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DIORA family of proteins and functional methods of investigation
No full textThis 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
Arrhythmias and antiarrhythmic therapy in patients with type 2 diabetes mellitus : Swedish registry based studies
No full textBackgroundType 2 diabetes mellitus (T2DM) is a well-established risk factor for cardiovascular complications such as coronary heart disease (CHD), heart failure (HF), and stroke. Diabetes mellitus (DM) has also been linked to an increased risk of certain cardiac arrhythmias, particularly atrial fibrillation (AF) and sudden cardiac death (SCD), presumably due to ventricular arrhythmias (VA). However, this association is not as well established as that of other cardiovascular diseases in the context of DM. Moreover, the need for cardiac device therapy among patients with T2DM, and its potential prognostic implications, remains insufficiently explored.AimsThe overall aim of this thesis was to broaden the knowledge regarding the association between T2DM and severe arrhythmias in need of treatment with cardiac implantable electronic device (pacemaker, implantable cardioverter defibrillator (ICD), cardiac resynchronization therapy defibrillator/pacemaker (CRT-D/P)) and to study prognosis after device implantation. Specifically, the four studies explored the:prevalence of bradyarrhythmias and incidence of pacemaker treatment in individuals with T2DM and without T2DM and potential factors associated with pacemaker implantation (Study I).prevalence of tachyarrhythmias and incidence of ICD/CRT-P/D treatment in individuals with T2DM and without T2DM and potential factors associated with ICD/CRT implantation (Study II).aetiology for pacemaker and ICD treatment in patients with T2DM and without T2DM (Study III-IV).complications and prognosis after a pacemaker and ICD implantation in patients with T2DM and without T2DM (Study III-IV).Material and MethodsThis thesis consists of four retrospective cohort studies based on data from several Swedish national registries: the Swedish National Diabetes Registry (NDR), the Swedish ICD and Pacemaker Registry, the Swedish Population Registry, the National Patient Registry, the Swedish Prescribed Drug Registry, the Longitudinal Integration Database for Health Insurance and Labor Market Studies and the Swedish Cause of Death Registry.Study I included patients with T2DM from the NDR and to each patient, five age-, sex-, and residence- matched controls from the general population based on registration in the population registry. Data on socioeconomic factors, comorbidities, and mortality were obtained from the registries mentioned above apart from the Swedish ICD and Pacemaker Registry and the Prescribed Drug registry. Participants were included from 1998 to 2012 and followed until 2013 with a mean follow-up of seven years after pacemaker implantation. Cox proportional hazards models were used to estimate the risk of needing a pacemaker during follow-up and predictors of pacemaker implantation.Study II shared the same study design as Study I, but with the focus on an ICD or CRT-P/D implantation. It included patients with T2DM from the NDR with five matched controls from the general population.Study III included patients who underwent de novo pacemaker implantation between 2010 and 2021 from the ICD and Pacemaker registry. The patient cohort was then linked with data from the other registries mentioned above except from the population registry. Information regarding a T2DM diagnosis was retrieved form the NDR. Patients with and without T2DM were compared regarding pacemaker implantation indications, peri- and postoperative complications (up to one year) using chi-square tests. Time to event analyses of major adverse cardiovascular events (MACE), and all-cause mortality were assessed using Cox proportional hazard regression models. The cumulative incidence of MACE and all-cause mortality was visualized using Kaplan-Meier curves and compared between groups with the log-rank test. Study IV share the same study design as Study III but included patients with de novo ICD implantation between 2010 and 2021. Indications, procedural complications, and outcomes were compared between patients with and without T2DM. Additional subgroup analyses regarding ICD implantation for primary or secondary prevention with T2DM and without T2DM were studied by pairwise comparisons using the log-rank test.ResultsStudy I and II included approximately 400,000 patients with T2DM and 2 million controls. Patients with T2DM had a higher prevalence of cardiovascular comorbidities and brady- and tachyarrhythmias than controls. During follow- up, device implantation was more frequent in patients with T2DM (pacemaker 242.2 vs. 152.5 per 100,000 person-years; pStudy III included approximately 95,000 patients with a de novo implanted pacemaker whereof 19% had T2DM. The study showed that sick sinus syndrome and atrio ventricular block III were the most frequent reported ECG findings in patients undergoing a de novo pacemaker implantation regardless of the presence of T2DM at the time of implantation. Cardiac fibrosis and CHD were the most common underlying aetiologies for pacemaker in patients with T2DM and without T2DM. Procedural complications were slightly lower in patients with T2DM, but the all-cause mortality and cardiovascular prognosis was impaired in this group even after adjustments for CHD and HF.Study IV included approximately 13,000 patients with a de novo implanted ICD whereof 22% had T2DM. The study showed that patients with T2DM received ICD implantations more often for primary prevention compared with patients without T2DM, and coronary heart disease was the dominant underlying cause in this group. Patients without T2DM more frequently had non-ischemic aetiologies as indications for ICD, such as dilated cardiomyopathy. After ICD implantation, patients with T2DM demonstrated a substantially higher adjusted risk of both all-cause mortality and MACE. Complication rates including infections within the first year after implantation were low and did not differ significantly between the two groups.ConclusionsThere is a significant association between T2DM and an increased risk of serious cardiac arrhythmias and the need for device treatments. The findings suggest that overall, patients with T2DM