and assess data for quantification, and (2) a statistical modelling approach to make the best use of the collected data.Through the development and application of AOP networks (Study I and III) I identified several opportunities for improving AOP development and functionality of the AOP-Wiki. The issues identified hinder topological analysis and application of data-driven approaches to connect mechanistic data to the network. In Study II, based on the standard ED assessment, I concluded that PFOS fulfils the scientific criteria as an ED based on thyroid disruption and developmental neurotoxicity. On the other hand, in the mechanism-based assessment, I could only identify endocrine activity and not endocrine-mediated adversity. There was a lack of quantitative understanding of the AOPs in the network, which was essential to inferring adversity based on mechanistic data. Study III revealed that both cadmium and PCB126 potentially have endocrine disrupting properties. Endocrine activity, and adversity likely mediated by EATS or non- EATS modalities, were observed. The data-driven approach to connect transcriptomics data to the AOP network yielded very few results, while the expert-driven approach provided many more connections. Gene expression measurements followed by enrichment analysis may be fit for connecting data to intermediate and late KEs, but not for early KEs and molecular initiating events (MIEs). In Study IV, the quantified KER can be used to predict decreased sperm count based on a decrease in circulating testosterone levels, including some uncertainty in the circulating testosterone levels. The model can potentially be used to reduce or replace animal experiments if coupled to e.g., in vitro testosterone synthesis experiments or physiologically based kinetic (PBK) models.To conclude, I have developed and applied novel risk assessment methodologies to maximize the use of mechanistic data for ED assessment. Both opportunities and challenges with these approaches have been explored, and future research needs have been identified. Moreover, endocrine disrupting properties of chemicals not yet classified as EDs (perfluorooctane sulfonic acid , cadmium, and 3,3',4,4',5- pentachlorobiphenyl) were investigated. Altogether, these results pave the way toward non-animal mechanism-based assessment of EDs and improved protection of human health.List of scientific papersI. Wiklund L, Caccia S, Pipal M, Nymark P, Beronius A. Development of a data-driven approach to Adverse Outcome Pathway network generation: a case study on the EATS-modalities. Front Toxicol. 2023 May 9; 5:1183824. https://doi.org/10.3389/ftox.2023.1183824II. Wiklund L, Pipal M, Weiss J, Beronius A. Exploring a mechanism-based approach for the identification of endocrine disruptors using Adverse Outcome Pathways (AOPs) and New Approach Methodologies (NAMs): A perfluorooctane sulfonic acid case study. Toxicology. 2024 May; 504:153794. https://doi.org/10.1016/j.tox.2024.153794III. Wiklund L, Wincent E, Beronius A. Using transcriptomics data and Adverse Outcome Pathway networks to explore endocrine disrupting properties of Cadmium and PCB-126. Environ Int. 2025 Mar; 197:109352. https://doi.org/10.1016/j.envint.2025.109352IV. Wiklund L, Ristovska M, Moe J, Beronius, A. Quantification of the Key Event Relationship "Decreased circulating testosterone leading to impaired spermatogenesis" based on data from peer-reviewed studies. [Manuscript]</p

Assessment and identification of cognitive changes after surgery in older adults

The overarching aim of this dissertation was to explore methods for assessing postoperative cognitive changes in older adults, including an evaluation of a digital cognitive test battery, and exploring psychological factors influencing cognitive outcomes.Study I was an observational, mixed-methods study with 40 older adult patients (mean age 73, 21 women) who underwent total joint arthroplasty. Postoperative cognitive functions and neuroinflammation were assessed using an analog cognitive test battery, blood biomarkers and qualitative interviews. Five patients met criteria for delayed neurocognitive recovery, however, many patients experienced cognitive issues, especially in executive functions and fatigue. Psychological factors like low mood and sense of control played a role in recovery and daily life, while biomarkers remained normal.Study II was a systematic review and meta-analysis synthesizing psychological factors associated with postoperative delirium, delayed neurocognitive recovery and postoperative neurocognitive disorder in older adults. In total, 27 studies involving 6146 patients were included: depression was slightly linked (OR 1.07 95% CI 0.96-1.18) to postoperative delirium, while anxiety, stress, and certain personality traits were also associated with cognitive outcomes (p Study III was a randomized cross-over study with 50 older adult volunteers (mean age 76, 28 women). Two performance-based measures were compared, an analog cognitive test battery with a self-administered digital one. Participants were interviewed regarding their perceptions of the cognitive assessments. Correlation analyses between the tests were weak to moderate, r=0.19-0.52 and Cohen's d effect sizes were small to large, ranging from 0.25 to 1.43. Qualitative analysis identified two themes: self-competing in a safe environment and experience with technology; participants felt comfortable during the tests, and they reported high usability.Study IV was an observational feasibility study with 24 older adult patients (mean age 77, 13 women) who underwent abdominal surgery, and 5 nurses involved in the recruitment or administration of a digital cognitive test. The feasibility, acceptability and usability of the test was assessed. The test was found feasible, acceptable, and with excellent usability (System Usability Scale mean 87), meeting criteria for a larger trial. Three patients were diagnosed with one-day postoperative delirium, and none were diagnosed with postoperative neurocognitive disorder. The participants considered the test important for assessing cognitive function and easy to use. However, several challenges regarding feasibility were expanded upon in the qualitative interviews including reasons behind low recruitment rates.Conclusions: This dissertation advances understanding of how postoperative cognitive changes can be assessed in older adults. A digital self-administered cognitive test was shown to be feasible and acceptable in healthy older adults and in a small group of highly educated, non-frail patients undergoing abdominal surgery. Depression and postoperative delirium were most consistently studied in the current evidence base. Overall, the findings indicate that postoperative cognitive changes cannot be fully understood by test performance alone; psychological factors and subjective experiences also shape cognitive trajectories after surgery.List of scientific papersThis dissertation is based on the following studies, referred to in text by their Roman numerals. As an author, I retain the copyright to our papers published in these open access journals and have the right to republish them.I. Amirpour, A., Bergman, L., Markovic, G., Liander, K., Nilsson, U., & Eckerblad, J. (2025). Understanding neurocognitive recovery in older adults after total hip arthroplasty-neurocognitive assessment, blood biomarkers and patient experiences: a mixed-methods study. BMJ open, 15(1), e093872. https://doi.org/10.1136/bmjopen-2024-093872II. Amirpour A, Bergman L, Markovic G, Eckerblad J, Nilsson, U, Falk, A. Psychological factors associated with postoperative cognitive outcomes - a systematic review and meta-analysis. [Submitted] III. Amirpour, A., Eckerblad, J., Bergman, L., & Nilsson, U. (2024). Comparing analog and digital neurocognitive tests with older adults: a study of the ISPOCD battery vs. a digital test battery from Mindmore. BMC geriatrics, 24(1), 34. https://doi.org/10.1186/s12877-023-04648-wIV. Amirpour A, Saarijärvi M, Eckerblad J, Markovic G, Thorell A, Nilsson U, Bergman L. Evaluation of a digital, self-administered, cognitive test battery in older adult patients undergoing abdominal surgery: a non-randomized feasibility trial. [Submitted]</p

