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Zygomaticomaxillary complex fractures : aspects of diagnosis, treatment and sequelae
No full textZygomaticomaxillary Complex (ZMC) fractures are common types of midfacial fractures and are mostly sustained by young males. Surgical repair consists of either anatomical reconstruction with plates and screws or reduction without internal fixation. In the absence of functional sequelae, many reconstructions are purely cosmetic and are performed to restore facial symmetry. The main purpose of this thesis was to present novel findings and new perspectives to aid surgeons in deciding whether to reconstruct ZMC fractures.Paper I presented the loss of lower eyelid bags as a previously unknown complication to orbital floor reconstructions. Sixty adult patients with surgical reconstruction of isolated blow-out fractures using either pre- or retro-septal transconjunctival incisions were included and compared with a control group of 47 adult patients with conservative management. The patients were all photographed and a grading system was created to describe bags of the lower eyelids. All 25 patients with surgical treatment and a visible lower eyelid bag on the non-injured side of the face suffered lower eyelid bag asymmetry compared to 1 out of 11 patients with conservative management (p Paper II introduced the Volume Difference Along the External Surface (VDAES) as a novel method of measuring zygomatic bone asymmetry. The computer tomography (CT) scans of 50 male and 50 female patients without fractures or deformities to the facial skeleton were analyzed using iPlan Cranial (Brainlab AG, Germany). Measuring consisted of image alignment, creation of a 3-dimensional (3D) object of the zygomatic bone, mirroring across the midline and manual highlighting of asymmetry on axial image cuts. Median VDAES was 1.48 cm3 with a significant difference between males and females (p=0.003). There was no significant association with zygomatic bone volume. The inter-class coefficient (ICC) value for both intra- and inter-rater reliability was 0.99, suggesting that the method, if further developed, has the potential of aiding surgeons in evaluating zygomatic bone asymmetry.Paper III described long-term outcomes of patients with ZMC fractures and different treatments by accounting for the perspectives of both patients and surgeons. Patients with unilateral ZMC fractures between 2007-2018 were invited to follow-ups where they submitted a questionnaire, underwent a clinical examination and were photographed. A review panel consisting of three experienced facial fracture surgeons evaluated all photographs and CT scans. The study sample had a follow-up duration of more than 6 years and consisted of 180 patients, of which 137 were treated by surgery. There were marked differences in the ways patients and surgeons perceived sensory disturbance, trismus and diplopia. Surgeons were more prone to detect malar asymmetry on photographs (28.9%) or to predict asymmetry on CT scans (28.9%) compared to patient's reports (11.7%). Predicted asymmetry on CT scans did not correlate to photo-detected or patient-reported asymmetry.Paper IV described long-term outcomes of patients with ZMC fractures and surgical treatment. Patients with unilateral ZMC fractures between 2007-2018 were invited to follow-ups where they submitted a questionnaire, underwent a clinical examination and were photographed. A review panel consisting of three experienced facial fracture surgeons evaluated all photographs and CT scans. The study sample had a follow-up duration of more than 6 years and consisted of 137 patients, of which 108 were treated by open reduction with internal fixation (ORIF) and 28 by closed reduction (CR). Patient satisfaction was high (97.8%) and dissatisfaction was mainly related to hypesthesia. On CT scans, surgeons predicted higher rates of malar asymmetry in patients with 1-point fixations (31.6%). On questionnaires and photographs, malar asymmetry was more common in patients with 3-point fixations (14.5% and 38.2%, respectively).Paper V explored the loss of lower eyelid bags and resulting lower eyelid bag asymmetry in patients with ZMC fractures and different treatments. Patients with unilateral ZMC fractures between 2007-2018 were invited to follow-ups where they submitted a questionnaire and were photographed. The study sample had a follow-up duration of more than 6 years and consisted of 104 patients with a visible lower eyelid bag on the non-injured side of the face. Lower eyelid bag asymmetry was seen in 42 patients (40.4%) and was significantly associated with a lower eyelid incision (p=0.008). Among patients with lower eyelid bag asymmetry, 16 were not treated with a lower eyelid incision.In conclusion, this thesis presented lower eyelid bag loss as a cosmetic complication that is primarily associated with the lower eyelid incision and introduced a concept by which zygomatic surface anatomy can be evaluated. Furthermore, differences