carry a heavier burden of arrhythmias and a more dismal prognosis than patients without T2DM, even when device therapy is provided. These associations were partly, however not entirely driven by CHD and HF but other factors that might relate to T2DM, may directly promote arrhythmogenesis and the subsequent need for device therapy in this population. This underscores that while cardiac device implantation remains important for arrhythmic risk reduction in general, additional strategies addressing T2DM specific and comorbidity related risks are essential to optimize outcomes in this high-risk population.List of scientific papersI. Elina Rautio, Fredrik Gadler, Soffia Gudbjörnsdottir, Stefan Franzén, Lars Rydén, Ann-Marie Svensson, Linda Mellbin. Patients With Type 2 Diabetes Have an Increased Demand for Pacemaker Treatment: A Comparison With Age- and Sex-Matched Control Subjects From the General Population. Diabetes care. 2020;43(11):2853-8. https://doi.org/10.2337/dc20-0084II. Elina Rautio, Fredrik Gadler, Soffia Gudbjörnsdottir, Stefan Franzén, Lars Rydén, Gianluigi Savarese, Ann-Marie Svensson, Linda Mellbin. Implantable Cardioverter Defibrillator and Cardiac Resynchronization Treatment in People with Type 2 Diabetes: a Comparison with Age- and Sex Matched Controls From the General Population. Cardiovasc Diabetol. 2024 Jan 6;23(1):18. https://doi.org/10.1186/s12933-023-02084-zIII. Elina Rautio, Fredrik Gadler, Paolo Gatti, Soffia Gudbjörnsdottir, Lars Rydén, Per Näsman, Anne Wang, Yiran Zhou, Linda Mellbin. Indications for Pacemaker Implantation, Prognosis and Peri/Postoperative Complications in Patients with and without Type 2 Diabetes Mellitus. [Manuscript]IV. Yiran Zhou/Elina Rautio, Fredrik Gadler, Soffia Gudbjörnsdottir, Per Näsman, Lars Rydén, Tigist Wodaje, Linda Mellbin. Indications, Prognosis, and Complications of De Novo Implantable Cardioverter Defibrillators in Patients with and without Type 2 Diabetes. [Submitted]</p
The role of the insulin receptor in mature human adipocytes
No full textObesity is a global health crisis, affecting millions of individuals worldwide and imposing substantial burdens on healthcare systems. The prevalence of obesity has reached epidemic proportions, with roughly 12.5% of adults worldwide classified as obese. Obesity is closely linked to the development of insulin resistance, type 2 diabetes (T2D) and other metabolic disorders. While adipose tissue plays a crucial role in metabolic homeostasis, the precise molecular mechanisms underlying insulin resistance in adipocytes remain incompletely understood. This thesis presents three interconnected studies that provide new insights into adipocyte biology, with a focus on developing advanced methodologies to investigate insulin receptor (INSR) dynamics in the context of obesity and metabolic dysfunction.(I) The first study introduces a novel approach for detecting cellular senescence using reflected light microscopy. Senescence is an important cellular program involved in development, tissue repair, cancer and aging. More recently, a premature, metabolically induced senescence has been described in multiple tissues and is associated with obesity and T2D. However, characterizing and quantifying senescent cells at the single-cell level has been challenging, particularly in large primary cells like human adipocytes. This study introduces a technique that utilizes reflected light for accurate senescence-associated beta- galactosidase (SABG) staining measurements in adipocytes. By leveraging confocal microscopy to detect X-gal crystals using reflected light, this approach achieves superior sensitivity over traditional brightfield techniques. It allows for the capture of all X-gal precipitates in SABG-stained samples and can detect diverse staining patterns. The method can be integrated with immunofluorescence and is compatible with primary mature adipocytes from both human and mouse, as well as differentiated 3T3-L1 cells. Importantly, this technique outperforms western blot analysis for detecting and quantifying senescence in mature human adipocytes. This methodological advancement provides a powerful tool for investigating the role of cellular senescence, or other metabolic phenotypes, in adipose tissue dysfunction at a single-cell level.(II) The second study addresses methodological challenges in protein quantification using western blotting, a crucial technique for analyzing protein expression in adipocyte research. This study investigates the robustness of housekeeping proteins and total protein (TP) as normalization references for western blotting in primary mature human adipocytes. TP exhibited the lowest variance among technical replicates and was superior as a normalization reference for glucose transporter 4 (GLUT4), a key protein involved in insulin- stimulated glucose uptake. TP also demonstrated the closest alignment with expected values when loaded as a protein gradient and showed lower intra- and inter-patient variability compared to housekeeping proteins across metabolically similar patients. The study concludes that TP normalization is the preferred method for reliable protein expression analysis in primary mature human adipocytes. This methodological improvement enhances the accuracy of protein quantification in adipocyte research, which is crucial for understanding the nuanced molecular changes associated with obesity and insulin resistance.(III) These two methodological advancements (I, II) serve as foundational tools for the third and principal study, which investigates INSR and insulin-like growth factor 1 receptor (IGF1R) expression and dynamics in primary mature human adipocytes from individuals with various metabolic profiles. By leveraging the high-precision confocal imaging technique (I), plasma membrane INSR negative (INSR-) adipocytes were observed primarily in obese hyperinsulinemic (OB/HI) males at a single-cell resolution. Further analysis of protein lysates using the optimized western blot technique (II) revealed that although total INSR levels remained unchanged, phosphorylated INSR and the activation of downstream signaling pathways were elevated in OB/HI males compared to non-obese (NOB) male controls. Notably, these patterns were sex-specific, with significant correlations between the fraction of INSR+ adipocytes and markers of insulinemia observed exclusively in obese males. In