HIV prevention and sexual health among men who have sex with men in Vietnam

Introduction:Men who have sex with men (MSM) in Vietnam remain a key population disproportionately affected by HIV and emerging infections such as mpox. Despite the availability of prevention tools such as pre-exposure prophylaxis (PrEP) and digital health technologies, uptake remains low due to complex psychosocial and structural barriers. In this context, understanding behavioral, informational, and systemic factors influencing prevention readiness is essential to strengthen Vietnam's public health response.Aim:This thesis aims to explore knowledge, perceptions, and access to HIV and mpox prevention among MSM in Vietnam. Using a multi-method approach, the research investigates the role of eHealth, behavioral predictors, stigma, and healthcare trust in shaping uptake and engagement with prevention strategies such as PrEP and vaccination.Methods:This thesis employed a multi-method research design, integrating systematic review, qualitative inquiry, and quantitative surveys to generate a comprehensive understanding of HIV and mpox prevention dynamics among MSM in Vietnam. Study I was a systematic review conducted following PRISMA guidelines, incorporating 54 eHealth interventions targeting HIV/STI prevention among MSM globally. These interventions were assessed using the ICROMS quality appraisal tool, with thematic synthesis used to analyze behavioral, psychosocial, and technological outcomes. Particular attention was paid to the duration, mode of delivery, and sustainability of behavior change effects.Studies II and III utilized qualitative focus group discussions (FGDs) in Hanoi, Vietnam. Study II explored eHealth preferences with 35 MSM across five stratified FGDs, while Study III examined PrEP acceptability, perceived barriers, and service design preferences among 30 HIV-negative or status-unknown MSM. The FGDs were transcribed, translated, and analyzed using directed content analysis to identify themes related to digital engagement, privacy, stigma, and trust. Studies IV and V were nationwide online cross-sectional surveys conducted between August and November 2022. Study IV involved 1,422 MSM and focused on awareness, access, and willingness to use PrEP. Study V involved 1,549 MSM and assessed mpox-related knowledge, misconceptions, and vaccine willingness. Participants were recruited using a snowball sampling strategy via trusted MSM community leaders. Surveys were administered anonymously using structured online questionnaires covering sociodemographic, behavioral, and psychosocial domains. Multivariable logistic and Tobit regression models were applied to identify predictors of prevention behavior and knowledge.Findings:The systematic review (Study I) synthesized 54 eHealth interventions for HIV/STI prevention among MSM globally. Most were randomized controlled trials conducted in high-income countries, predominantly web-based, and aimed at reducing sexual risk behaviors and increasing HIV/STI testing. About two-thirds employed behavioral theories, but methodological quality varied. The review highlighted that sustained behavior change remains a challenge, with limited long- term follow-up and a lack of booster interventions. Interventions integrating interactivity, personalization, and multi-platform access were associated with higher engagement and effectiveness.Qualitative studies (Study II and III) conducted in Hanoi provided contextual depth on MSM's preferences for digital tools and perceptions of PrEP. MSM preferred smartphone-based, interactive, and discreet eHealth platforms, favoring private channels such as Facebook groups or encrypted chat over SMS, which was viewed as spam-like or privacy-threatening. Participants emphasized trust in content source, short and engaging formats, and integration with real-time services such as booking and counseling. Regarding PrEP, while participants acknowledged its value in reducing HIV risk and enhancing sexual agency, concerns about daily adherence, side effects, stigma, and cost were common. PrEP was seen as a complement-not a replacement-for condoms, and participants favored accessing PrEP through MSM-friendly CBOs or trusted clinics.Survey findings (Study IV and V) reinforced and extended these insights. Among 1,422 MSM across 62 provinces (Study IV), 56.1% were aware of PrEP, and 67.2% expressed willingness to use it. Willingness was higher among younger, better- educated MSM, those with higher income, and those open about their sexual orientation. However, recent experiences of sexual violence and stigma significantly reduced willingness, indicating psychological and structural barriers. Notably, inconsistent condom users and those engaging in group sex were more likely to express PrEP interest-suggesting risk-awareness as a motivating factor. In the context of emerging infections, Study V surveyed 1,549 MSM and found low mpox awareness despite high vaccine willingness (72.4%). Misconceptions about transmission were widespread, and only one in four participants knew where to get tested for mpox. Higher awareness was associated with youth, education, risk behaviors, and social support. Predictors of vaccine acceptance included general mpox knowledge, perceived necessity of vaccination, group sex engagement, and perceived HIV risk. These results emphasize the need for integrated communication strategies that align sexual health education with outbreak preparedness, particularly using trusted digital and peer-based channels.Together, the five studies revealed that while prevention interest among MSM in Vietnam is high-especially for PrEP and mpox vaccination-significant gaps in digital engagement, structural access, and trust must be addressed to translate willingness into action.Conclusion:This thesis provides an integrated understanding of HIV and emerging infectious disease prevention among MSM in Vietnam, drawing on findings from a systematic review, two qualitative studies, and two national surveys. Together, the studies highlight the readiness of MSM to engage with digital and biomedical prevention tools-such as eHealth platforms, PrEP, and vaccines-while also revealing substantial barriers related to stigma, disclosure, service trust, and knowledge gaps. The systematic review affirmed the short-term effectiveness of eHealth interventions when grounded in behavioral theory, but also pointed to limitations in long-term impact and sustainability. Qualitative findings showed that MSM valued credible, interactive, and private online interventions, yet remained concerned about information overload and inadvertent disclosure. Surveys confirmed a high willingness to use PrEP and accept mpox vaccination but revealed low awareness and structural obstacles to access. Psychosocial factors such as stigma, mental health distress, and sexual violence were consistently associated with lower prevention engagement. To close the gap between willingness and action, future health programs must prioritize integrated, stigma- free, and community-centered service models. This thesis offers key recommendations for designing inclusive digital and biomedical interventions, improving policy, and ensuring that MSM in Vietnam are equitably reached by national HIV and infectious disease prevention strategies.List of scientific papersI. A systematic review of eHealth interventions addressing HIV/STI prevention among men who have sex with men. Long Hoang Nguyen, Bach Xuan Tran, Luis EC Rocha, Huong Lan Thi Nguyen, Cui Yang, Carl A Latkin, Anna Thorson, Susanne Strömdahl. AIDS and behavior, 23(9), 2253-2272. https://doi.org/10.1007/s10461-019-02626-1II. An exploratory assessment of the preference for eHealth interventions to prevent HIV and sexually transmitted infections among men who have sex with men in Hanoi, Vietnam. Long Hoang Nguyen, Huong Lan Thi Nguyen, Mattias Larsson, Bach Xuan Tran, Mart L. Stein, Luis EC Rocha, Susanne Strömdahl. BMC public health, 20(1), 1387. https://doi.org/10.1186/s12889-020-09449-zIII. A qualitative assessment in acceptability and barriers to use pre- exposure prophylaxis (PrEP) among men who have sex with men: implications for service delivery in Vietnam. Long Hoang Nguyen, Huong Lan Thi Nguyen, Bach Xuan Tran, Mattias Larsson, Luis EC Rocha, Anna Thorson, Susanne Strömdahl. BMC infectious diseases, 21(1), 472. https://doi.org/10.1186/s12879-021-06178-5IV. Empowering Wellness: Exploring PrEP Access, Utilization, and Willingness among Men Who Have Sex With Men in Vietnam. Long Hoang Nguyen, Huong Lan Thi Nguyen, Tham Thi Nguyen, Mattias Larsson, Luis E. C. Rocha, Anna Thorson, and Susanne Strömdahl. [Manuscript]V. Exploring Awareness of the Disease, Attitude, and Acceptance Towards Vaccine against Monkeypox among Men Who Have Sex With Men in Vietnam. Long Hoang Nguyen, Huong Lan Thi Nguyen, Mattias Larsson, Luis E. C. Rocha, Anna Thorson, Susanne Strömdahl. [Manuscript]</p