vitro differentiated adipocytes partially recapitulated the INSR- phenotype, and subsequent mass spectrometry indicated alterations in the endosomal trafficking proteins, providing a potential mechanistic explanation for the observed INSR- adipocytes.Together, these three studies advance our understanding of adipocyte biology and explore the molecular mechanisms underlying obesity-associated metabolic dysfunction. The novel senescence method and improved western blotting normalization strategy serve as powerful tools for investigating adipocyte biology with enhanced precision and accuracy, enabling the exploration of INSR dynamics and revealing a potential new mechanism of insulin resistance in mature human adipocytes. These insights open new avenues for targeted therapies, and future research based on these methodological and mechanistic advances may improve strategies for preventing and treating obesity-related metabolic dysfunction in adipose tissue. Moreover, the techniques developed here may find broader application in studying other aspects of adipocyte biology and disease mechanisms beyond insulin resistance, T2D, and obesity.List of scientific papersI. Dedic, B., Westerberg, L., Mosqueda Solís, A., Dumont, K. D., Ruas, J. L., Thorell, A., Näslund, E., & Spalding, K. L. (2024). Senescence detection using reflected light. Aging cell,23(11), e14295. https://doi.org/10.1111/acel.14295II. Westerberg, L. J. S., Dedic, B., Näslund, E., Thorell, A., & Spalding, K. L. (2025). Superior normalization using total protein for western blot analysis of human adipocytes. PloS one, 20(7), e0328136. https://doi.org/10.1371/journal.pone.0328136III. Dedic, B., Westerberg, L. J. S., Cutler, H. B., Thorell, A., James, D. E., & Spalding, K. L. Plasma Membrane Insulin Receptor-Negative Adipocytes in Hyperinsulinemic Males Exhibit Persistent Metabolic Activation. [Manuscript]</p
A mixed methods study of self-directed learning in clinical practice using a mobile skills training system
No full textAbstractBackgroundThe challenge of increasing demands on healthcare and a less available workforce requires new approaches to retain and develop healthcare professionals. One way of addressing this challenge is by instituting competency-based continuing professional development based on self-directed learning (SDL) principles. We investigated the feasibility of a mobile training system in a clinical and peer-to-peer, SDL setting. MethodsThe study used mixed methods with surveys, observations, and interviews to gain comprehensive insights into how healthcare professionals (nurse, assistant nurse) and managers experienced the investigated educational tool. ResultsHealthcare professionals’ experiences of SDL were illustrated in four main themes: (1) leadership required for learning, (2) conditions for learning, (3) effects of continuing professional development, and (4) suggestions for improving training methods. In surveys, healthcare professionals self-assessed their confidence in executing care tasks before and after undergoing SDL for those tasks. The results indicated a significant increase in self-efficacy post-SDL training. Engaging in care tasks through SDL, which includes checklists aligned with evidence-based practices, enhances healthcare professionals’ awareness of their own competence. Additionally, it provides managers with the means to ensure that employees meet core competence requirements. The role of a clinical skills centre to support managers and staff in their learning efforts was seen as important for sustained learning. Conclusion A mobile training system, implemented within a clinical and peer-to-peer SDL framework, facilitates and complements competency-based continuing professional development and reduces the perceived length of time healthcare professionals spend away from direct patient care. Access to clinical educators for guidance during implementation, along with support from the clinical skills centre during the planning and follow-up stages, proved essential for facilitating accessible learning. Managers play a crucial role in prioritizing continuous learning for their employees and establishing a culture of ongoing education among managers and healthcare professionals.</p
COVID-19 disruptions to essential health services in Uganda : access, interventions and lessons for emergency
No full textBackgroundPublic health emergencies like disease outbreaks cause disruption to delivery of, and access to essential health services. Prior to the COVID-19 pandemic, existing frameworks for preparedness and response to public health emergencies did not explicitly highlight the maintenance of essential health services as an important strategic operation of an emergency response. Previous researchers have also noted that the morbidity and mortality due to these disruptions can outstrip the disease outbreak specific morbidity and mortality.AimsThe overall aim was to investigate how responses to public health emergencies can minimize disruptions to the delivery of and access to essential health services while strengthening systems for service delivery. Specific aims were:1. To describe the interventions and strategies that were implemented for continuity of HIV/AIDS, TB and Malaria service delivery during public health emergencies (Study I).2. To investigate the challenges to access and delivery of essential health services during the response to the COVID-19 pandemic in Uganda (Study II).3. To describe the interventions and innovations that were implemented to ensure the continuity of health service delivery during the COVID-19 pandemic in Uganda (Study II).4. To investigate the effects of the COVID-19 vaccine rollout on the continuity of routine childhood immunization services in Uganda (Study III).5. To describe the innovations and adaptations for continuity of services that were adopted for delivery of routine maternal and child health services in Uganda (Study IV).Materials and MethodsIn this thesis, I used both quantitative and qualitative research methods.Quantitative Methods Analysis of Routine Data: Interrupted times series analysis was conducted using monthly data at national (Study II) and district (Study III) level to estimate the magnitude of change in service use before and during the COVID-19 pandemic. Qualitative Research Methods Systematic Review (Study I): Articles describing interventions to maintain the delivery of or demand for HIV/AIDS, TB and/ or malaria services in low- and middle-income countries were searched in Medline, Web of Science, Embase, Cochrane and Global Index Medicus and thematically analyzed.Document Review (Study II): Uganda Ministry of Health documents and reports were reviewed to synthesize the challenges to service delivery and interventions for continuity of services during the COVID-19 response.Key Informant Interviews (Study II, Study III, Study IV): Study II participants were purposively selected from all levels of the health system. Study III and Study IV included health workers at public and private health facilities in Wakiso District. Focus Group Discussions (FGD) (Study III and Study IV): Three FGDs were conducted with mothers if they had at least 2 children below the age of 3 years to understand the effect of the COVID-19 vaccine rollout on routine immunization services and experiences regarding use of maternal and child health services. Qualitative data were thematically analysed using inductive (Study I), deductive (Study II and Study IV) and abductive (Study III) approaches.ResultsInterventions to maintain HIV/AIDS, TB and Malaria services included leveraging existing service delivery platforms for service delivery, modifying existing logistics operations systems, combining operational workflows and scaling up self-care. New interventions included population-based interventions (e.g. mass administration of antimalarials) and policy level interventions (e.g. removing user fees).Using the interrupted times series analysis, I found that there were significant differences in the pre-COVID-19 trends compared with the post-COVID-19 trends of service use for facility deliveries and new attendances in out-patient departments. During the COVID-19 pandemic in Uganda, challenges to service delivery included movement restrictions and concerns for health worker safety, shortage of resources for continuity efforts, shortage of equipment, commodities and space as well as mobility restrictions affecting service delivery and use. Interventions to address the challenges included establishment of coordination mechanisms for essential health services continuity, provision of infection control commodities and stocking essential health products, provision of financial resources for continuity efforts, strengthening existing health workforce through temporary hires and leveraging the community health workforce and modifying existing service delivery strategies.Between March and April 2021, Diptheria, Pertussis and Tetanus (DPT3) vaccine doses administered reduced by 4.3%, Polio 1 vaccine doses reduced by 5.5%, Polio 2 doses reduced by 5.8% and Polio 3 doses reduced by 5.6%. The challenges to continuity of routine childhood vaccination services included increased workload, competition for cold chain and storage capacity and impact on perceptions about vaccination. Interventions to sustain demand included engaging community health workers, community mobilization, health education, and prioritizing routine immunization services. Interventions to maintain delivery included integration of services and increasing health workforce. Interventions to maintain essential health services led to sustained changes like new infrastructure and supplies e.g ambulances, new roles involving infection control, increased role of community health workers and outcomes like better workplace safety and teamwork.ConclusionsIn this thesis, I showed that the capacity to maintain essential health services during emergencies is dependent on baseline capacities and therefore its important to invest in the policies and infrastructure for service delivery before emergencies. Multisectoral engagement to balance response strategies with continuity efforts is critical to forestall service disruptions. Also, in low- and middle-income countries, community structures and systems are important for response and maintenance of services. Ultimately, services are provided by health workers and its important to prioritize their safety and wellbeing during emergency response. Finally, there should be deliberate efforts to document lessons to inform sustained system improvements that result from the innovation, flexibility and dynamism required to maintain service delivery during an emergency.List of scientific papersI. Interventions to Maintain HIV/AIDS, TB and Malaria Service Delivery During Public Health Emergencies in Low- and Middle-Income Countries: A Systematic Review. Steven N Kabwama, Rawlance Ndejjo, Tobias Alfvén, Neda Razaz, John M Ssenkusu, Helena Lindgren, Rhoda K Wanyenze. [Submitted]II. Interventions for Maintenance of Essential Health Service Delivery during the COVID-19 Response in Uganda, between March 2020 and April 2021. Steven N Kabwama, Rhoda K Wanyenze, Suzanne N Kiwanuka, Alice Namale, Rawlance Ndejjo, Fred Monje, William Wang, Siobhan Lazenby, Susan Kizito, Christopher Troeger, Anne Liu, Helena Lindgren, Neda Razaz, John Ssenkusu, William Sambisa, Rebecca Bartlein, Tobias Alfvén. International Journal of Environmental Research and Public Health. 2022, 19, 12522. https://doi.org/10.3390/ijerph191912522III. Maintenance of Service Delivery During Medical Countermeasures Deployment: The Association between the COVID-19 Vaccine Rollout and Continuity of Routine Childhood Immunization Services in Uganda. Steven N Kabwama, Neda Razaz, John M Ssenkusu, Helena Lindgren Rhoda K Wanyenze, Tobias Alfvén. PLOS Global Public Health. 2025, 5(6): e0004731. https://doi.org/10.1371/journal.pgph.0004731IV. How interventions to maintain services during the COVID-19 pandemic strengthened systems for delivery of maternal and child health services: a case-study of Wakiso District, Uganda. Steven N Kabwama, Rhoda K Wanyenze, Neda Razaz, John M Ssenkusu, Tobias Alfvén, Helena Lindgren. Global Health Action. 2024 Dec 31;17(1):2314345. https://doi.org/10.1080/16549716.2024.2314345</p
Dosage compensation of sex- and autosomal chromosomes