Unraveling the impact of bacterial peptidoglycans from gut microbiota on brain development, function and behavior

Over recent decades, the microbiota-gut-brain axis has emerged as a central regulator of neurodevelopment, influencing lifelong brain functions. Increasing preclinical and clinical evidence implicates the gut microbiota as a susceptibility factor for neurodevelopmental and psychiatric disorders, including autism spectrum disorder (ASD). Additionally, mounting evidence highlights the pivotal role of the maternal gut microbiota during pregnancy and lactation in shaping early-life neural circuits and influencing long-term brain health in offspring. However, the molecular mechanisms through which maternal microbial signals affect fetal brain development remain poorly understood. This thesis investigates how alterations in the maternal and adult gut microbiota influence brain development, function, and behavior, with a particular focus on bacterial peptidoglycan (PGN) fragments as key signaling molecules.Paper I investigated whether disrupting the maternal gut microbiota with antibiotics during late pregnancy and early postnatal life affects early neurobehavioral outcomes relevant to ASD in offspring. Neonatal mice born to treated dams exhibited sex-specific alterations in ultrasonic vocalizations. As juveniles, these mice showed reduced social motivation, impaired social interactions, and altered anxiety-like behavior. These behavioral alterations were accompanied by reduced expression of oxytocin receptor and tight-junction proteins in the prefrontal cortex, low-grade colonic inflammation, and shifts in gut microbiota composition. These findings underscore the vulnerability of the maternal gut microbiota during the perinatal period and its critical role in shaping offspring social and emotional development.Papers Il and Ill explored the role of PGN fragments-specifically muramyl dipeptide (MDP)-as maternal gut microbiota-derived signals that influence fetal brain development. PGN fragments administered during mid- or late pregnancy were detected in the amniotic fluid, confirming their translocation to the fetal compartment in the absence of overt inflammation. Mid-gestation PGN exposure induced widespread transcriptomic alterations in fetal brains, affecting genes involved in chromatin remodeling, estrogen receptor signaling, synaptic plasticity, and glutamatergic transmission. Male offspring exhibited altered neonatal vocalizations and impaired social behavior, accompanied by changes in gut microbiota composition and dysregulated expression of synaptic (e.g., Ppp1r9b, Bdnf, Oxtr) and glutamatergic (Gria1) genes in the prefrontal cortex.In contrast, late gestational PGN exposure led to distinct, sex-specific behavioral outcomes. Male offspring displayed reduced social interaction, while females showed increased social engagement, reduced locomotion, and impaired social recognition. These effects were associated with altered expression of microglial (Trem2, Cx3cr1) and synaptic (Dlg4, Darpp-32) genes. Together, these findings highlight that the timing of PGN exposure and fetal sex critically shape neurodevelopmental responses, likely reflecting differences in developmental stage and hormonal milieu.Paper IV extended these findings into adulthood, examining how short-term exposure to B-lactam antibiotics alters PGN translocation and impacts brain function and behavior. Adult male mice treated with ampicillin showed region- specific increases in brain PGN levels. Expression of PGN transporters (Slc15a4 and Slc46a3) in the brain varied in response to changes in PGN levels. Within 72 hours, antibiotic treatment triggered rapid changes in synaptic and microglial gene expression, disrupted functional brain connectivity, and impaired sociability and social recognition. These effects were accompanied by pronounced shifts in gut microbial composition. Notably, administration of DAP-type PGN fragments (i.e., iE-DAP) derived from Gram-negative bacteria recapitulated key behavioral and molecular effects observed in antibiotic-treated mice, suggesting a direct role of PGN in antibiotic-associated neurobehavioral alterations.Collectively, this thesis identifies PGN as a critical molecular messenger in the microbiota-gut-brain axis, capable of influencing fetal brain programming and shaping long-term neurodevelopmental trajectories. It provides compelling evidence that maternal microbial signals-particularly during sensitive developmental windows-can shape neurodevelopment in a sex- and timing- dependent manner. By linking maternal microbiota alterations to specific molecular and behavioral outcomes, this work offers novel insights into how antibiotic use or dysregulated PGN levels during pregnancy may contribute to atypical neurodevelopment. Ultimately, these findings open new avenues for therapeutic strategies targeting PGN signaling to mitigate neurodevelopmental disorders and promote brain health.List of scientific papersI. Morel C, Martínez Sánchez I, Cherifi Y, Chartrel N, Diaz Heijtz R. Perturbation of maternal gut microbiota in mice during a critical perinatal window influences early neurobehavioral outcomes in offspring. Neuropharmacology. 