No full textGene dosage imbalances that arise from imbalanced chromosome copy numbers (aneuploidy) are disruptive to organismal fitness and homeostasis. An exception to that can be found in the case of heteromorphic sex chromosome systems such as the mammalian XX/XY and avian ZZ/ZW. Both of these systems independently evolved from distinct pairs of ancient autosomal chromosomes following the gain of a sex-benefitting factor on one of the two copies and eventually, the degeneration of the sex-specific chromosome (i.e. the mammalian male-specific Y chromosome and the avian female- specific W chromosome). This degeneration therefore rendered the heterogametic mammalian XY males and avian ZW females practically monosomic for the X and Z chromosomes, respectively, creating significant dosage imbalance between the single large chromosome and the pairs of autosomes. Pioneering geneticist, Susumu Ohno, hypothesized that such dosage imbalances are resolved through the transcriptional hyperactivation of the single large sex chromosome. While Ohno's hypothesis has been shown to be true in many species, sex chromosome dosage compensating mechanisms have been revealed to be complex and often, to rely on the master regulatory functions of long non-coding RNAs (lncRNAs), such as roX1 and roX2 in Drosophila, RSX in marsupials and the newly identified MAYEX and FEREX in the green anole lizard.In mammals, two X-chromosome dosage compensating mechanisms are present: X- chromosome inactivation, mediated by the lncRNA Xist, which results in the transcriptional silencing of one of the two X-chromosomes in female cells and X- chromosome upregulation, which transcriptionally upregulates the single active X chromosome in both male and female cells. However, the regulatory link between these processes and the dynamics of how they act in concert to maintain the correct X-linked dosage during development remain largely uncharacterised. On the other hand, avian sex chromosome dosage compensation does not rely on a Z-chromosome inactivation mechanism. While some genes on the female Z chromosome have been proposed to be upregulated to compensate for the dosage discrepancy, avian dosage compensating mechanisms have been suggested to be either absent or highly inefficient, begging the question of how the stoichiometric imbalance is resolved.Similarly, sex chromosome dosage compensation in the vast majority of non-model animals and alternative sex chromosome systems remains elusive. Finally, whether any similar dosage compensating mechanisms may act to correct for autosomal chromosome aneuploidies to any degree in karyotypic disorders such as cancer, remains largely uncharacterised.In Paper I, we investigate the dynamics of X-chromosome upregulation in vivo during mouse pre-and peri-implantation embryonic development and in vitro during mouse embryonic stem cell priming. Using allele-resolved single-cell RNA-sequencing data and multi-modal joint single-cell RNA and ATAC-seq data, we dissect the separate effects of X-chromosome inactivation and X-chromosome upregulation on RNA levels during mouse development. We find that X-chromosome upregulation is an elastic, dosage- compensating mechanism tuning expression dosage in a sex- and lineage-specific manner in concert with the degree of X-chromosome inactivation. Furthermore, we report that while male blastocyst cells achieve X-chromosome upregulation upon zygotic genome activation, female cells experience two waves of X-upregulation, one during imprinted X-inactivation and one during random X-inactivation, with ablation of Xist impeding X-upregulation. In contrast to the conventional and widely believed model of X- upregulation proposing that X-inactivation acts to silence one of the already hyperactive X chromosomes, we challenge this model, finding that naïve female cells lack X- upregulation, which only initiates following the initiation of X-inactivation. These results demonstrate that X-upregulation is an elastic process, dynamically controlling X- chromosome dosage during development.In Paper II, we characterise the presence, degree and mechanistic aspects of avian Z- chromosome upregulation. While the avian ZZ/ZW sex chromosome system has been widely considered to lack efficient dosage-compensating mechanisms, we find that the female Z chromosome is dosage compensated at multiple regulatory layers. Using an array of allele-resolved multi-omics approaches, we report that the single female Z chromosome is transcriptionally hyperactivated through increased transcriptional burst frequency mechanistically resembling mammalian X-chromosome upregulation. Furthermore, ribosomal profiling and mass spectrometry-based proteomics revealed that this upregulation is additionally enhanced via elevated translational efficiency of Z- linked transcripts, achieving significant, yet incomplete rebalancing between the heterogametic ZW female and the homogametic ZZ males.In Paper III, we explore the potential dosage compensating mechanisms employed by the newly identified cephalopod ZZ/Z0 sex chromosome system. Using RNA-sequencing data from two Octopus species, Octopus vulgaris and Octopus sinensis, we find evidence for partial sex chromosome dosage compensation between the sexes and identify a novel a male-specific Z-linked long non-coding RNA we termed "Zmast", conserved in both species. Furthermore, using high-sensitivity, bulk RNA-seq data in O. vulgaris paralarvae, we determine that partial Z-chromosome dosage compensation may be achieved by transcriptional upregulation of the single female Z chromosome and identify a second, female-biased long non-coding RNA, we termed "Zfest", conserved in both Octopus species, and displaying sex-specific splicing patterns. These findings suggest that long non-coding RNAs may be implicated in the regulation of the ancient cephalopod sex chromosome dosage compensation, mirroring some of the mechanisms found in mammals and other non-mammalian species.In Paper IV, we explore if, and how, autosomal chromosome aneuploidies might be dosage compensated. Using allele-resolved, high-sensitivity, single-cell RNA-sequencing in monoclonal fibroblast lines with different degrees of aneuploidy, we determine that autosomal chromosome loss is transcriptionally compensated through burst-frequency- mediated transcriptional upregulation of the remaining intact allele. We find that this effect operates in a region-specific manner in both complete and segmental (partial) aneuploidies, highlighting the previously unappreciated flexibility of this mechanism. Proteomics measurements further revealed an additional layer of dosage compensation, resulting in considerable stoichiometric rebalancing across autosomal aneuploid chromosomes. These findings highlight that