2023 May 15;229:109479. https://doi.org/10.1016/j.neuropharm.2023.109479II. Perego S#, Martínez Sánchez# I, Hökfelt T, Diaz Heijtz R. Maternal Peptidoglycan overexposure in mid-pregnancy alters neurodevelopmental trajectories and behavioral outcomes in mice. #: These authors contributed equally to the work. [Manuscript]III. Martínez Sanchez I, Spielbauer J, Diaz Heijtz R. Maternal peptidoglycan overexposure during late pregnancy alters neurodevelopment and behavior in juvenile offspring. Brain Behavior and Immunity. 2025 Mar 7;127:96-102. https://doi.org/10.1016/j.bbi.2025.03.014IV. Martínez Sánchez I, Kim WS, Heather C, Nylén S, Shapiro MG, Diaz Heijtz R. Short-term exposure to B-lactam antibiotics enhances peptidoglycan translocation to the brain, disrupting functional connectivity and social recognition in mice. [Submitted]</p

Heterogeneity in platelet differentiation pathways

Hematopoietic stem cells replenish billions of mature blood cells every day through a series of increasingly lineage-restricted progenitor cells and are therefore vital for immune response, oxygen transport and hemostasis. Upon acute depletion of the hematopoietic system, such as after cytotoxic treatment for blood cancer, one of the most troublesome complications is bleeding due to thrombocytopenia.Through mouse models, our lab has characterized a novel and direct pathway of platelet generation, initiated by a hitherto unrecognized subset of HSCs, that are activated in response to cytotoxic stress. Study I investigates the emergence of lineage restriction and bias in perinatal HSCs. Study II describes the non- hierarchical relationship of two distinct types of HSCs, von Willebrand factor positive platelet-restricted and -biased HSCs (P-HSCs) and von Willebrand factor negative multilineage HSCs (M-HSCs). The transcriptional expression of FMS-like tyrosine kinase (Flt-3) was found to be a marker that distinguishes the differentiation pathways of P-HSCs and M-HSCs. This finding was validated by fate mapping using a Flt3-Cre/Rosa26-tdTomato mouse model. Furthermore, we performed molecular characterization of the megakaryocyte progenitors, the unilineage progenitors responsible for platelet generation, finding distinct molecular profiles depending on the type of HSC that generated them. We therefore speculate that the different pathways can in part explain platelet heterogeneity, an issue that has gained interest due to expansion of prothrombotic subsets upon aging. To this end, Study III describes a protocol for RNA-seq of sorted platelet populations allowing for molecular studies of platelet heterogeneity within the same individual.Taken together, these studies show that a mature cell can be generated through more than one differentiation pathway, suggesting a revision of the established hematopoietic hierarchy that currently posits only one unique sequence of differentiation steps for each mature cell type.List of scientific papersI. Platelet and myeloid lineage biases of transplanted single perinatal mouse hematopoietic stem cells. (Cell Research. 2023, 33, 883-886). Karin Belander Strålin*, Joana Carrelha*, Axel Winroth, Christoph Ziegenhain, Michael Hagemann-Jensen, Laura M. Kettyle, Amy Hillen, Kari Högstrand, Ellen Markljung, Francesca Grasso, Masafumi Seki, Stefania Mazzi, Yiran Meng, Bishan Wu, Edwin Chari, Madeleine Lehander, Rickard Sandberg, Petter S. Woll, Sten Eirik W. Jacobsen. https://doi.org/10.1038/s41422-023-00866-4II. Alternative platelet differentiation pathways initiated by non-hierarchically related hematopoietic stem cells. (Nature Immunology. 2024, 25, 1007-1019). Joana Carrelha*, Stefania Mazzi*, Axel Winroth*, Michael Hagemann-Jensen, Christoph Ziegenhain, Kari Högstrand, Masafumi Seki, Margs S. Brennan, Madeleine Lehander, Bishan Wu, Yiran Meng, Ellen Markljung, Ruggiero Norfo, Hisahi Ishida, Karin Belander Strålin, Francesca Grasso, Christina Simoglu Karali, Affaf Aliouat, Amy Hillen, Edwin Chari, Kimberly SiIletti, Supat Thongjuea, Adam J. Mead, Sten Linnarsson, Claus Nerlov, Rickard Sandberg, Tetsuichi Yoshizato, Petter S. Woll, Sten Eirik W. Jacobsen. https://doi.org/10.1038/s41590-024-01845-6III. The platelet transcriptome is stable across age and is minimally responsive to spontaneous CD62 externalization. Masafumi Seki*, Axel Winroth*, Margs Brennan, Kari Högstrand, Tetsuichi Yoshizato, Michael Hagemann-Jensen, Christoph Ziegenhain, Amie Waller, Holly Foster, John W. Semple, Cedric Ghevaert, Sten Eirik W. Jacobsen^, Petter S. Woll^. [Manuscript](*Equal contribution, ^Shared senior authorship)</p

Mobile phone technology for reduced drinking : novel methods for measuring alcohol consumption and treating alcohol dependence

Background: Studies on the use of smartphone applications (apps) to curb alcohol consumption are limited but increasing. Previous studies show promising effects, especially on samples in the general population. The aim of this thesis was to enhance the understanding of using apps within addiction treatment. The research questions ranged from estimating the effectiveness (Study I) and examining the perceptions of the users (Studies II and IV), to testing the agreement between app reports and traditional methods for measuring alcohol consumption (Study III), and assessing factors needed for successful implementation (Study IV).Methods: The project was based on a randomised controlled trial (RCT). Alcohol-dependent patients with recent patterns of heavy drinking, no co- occurring drug dependence and limited psychosocial problems were recruited at an alcohol addiction clinic or by social media advertisements and were randomised to either treatment as usual (TAU) or TAU complemented with a drink-counting or breathalyser-coupled app for 12 weeks (Study I). TAU comprised four therapy sessions for 12 weeks, that could also be complemented with pharmacotherapy. Effects were measured at 12 weeks (post-intervention) and 26 weeks after randomisation (follow-up). The primary outcome was heavy drinking days in the past month at 26 weeks. A purposive sample of 21 participants that had used the apps later partook in individual interviews on their experiences (Study II). The apps were further tested in a subsequent study where app-based data (e.g., number of drinks, drinking days and heavy drinking days) were compared with traditional methods for assessing consumption (e.g., Timeline FollowBack) (Study III). Since the two apps were linked to online portals that shared data with clinic staff, focus group discussions were undertaken to explore clinicians' views on incorporating the apps into their treatment delivery (Study IV).Results: Results from Study I showed that the breathalyser-coupled app given as a complement to TAU was more effective than TAU alone in reducing heavy drinking days at 26 weeks (30-33 % lower rate). In Study II, results showed that the apps were considered relevant and helpful adjuncts to TAU by the patients. Nonetheless, app usage was hindered by primarily technical problems but also non-personalised procedures and features and usage among other people. Similarly, in Study IV, the clinicians believed that the apps offered several advantages for their patients, and that the portals could provide valuable support during treatment sessions. However, the breathalyser was considered unreliable, which further was believed to affect both their own, and their patients', motivation for sustained use. In Study III, it was found that the data from the drink-counting app, but not the breathalyser, well agreed with consumption reported in TLFB and PEth. For the successful implementation of the apps, Study IV found that ensuring their technical stability, protecting data security, and identifying patients at risk of adverse events were considered crucial. Additionally, several other factors, including education, support, additional time, and continuity, were deemed essential.Conclusion: Taken together, the results of this thesis show that a breathalyser- coupled smartphone application may be effective in reducing heavy drinking, and that a drink-counting app may be a useful and available tool for reporting alcohol consumption. Both apps have relevant features considered useful in treatment and provide an opportunity to enhance patient accountability in the change process. A person-centred approach is essential when using the smartphone-based interventions within addiction treatment, along with ensuring the necessary conditions (e.g., guidance, support, workflow and time management) for clinicians to effectively utilise the tools. Technical issues with the apps are a main barrier to their reliability and use and must be resolved before they can be integrated into routine care.List of scientific papersI. Östh, J., Lundin, A., Wennberg, P., Andréasson, S., & Danielsson, A. K. (2025). The effectiveness of a drink-counting and a breathalyser- coupled smartphone application for reduced heavy drinking among alcohol-dependent adults in Sweden: a randomised controlled trial. Addiction.https://doi.org/10.1111/add.16769II. Östh, J., Danielsson, A. K., Lundin, A., Wennberg, P., Andréasson, S., & Jirwe, M. (2024). Keeping Track of My Drinking - Patient Perceptions of Using Smartphone Applications as a Treatment Complement for Alcohol Dependence. Substance Use & Misuse, 59(2), 291-299.https://doi.org/10.1080/10826084.2023.2269578III. Östh, J., Lundin, A., Wennberg, P., Andréasson, S., & Danielsson, A. K. Measuring alcohol use: Comparing instant smartphone application- based reports with Timeline Followback, Alcohol Use Disorders Identification Test Consumption, and Phosphatidylethanol. [Manuscript] https://doi.org/10.1016/j.abrep.2025.100643IV. Östh, J., Lundin, A., Wennberg, P., Andréasson, S., & Danielsson, A. K. Clinicians' Perspectives on Integrating Smartphone Application Data into Routine Alcohol Dependency Treatment: Factors Influencing Implementation. [Manuscript] https://doi.org/10.1186/s13722-025-00597-4</p