dosage compensation is not a sex- chromosome-specific feature, but a fundamental, genome-wide molecular mechanism in response to gene dosage imbalance.List of scientific papersI. Antonio Lentini, Huaitao Cheng, J.C. Noble, Natali Papanicolaou, Christos Coucoravas, Nathanael Andrews, Qiaolin Deng, Martin Enge & Björn Reinius#. Elastic dosage compensation by X-chromosome upregulation. Nat Commun 13, 1854 (2022). https://doi.org/10.1038/s41467-022-29414-1II. Natali Papanicolaou*, Antonio Lentini*, Sebastian Wettersten, Michael Hagemann- Jensen, Annika Kruger, Jilin Zhang, Christos Coucoravas, Ioannis Petrosian, Xian Xin, Ilhan Ceyhan, Joanna Rorbach, Dominic Wright & Björn Reinius#. Multi-layered dosage compensation of the avian Z chromosome by increased transcriptional burst frequency and elevated translational rates. Nat Commun 16, 9088 (2025). https://doi.org/10.1038/s41467-025-64817-wIII. Natali Papanicolaou#, Sebastian Wettersten, Alexander Kloosterman, Eduardo Almansa, Eve Seuntjens & Björn Reinius#. Z-chromosome dosage compensation and sex-specific long non-coding RNAs in octopus. https://doi.org/10.1101/2024.12.09.627507 [Manuscript Preprint]IV. Natali Papanicolaou*, Sebastian Wettersten*, Guilherme Maia, Antonio Lentini# & Björn Reinius#. Dosage responses of aneuploid autosomal chromosomes. https://doi.org/10.1101/2025.09.18.677044 [Manuscript Preprint]*Equal contribution#Corresponding author</p
Navigating uncertainty : trauma quality improvement in urban India
No full textBackground and aimImproving the quality of health care is a global priority, yet many conventional quality improvement approaches, rooted in industrial models of standardisation and control, struggle to account for the uncertainty and complexity of clinical care. Trauma, defined as external injury and the body's physiological response, causes about 4.4 million deaths each year. The impact is greatest in low- and middle-income countries, where trauma is a leading cause of quality-related deaths. Trauma care involves time pressure, interdisciplinary teams, and high variability. This thesis investigates if, and how, a trauma quality improvement programme improves patient outcomes. The work is based on four empirical studies embedded in the Trauma Audit Filter Trial, a multicentre study evaluating a programme using audit filters and multidisciplinary case reviews in urban India. Audit filters are statements of agreed standards of care; when care deviates from these, the case is selected for review.Methods and resultsStudy I used a Delphi method to identify audit filters appropriate for the local context. Filters developed in other low- and middle-income countries were perceived as highly useful, and perceived usefulness was linked to medical relevance and feasibility.Study II, a prospective controlled interrupted time series trial, showed an 11% reduction in in-hospital mortality after programme implementation, while also highlighting confounding external factors and the limits of attributing causality in complex systems.Study III assessed the effect on health-related quality of life, showing that while survival improved, health-related quality of life slightly declined, likely because more severely injured patients survived, highlighting rehabilitation needs.Study IV used reflexive thematic analysis of interviews with participants in multidisciplinary review meetings to identify mechanisms through which the programme contributed to improvement. These included system-level awareness, shared understanding, navigation of hierarchical structures, and ethical sensitisation.ConclusionA trauma quality improvement programme based on case review and multidisciplinary discussion may reduce mortality in settings with a high burden of preventable deaths. Quality improvement in complex systems depends on structures that enable continuous learning, strengthen the forms of knowledge used in medical decision-making, and increase practitioners' understanding of the systems in which they work. Such approaches support better decision-making, enhance adaptability, and help identify and address areas for improvement within local environments. This thesis contributes to understanding how quality improvement can lead to change in trauma care and may help inform the design and implementation of similar programmes in other contexts. The knowledge gained about the mechanisms of improvement may also inform quality improvement efforts in other areas of health care.List of scientific papersI. Berg J, Alvesson HM, Roy N, Ekelund U, Bains L, Chatterjee S, Bhattacharjee PK, David S, Gupta S, Kamble J, Khajanchi M, Lal P, Malhotra V, Meher R, Mishra A, Mohan LN, Petzold M, Saxena R, Shrivastava P, Singh R, Soni KD, Sural S, Gerdin Wärnberg M. Perceived usefulness of trauma audit filters in urban India: a mixed-methods multicentre Delphi study comparing filters from the WHO and low- and middle-income countries. BMJ Open. 2022;12:e059948. DOI: https://doi.org/10.1136/bmjopen-2021-059948II. Berg J, David S, Bakhshi GD, Basak D, Chatterjee S, Soni KD, Ekelund U, Felländer-Tsai L, Joshipura M, Khan T, Khajanchi M, Mohan LN, Mishra A, Petzold M, Rajan S, Roy N, Singh R, Gerdin Wärnberg M. Effects of trauma quality improvement programme implementation on mortality: A nonrandomised controlled trial. DOI: https://doi.org/10.1101/2024.05.27.24307748 [Manuscript Preprint]III. Kapitan E, Berg J, David S, Mohan LN, Felländer-Tsai L, Chatterjee S, Ekelund U, Roy N, Petzold M, von Schreeb J, Soni KD, Rajan S, Khajanchi M, Gerdin Wärnberg M. The effect of trauma quality improvement programme implementation on quality of life among trauma patients in urban India. Injury. 2025;56:112333. DOI: https://doi.org/10.1016/j.injury.2025.112333IV. Berg J, Alvesson HM, Gerdin Wärnberg M, Roy N, Ekelund U, David S. "We saw them as stories" - Understanding how multidisciplinary case review contributes to quality improvement in trauma care. DOI: https://doi.org/10.1101/2025.08.08.25333298 [Manuscript Preprint]</p
International classification of functioning, disability and health in the sickness certification process