Modeling early human neural development using iPS cells

Human induced Pluripotent Stem Cells (iPSCs) have long been known for their great potential in disease and development modeling as well as their possible use for cell transplantations. This thesis investigates the advantages and limitations of human iPSCs. We investigated the fundamentals of neural lineage specifications during neural induction. In addition to modeling human neural development, we assessed the therapeutic efficiency of iPSCs derived neural epithelial stem (NES) cell doses in a pre-clinical study of spinal cord injury (SCI). Modeling development and the generation of cells for transplantations is dependent on the generation of standardized and trustable cells. NES cells are a great tool for both fields. They provide unlimited supply of neural progenitor cells and are able to generate into all major neural lineages. This thesis consists of one protocol for the standardized generation of NES cells, two studies using these cells for pre-clinical cell transplantation studies, and one paper to investigate the role of an adhesion molecule during early neural development. All included papers provide deeper insights into development in health and disease.In paper I we develop a protocol for the standardized and robust generation of NES cells. This neural induction protocol can be used to generate cells relevant for transplantations and for modeling human development in 2D.In paper II we prove that the generation of big batches of transplantable cells is possible and has many advantages. The cells have robust viability and keep differentiation potential even after prolonged periods of freezing. The quality of the cells can be assed prior to transplantation.In paper III off-the-shelf NES cell doses were transplanted into a rat SCI model. The cells were able to survive and differentiate into relevant neural cell types. Transplantation showed positive effects regarding regeneration tissue integrity.In paper IV we identified changes in relevant signaling pathways and early development in cells with bi-allelic NRXNla deletion. We thereby identified a possible mechanism of action for this adhesion molecule early in development. Developmental changes included change in regionalization, switch in cell fate, further progression in EMT, and higher levels of TGFB and BMP signaling.In total, this thesis provides methods and protocols for generation of possible cell transplantation products in the future. We also show that these cells can be used in disease modeling in 2D and to identify new roles of proteins beyond their known function.List of scientific papersI. Protocol for the derivation, culturing and differentiation of human iPS-cell-derived neuroepithelial stem cells to study neural differentiation in vitro. Javier Calvo-Garrido, Dania Winn, Camilla Maffezzini, Anna Wedell, Christoph Freyer, Anna Falk, Anna Wredenberg. STAR Protocols. (2021). https://doi.org/10.1016/j.xpro.2021.100528II. Pre-clinical evaluation of clinically relevant iPSC derived neuroepithelial stem cells as an off-the-shelf cell therapy for spinal cord injury. Dania Winn, Elias Uhlin, Malin Kele, Ilse Eidhof, Anna Falk Frontiers. Pharmacology. (2024). https://doi.org/10.3389/fphar.2024.1390058III. Multiple therapeutic effects of human neural stem cells derived from induced pluripotent stem cells in a rat model of post-traumatic syringomyelia. Tingting Xu, Xiaofei Li, Yuxi Guo, Elias Uhlin, Lena Holmberg, Sumonto Mitra, Dania Winn, Anna Falk, Eriks Sundström. eBioMedicine. (2022). https://doi.org/10.1016/j.ebiom.2022.103882IV. Multi-Omic Profiling reveals the Impact of NRXNa Deletion on early Neural Development in an iPSC Model. Dania Winn, Alireza Ghahramani, Sarfraz Shafiq, Yan Jiang, Ilse Eidhof, Nathalie Bérubé, Anna Falk. [Manuscript]</p

Anaemia and iron deficiency anaemia during pregnancy in Nigeria : risk factors, treatment options and impact on maternal and perinatal outcomes

Anaemia in pregnancy is a public health burden especially in low-middle-income countries with prevalence above 40% in many parts of sub-Saharan Africa and South-East Asia. It is a major contributor to the high maternal mortality in the regions. Anaemia is associated with an increased risk of preterm births, intrauterine growth restriction, intrauterine foetal death, and a higher risk of having postpartum haemorrhage. In addition, it has recently been associated with a higher risk of antenatal and postpartum depression.Nigeria has one of the highest maternal and perinatal mortality rates in the world. Among the leading causes of maternal mortality are haemorrhage which consequence is anaemia, and anaemia from other causes including nutritional deficiencies. To reduce the mortality burden, anaemia prevention and care of the condition needs to be clearly understood. It is important to identify risk factors for the condition in Nigeria, know the current screening and treatment methods in Nigeria, identify an effective and safe alternative to oral iron which is commonly used during pregnancy for prophylaxis and treatment, and understand the effect of improving anaemia severity or iron levels during pregnancy on the risk of postpartum depression. This doctoral thesis provides answers to these gaps in knowledge and its findings will impact maternal healthcare worldwide.The doctoral research comprised four studies. The Study I, as well as Studies III and IV were conducted within a clinical trial on intravenous versus oral iron in Nigeria (IVON) TRIAL. Study I included 872 pregnant women with moderate or severe anaemia (haemoglobin concentration We found a prevalence of 41% for iron deficiency among pregnant women with moderate or severe anaemia. Diet rather than sociodemographic factors played dominant role in the occurrence of iron deficiency anaemia. The study indicated that consumption of green leafy vegetables has a protective effect on the occurrence of iron deficiency anaemia while consumption of edible kaolin clay daily and soybeans three to four times a week increased the odds of having iron deficiency anaemia during pregnancy. Screening of anaemic pregnant women for iron deficiency in Nigeria is suboptimal, and oral iron is mostly used for iron deficiency anaemia treatment during pregnancy. The commonly available parenteral iron preparations were identified as iron dextran and iron sucrose with only 30% of the skilled healthcare providers having ever prescribed intravenous iron for iron deficiency anaemia treatment. We found intravenous ferric carboxymaltose which is a new medicine entering Nigeria for the first time for the IVON TRIAL, given as a single loading dose, to be a suitable alternative to oral iron for iron deficiency anaemia treatment during pregnancy. In the clinical trial, intravenous ferric carboxymaltose demonstrated better effectiveness for iron deficiency anaemia treatment and caused a faster rise in haemoglobin concentration by four weeks post-treatment compared to the standard of care, oral ferrous sulphate given thrice daily all through pregnancy. Furthermore, intravenous ferric carboxymaltose is as safe as oral ferrous sulphate when administered during pregnancy. Regarding long term sequelae of delayed correction of anaemia severity and iron levels on mental health disorders like depression, there was no evidence to suggest that an improvement in anaemia severity or iron levels during pregnancy significantly alters the risk for postpartum depression. However, an increased risk of postpartum depression in women who had postpartum haemorhage was observed.The findings of the doctoral research underscore the need to modify practice regarding anaemia treatment in Nigeria and other regions of the world where the disease burden is high to promote the health and well-being of pregnant and postpartum women.List of scientific papersI. Babah OA, Akinajo OR, Beňová L, Hanson C, Abioye AI, Adaramoye VO, Adeyemo TA, Balogun MR, Banke-Thomas A, Galadanci HS, Sam-Agudu NA, Afolabi BB, Larsson EC. Prevalence of and risk factors for iron deficiency among pregnant women with moderate or severe anaemia in Nigeria: a cross-sectional study. BMC Pregnancy Childbirth. 2024 Jan 5;24(1):39. https://doi.org/10.1186/s12884-023-06169-1II. Babah OA, Beňová L, Hanson C, Abioye AI, Larsson EC, Afolabi BB. Screening and treatment practices for iron deficiency in anaemic pregnant women: A cross-sectional survey of healthcare workers in Nigeria. PLoS One. 2024;19(11):e0310912. https://doi.org/10.1371/journal.pone.0310912III. Afolabi BB, Babah OA, Adeyemo TA, Balogun M, Banke-Thomas A, Abioye AI, et al. Intravenous versus oral iron for anaemia among pregnant women in Nigeria (IVON): an open-label, randomised controlled trial. The Lancet Global Health. 2024;12(10):e1649-e59. https://doi.org/10.1016/s2214-109x(24)00239-0IV. Babah OA, Beňová L, Larsson EC, Hanson C, Afolabi BB. Is an improvement in anaemia and iron levels associated with the risk of early postpartum depression? A cohort study from Lagos, Nigeria. BMC Public Health. 2025;25(1):808. https://doi.org/10.1186/s12889-025-21942-x</p

Groin hernia surgery in women : outputs, factors, methods and cost-effectiveness

There is inequity in access to surgical treatment and quality of care globally and this is more pronounced in low-and middle-income countries (LMICs) especially among women. Prioritizing treatment of common surgical conditions like groin hernias in LMICs would improve this aspect.Aims. The present aims were to assess groin hernia surgery outputs and practices in Uganda, to evaluate the relationship between parity and the incidence rate of groin hernia repair among women born in Sweden between 1956 and 1983, to compare the safety and effectiveness of open anterior mesh repair for groin hernia in adult women in Uganda to modified open anterior mesh repair through which both the inguinal and the femoral canal can be accessed and covered with a mesh flap. A further aim was to assess the cost and cost-effectiveness of the open anterior mesh repair in comparison to the modified open anterior mesh repair.Methods. Studies I, III and IV were carried out in Uganda. Study II was carried out in Sweden. Study I was a hospital-based observational study to retrospectively review theatre records for groin hernia repairs in 2013 and 2014, and a questionnaire interview for the surgical providers. Study II was a population-based cohort study among women born in Sweden between 1956 and 1983 in which the Swedish Hernia Register, and the National Medical Birth Register were cross- matched to identify groin hernia repairs after childbirth. The follow up period was until 31st December 2022. Incidence rates and incidence rate ratios for inguinal and femoral hernia repairs were determined. Study III was a double- blinded randomized controlled trial among adult women in Uganda in which the safety and effectiveness of open anterior mesh repair were compared with those of modified open anterior mesh repair. Study IV was a cost-effectiveness study of the trial in Study III to compare the cost and cost-effectiveness of the two groin hernia repair methods.Results. Study I, found that 5518 groin hernia repairs were performed in 2013 and 2014, with an annual repair rate of 7/100, 000 population. Most of the repairs were performed by non- specialized surgeons using suture techniques and almost 16% of the repairs were performed in women. In Study II, the mean incidence of inguinal hernia repair was 10.7 per 100,000 person-year and 2.6 per 100,000 person-year for femoral hernia repairs. The incidence rate and the incidence rate ratios for both inguinal and femoral hernia repairs increased with the number of births. In Study III, altogether, 200 participants were included. Nearly 45% had a femoral hernia. At one-year follow-up, overall recurrence was 5.6% and overall wound complications at two weeks follow-up were 10.1%. In Study IV, the cost per DALY averted in the open anterior mesh repair was 292.0 (SD, 264.4) USD compared to 328.8 (SD, 284.2) USD in the modified open anterior mesh repair.In conclusion, groin hernia repair outputs in Uganda need to improve along with mesh-based methods. High parity levels may increase the risk of groin hernias. Opening of the transversalis fascia to inspect the femoral canal should be routinely done among women to detect femoral hernias. The modified open anterior mesh repair is an option to treat inguinal and femoral hernias in the absence of laparoscopy. While the open anterior mesh repair and its modified version are cost-effective, their application should depend on patient needs. However, the high possibility of femoral hernias among women in the study setting, calls for promotion of the modified open anterior mesh method.List of scientific papersI. Groin hernia Surgery in Uganda. Caseload and Practices at Hospitals Operating Within the Publicly Funded Health Care Sector. Alphonsus Matovu, Pär Nordin, Andreas Wladis, Mary Margaret Ajiko, Jenny Löfgren. World J.Surg (2020) 44:3277-3283. https://doi.org/10.1007/s00268-020-05633-9II. Incidence of groin hernia repairs in women and parity. A population - based cohort study among women born in Sweden between 1956 and 1983. A. Matovu, J. Lofgren, A. Wladis, P. Nordin, G. Sandblom, H.J. Pettersson. Hernia (2024) 28:1231-1238. https://doi.org/10.1007/s10029-024-03011-1III. Open anterior mesh repair versus modified open anterior mesh repair for groin hernia in women. A randomized clinical trial. Alphonsus Matovu, Par Nordin, Andreas Wladis, Gabriel Sandblom, Moses Elaju, Fredrik Lindmark, Olof Bladin, Jenny Löfgren. [Manuscript]IV. Cost-effectiveness of open anterior mesh repair compared to modified open anterior mesh repair for groin hernia repair in women in a low resource setting. A randomized clinical trial. Alphonsus Matovu, Zin Min Thet Lwin, Geoffrey Okello, Francis Lulu, Gabriel Sandblom, Moses Elaju, Olof Bladin, Filip Westling, Andreas Wladis, Pär Nordin, Jenny Löfgren. [Manuscript]</p