No full textBackground: In Sweden's healthcare system, sickness certification has long been a routine responsibility of general practitioners-yet its complexity continues to challenge patients, clinicians, and policymakers. To improve assessments of work-related disability and address rising sick leave rates, the International Classification of Functioning, Disability and Health (ICF) was introduced into the Swedish sickness certification process in the early 2000s. This thesis aims to examine how the quality of information in sickness certificates has changed over time and to explore whether, and how, the ICF can support improvements of the sickness certification process, with a particular focus on the documentation and assessment of work-related disability due to depression and chronic pain.Methods: The thesis comprises four descriptive studies using both quantitative and qualitative methods.Study I is a repeated cross-sectional study of 783 sickness certificates issued in primary health care in 2004, 2009 and 2012. A structured, expert-based protocol was used to assess changes in certificate quality. Logistic regression models were applied to examine associations between certificate quality and patient-, sick-leave-, and physician-related factors.Studies II and III evaluated the applicability of the ICF and selected ICF Core Sets for coding information on work-related disability in certificates for depression, long-term musculoskeletal pain and fibromyalgia. The qualitative method of ICF linking was used. Study II used a small local sample of sickness certificates (n = 59), while Study III used a large national sample (n = 400).Study IV applied the same ICF linking method to analyse the content of the Test Instrument for Profile of Physical Ability (TIPPA), evaluating its relevance in relation to ICF Core Sets for chronic widespread pain.Results: Study I showed a statistically significant improvement in the quality of sickness certificates during the eight years. Despite these improvements, the overall quality remained low. Diagnosis and sick leave duration were associated with the quality in 2004/2009, and patient's sex in 2012. The structured method developed for assessing certificate quality has the potential to be useful in future evaluations.Study Il revealed that the ICF was suitable for coding 75-83 percent of the information in the sample. However, some relevant categories were missing from the ICF Core Set for disability evaluation in social security, including b130 Energy and drive functions and b134 Sleep functions.In Study III, coding to the ICF remained consistent (78-85 percent), and additional relevant but missing categories in the corresponding ICF Core Sets were identified. For the ICF Core Set for depression, these included b455 Exercise tolerance functions and b230 Hearing functions. For the ICF Core Set for chronic widespread pain, missing categories included b144 Memory functions, b770 Gait pattern functions, and b230 Hearing functions.Study IV showed that TIPPA effectively captured key physical aspects of functioning, particularly within the activity and participation component. However, psychological domains essential for assessing work ability-such as problem-solving and stress management-were not covered.Conclusion: The findings demonstrate an overall improvement in the quality of sickness certificates over the study time, with patient's sex emerging as a factor associated with the quality in the latest sample. The ICF framework proved suitable for capturing work-related disability information in certificates for depression, long-term musculoskeletal pain, and fibromyalgia, covering the majority of relevant concepts. Adjustments to existing ICF Core Sets are recommended to enhance their applicability in sickness certification. While TIPPA assesses physical functioning, it does not fully address psychological domains critical for evaluating work ability in cases of long-term musculoskeletal pain.List of scientific papersI. Fresk M., Grooten, W., Backlund, L., Arrelöv, B., Skånér, Y., Henriksson, P., Kiessling, A. Quality Assessment in Sickness Certificates - Changes Over an Eight-Year Period in Sweden and Associated Factors. [Manuscript]II. Fresk, M., Grooten, W., Brodin, N., Backlund, L., Arrelöv, B., Skånér, Y., Kiessling, A. (2023). Mapping information regarding the work-related disability of depression and long-term musculoskeletal pain to the International Classification of Functioning, Disability and Health and ICF Core Sets. Frontiers in rehabilitation sciences, 4, 1159208. https://doi.org/10.3389/fresc.2023.1159208III. Fresk, M., Grooten, W., Brodin, N., Backlund, L., Arrelöv, B., Skånér, Y., Kiessling, A. (2024). Exploring international classification of functioning, disability and health applicability for coding work-related disability: a study on depression and fibromyalgia in Swedish sick leave certificates. Journal of rehabilitation medicine, 56, jrm36886. https://doi.org/10.2340/jrm.v56.36886IV. Fresk, M., Brodin, N., Grooten, W. J., Joseph, C., Kiessling, A. (2019). Mapping a measure of physical ability for persons with long-term musculoskeletal pain to the ICF and ICF Core Sets. European journal of public health, 29(2), 286-291. https://doi.org/10.1093/eurpub/cky135</p
Detection and prevention of bacteria in bone and joints - on microbial diagnostics and cloxacillin prophylaxis
No full textBone and joint infections (BJI) are challenging concerning diagnostics, treatment, and outcome. While physicians struggle to diagnose and manage BJI, patients often suffer pain, disability and require long-term antibiotic treatments.To increase the knowledge on which bacteria cause BJIs, a retrospective study on 363 patients from Södersjukhuset was performed. Staphylococcus aureus was the dominating cause, even in patients receiving antibiotics to avoid staphylococci, through cloxacillin prophylaxis. However, in 25% of the cases the bacterial aetiology could not be defined. 16S rDNA sequencing has been proposed to reduce the proportion of cases where the bacterial cause of BJI is not found. To assess this, a prospective study was designed, analysing 28 episodes of post-operative BJI, comparing conventional culture with sequencing, in patients on antibiotics. Sequencing was helpful in 5/28 cases (18%). Multiple samples per patient helped distinguishing contaminating bacteria from bacteria causing infection .Cloxacillin prophylaxis is dosed with little consideration of kidney function, duration of surgery or weight. Hypothesising that this dosing manner leads to insufficient concentrations to inhibit the growth of methicillin susceptible staphylococci in certain individuals, 204 patients subjected to primary hip or knee arthroplasty were prospectively assessed concerning free cloxacillin concentrations throughout surgery. Fifteen percent of the patients had concentrations 90, weight >100 kg and duration of surgery 90-120 minutes from the pre-operative dose of cloxacillin. Results also showed that the present guideline for dosing was poorly followed. To propose a new guideline for cloxacillin dosing in joint arthroplasty, safeguarding adequate levels throughout surgery in all patients, pharmacodynamic modelling was done, showing that even with optimal guideline adherence, patients would be at risk of inadequate cloxacillin levels during surgery. Monte Carlo simulation concluded that administering cloxacillin via a short bolus infusion of 1 to 2 grams before surgery, followed by a continuous infusion of 1 gram/hour , could ensure adequate levels in all patients. This without increasing the total dose given, compared to present dosing guidelines.List of scientific papersI. Wallander K, Jorup-Rönström C, Ullberg M, Törnblom I, Ottosson C, Giske CG. Etiology of bone and joint infections: a case series of 363 consecutive patients from an orthopaedic infection unit. Infect Dis (Lond). 2016 Aug, 48(8), 618-625. https://doi.org/10.1080/23744235.2016.1183814II. Wallander K, Vondracek M, Giske CG. Evaluation of multi-sample 16S ribosomal DNA sequencing for the diagnosis of postoperative bone and joint infections during antimicrobial treatment. BMC Research Notes. 2022 Mar(22);15(1):113. https://doi.org/10.1186/s13104-022-05992-7III. Wallander K, Beijer G, Eliasson E, Giske CG, Ponzer S, Söderquist B, Eriksen J. Is current guidance for cloxacillin prophylaxis dosages in hip and knee arthroplasty adequate? Evidence from a prospective Swedish cohort. Journal of antimicrobial chemotherapy. [Submitted]IV. Beijer G, Wallander K, Söderquist B, Giske CG, Breuer O, Eriksen J, Eliasson E. Optimising cloxacillin prophylaxis in hip and knee arthroplasty based on population pharmacokinetics of unbound plasma concentrations. [Manuscript]</p
Cell signaling in blood and lymphatic vessel malformation : insights into the mechanisms of human genetic diseases
No full textDysregulation of mechanisms controlling the development of the blood and the lymphatic vascular systems may cause structural abnormalities known as vascular malformations. These vascular malformations can be very limiting to the patients, not only because of symptomatology but also because of the social impact these anomalies have in their lives. Therefore, it is important to develop new therapies that can improve the quality of life of these patients.An example of vascular malformation affecting the blood vasculature is the arteriovenous malformation (AVM), which is a direct shunt between arteries and veins. AVMs are a hallmark of Hereditary Hemorrhagic Telangiectasia (HHT), an autosomal dominant disease where patients suffer from frequent bleedings and cardiac problems. HHT is caused by mutations in different members of the BMP9/10 signaling cascade, that regulates homeostatic angiogenesis.An abnormal vascular development can also cause lymphatic vessel overgrowth, like in Lymphangioleiomyomatosis (LAM), where patients can suffer from respiratory failure. LAM is caused by mutations in TSC1/2, an endogenous inhibitor of the mTORC1 pathway, that controls metabolism, cellular growth and proliferation.Despite knowing some of the mutations causing vascular abnormalities, it remains unclear how these abnormalities arise. Increasing the knowledge about how vascular anomalies emerge is key to developing effective therapeutic approaches.This thesis contributes to understanding the molecular mechanisms underlying AVM development. In Paper I, by using different mouse models of HHT and targeting the expression of different components of mTORC1 in endothelial cells (EC), we studied the contribution of EC mTORC1 to the development of AVMs and we concluded that, despite AVMs presented increased mTORC1 activity, EC mTORC1 is not driving either initiation or expansion of these malformations. In Paper III, by using a mosaic mouse model of HHT targeting endoglin (ENG) expression in ECs and single cell RNA sequencing, we studied the transcriptional changes associated with AVM development and we concluded that ENG modulation has a differential impact in the transcriptomes of arterial cells, tip cells and capillary ECs. The capillary ECs were the most affected by ENG modulation, which suggests they drive AVM development.In Paper IV, we refined a clearing protocol for the eye and, through Light-Sheet Fluorescence Microscopy, obtained a 3D picture of the complete eye vasculature. In Paper II, we studied the development of abnormalities in lymphatic vessels. By targeting the expression of Tsc1 in ECs and lymphatic ECs (LECs), we studied the role of EC mTORC1 in the development of the dermal lymphatic vasculature and concluded that increased EC or LEC mTORC1 activity can cause overgrowth of the dermal lymphatic vasculature and abnormal collecting vessel development.Taken together, the results presented in this thesis contribute to a better understanding of the molecular mechanisms underlying vascular malformations.List of scientific papersI. Antonio Queiro-Palou, Yi Jin and Lars Jakobsson. Genetic and pharmacological targeting of mTORC1 in mouse models of arteriovenous malformation expose non-cell autonomous signalling in HHT. Angiogenesis, 2025, 28, 6. https://doi.org/10.1007/s10456-024-09961-5II. Antonio Queiro-Palou, Anna Kawaguchi, Taija Mäkinen and Lars Jakobsson. mTORC1-driven lymphatic vessel hyperplasia relies on active lymphangiogenesis. [Manuscript]III. Maria Garcia-Collado, Antonio Queiro-Palou, Jaromir Mikes, Liqun He, Yi Jin, Christer Betsholtz, Lars Muhl, and Lars Jakobsson. Dissecting mechanisms of HHT-related arteriovenous malformation through single-cell transcriptomics. [Manuscript]IV. Luc Thomas Krimpenfort, Maria Garcia-Collado, Tom van Leeuwen, Filippo Locri, Anna-Liisa Luik, Antonio Queiro-Palou, Shigeaki Kanatani, Helder André, Per Uhlen and Lars Jakobsson. Anatomy of the complete mouse eye vasculature explored by light- sheet fluorescence microscopy exposes subvascular-specific remodeling in development and pathology. Experimental Eye Research, 2023, 237. https://doi.org/10.1016/j.exer.2023.109674</p