Flame-synthesized nanomaterials for drug delivery and immunostimulation

Calcium phosphate (CaP) nanoparticles are highly attractive for biomedical applications due to their inherent biocompatibility, biodegradability, and close compositional similarity to mineral components of human bone. However, "CaP" is an umbrella term encompassing multiple polymorphs and crystalline phases, each exhibiting distinct biological behaviors. This structural variability has severely limited the establishment of clear structure-function relationships, impeding the rational design and clinical translation of CaP-based nanomedicines. To address this bottleneck, there is a need for a nanofabrication method capable of producing CaP nanoparticles with precise, tunable, and reproducible physicochemical properties. These criteria are well met by flame spray pyrolysis (FSP), a continuous and scalable synthesis platform.Across four integrated studies in this thesis, FSP is employed to engineer CaP-based nanomaterials for immunomodulation, protein antigen, and nucleic acid delivery. As well as synthesize morphology-matched luminescent nanoprobes for studying the biodistribution of FSP-made phosphate nanoparticles. Collectively, these studies establish mechanistic relationships between nanoparticle structure and biological function.In Paper I, through systematic variation of flame residence time and precursor concentration, three CaP nanoparticle formulations with distinct sizes and crystallinity were synthesized. All formulations demonstrated excellent biocompatibility in A549 cells with no cytotoxicity observed. Surface area-dependent OVA adsorption revealed smallest CaP nanocarrier achieved the highest loading capacity. Proteinase K degradation assays highlighted the potential of flame-made nanocarriers in protecting the surface adsorbed antigen. CaP crystallinity dictated their immunostimulatory behavior. Amorphous CaP nanoparticles function as potent antigen-dependent immunopotentiators, promoting dendritic cell activation and costimulatory marker expression (CD86, CD80) primarily when conjugated with antigen, whereas crystalline hydroxyapatite particles demonstrate inherent adjuvant activity independent of antigen presence. Paper II extends this mechanistic understanding through single step co-oxidation of CaP with SiO2, yielding colloidally stable, amorphous CaP-SiO2 composite nanoparticles. Comparative immunological analysis reveals that increasing amorphous CaP size progressively shifts immune activation toward crystalline-like, antigen-independent behavior. On the other hand, SiO2 incorporation enhances colloidal stability but reduces antigen loading capacity and immunogenicity. This positions CaP-SiO2 as a promising low-immunogenicity carrier platform for biomolecule delivery applications where minimized immune activation is desired.Paper III explored flame-made CaP for nucleic acid delivery through two complementary surface-engineering strategies. First, inorganic modification via single-step SiO2 co-oxidation during FSP, and second, post-synthesis polymeric functionalization using poly-L-lysine (PLL). SiO2 incorporation with CaP reduced the hydrodynamic size and demonstrated good DNA loading. However, the negative surface charge impeded with the transfection efficiency. The PLL functionalization of CaP nanoparticles reversed the surface charge to positive thus aided in the transfection efficiency.Paper IV develops near-infrared luminescent neodymium-doped lutetium phosphate (LuPO4:Nd3+) nanoprobes that structurally mimic the fractal-like aggregates characteristic of flame-synthesized CaP particles. Nd3+ doping enabled deep-tissue fluorescent imaging and precise in vivo biodistribution assessment. These imaging studies reveal predictable liver and spleen accumulation patterns driven by nanoparticle aggregate morphology.List of scientific papersI. Anshika Maheshwari, Rebecca Dookie, Meztlli O. Gaytán, Birgitta Henriques-Normark and Georgios A. Sotiriou. In Vitro Evaluation of Flame-Made Calcium Phosphate Nanoparticles for Antigen Delivery and Immunostimulation. ACS Applied Nano Materials, 2025, Vol 8, 11986-11996. https://doi.org/10.1021/acsanm.5c01535II. Anshika Maheshwari, Rebecca Dookie, Thomas Thersleff, Dmytro Danilian, Inge K. Herrmann, Birgitta Henriques-Normark and Georgios A. Sotiriou. Flame Synthesis of CaP-SiO2 Nanoparticles with Tunable Immunogenicity: In Vitro Evaluation with Dendritic Cells. Powder Technology, 2026, Vol 468, 121645. https://doi.org/10.1016/j.powtec.2025.121645III. Anshika Maheshwari, Eleni Chronopoulou, Natalia Teixeira, Andrea Del Valle, Francesco Righetti, Reshma Ramachandran, Birgitta Henriques-Normark and Georgios A. Sotiriou. Surface engineering of flame-made calcium phosphate nanoparticles for nucleic acid delivery. [Manuscript]IV. Olof Eskilson, Padryk Merkl, Eleni Bletsa, Anshika Maheshwari, Abhilash Kulkarni, Uliana Kostiv, Anandi Narayana Moorthy, Jerker Widengren, Birgitta Henriques-Normark & Georgios A. Sotiriou. Morphology-Preserving Flame Synthesis of Fractal Like Nanoaggregates for Deep-Tissue Bioimaging. [Manuscript]</p