were observed in the ways long-term sequelae were perceived, in particular malar asymmetry. Overall, the findings suggest that selected ZMC fractures can be treated using less invasive methods. More knowledge is needed to understand the dynamics of soft tissue changes and the different ways skin and subcutaneous tissue react to trauma and surgery.List of scientific papersI. Rahbin S, Liakos A, Alinasab B. Loss of Malar Bags in Lower Eyelid in Orbital Blow Out Fracture Reconstruction Following Pre- or Retro-Septal Transconjunctival Incision. J Craniofac Surg. 2020 May/Jun;31(3):769-771. https://doi.org/10.1097/scs.0000000000006103II. Rahbin S, Toufani T, Al-Khabbaz AM, Lindblom J, Sunnergren O, Darabi H, Qureshi AR, Alinasab B. The Volume Difference Along the External Surface of the Zygomatic Bone: A Novel Method of Measuring Zygomatic Bone Asymmetry. J Craniofac Surg. 2022 Mar-Apr 01;33(2):463-468. https://doi.org/10.1097/scs.0000000000008186III. Rahbin S, Sunnergren O, McBride E, Darabi H, Alinasab B. Differences Between Patient and Surgeon Perspectives: A Long-Term Follow-Up of 180 Patients With Zygomaticomaxillary Complex Fractures Following Either Conservative or Surgical Treatment. Craniomaxillofac Trauma Reconstr. 2024 Dec;17(4):NP121-NP130. https://doi.org/10.1177/19433875231208463IV. Rahbin S, Sunnergren O, McBride E, Darabi H, Alinasab B. Does More Invasive Surgery Result in Higher Patient Satisfaction? A Long-Term Follow-Up of 136 Zygomaticomaxillary Complex Fractures. Craniomaxillofac Trauma Reconstr. 2024 Dec;17(4):NP271-NP280. https://doi.org/10.1177/19433875241286544V. Rahbin S, Sunnergren O, Darabi H, Alinasab B. Loss of lower eyelid bags in patients with zygomaticomaxillary complex fractures. [Manuscript]</p
Labour companionship in practice : systems, perspectives and pathways to scale from eight low- and middle-income countries
No full textLabour companionship is defined as the presence of a person of the woman's choice to support her during labour and birth. A labour companion can be a woman's partner, her relative such as mother, sister or in-laws, a friend, or a doula. A Cochrane review by Bohren and colleagues presented clinical and nonclinical benefits to having a labour companion both to the woman and her baby, including shorter labour durations, less use of analgesia, better 5 minutes APGAR scores for the baby and better reported experiences of care. Given its benefits, labour companionship is a recommended practice, for women who want it and is mentioned in several guidelines including the World Health Organization's recommendations on positive intrapartum care. While the practice offers several benefits, its implementation, especially across low-and- middle-income countries, varies and remains suboptimal in many settings.The aim of this thesis was to increase the understanding of the processes, factors and potential benefits that influence the integration and scalability of labour companionship practices across the macro, meso, and micro-levels of the maternity health system. This thesis was nested under two collaborative implementation projects implemented from 2020 until 2024. The ALERT project (Action Leveraging Evidence to reduce perinatal Mortality and morbidity in Sub- Saharan Africa) was implemented in 16 public and private not-for-profit hospitals in Benin, Malawi, Tanzania and Uganda and the QUALI-DEC project (QUALIty DECision making by women and providers for appropriate use of caesarean section) was implemented in 32 public and private hospitals in Argentina, Burkina Faso, Thailand and Viet Nam. This thesis included a mix of qualitative and quantitative methodologies.Study I was a multi-country study that aimed to understand the implementation maturity of labour companionship across the eight countries before implementation. This study used data from the formative research conducted in ALERT and QUALI-DEC projects to (1) analyse the content of policy documents pertaining to labour companionship, using directed content analysis and (2) describe labour companionship practices and resources available on a facility level. Based on these results, the eight countries were classified into three phases of implementation following subject-specific models by Bohren and colleagues and Bergh and colleagues. Additionally, the physical structure of the maternity ward was illustrated to visually present labour companionship practices across the implementation phases.Study II was a cross-sectional study conducted with 4006 women aiming to assess the associations between having a labour companion and respectful treatment as reported by women in 16 hospitals in Benin, Malawi, Uganda and Tanzania. A validated questionnaire, developed as part of the ALERT project, on respectful treatment and responsiveness was administered to all eligible women before discharge from December 2021 until March 2024. Women were eligible to participate if they had given birth at one of the 16 hospitals to a baby weighing more than 1000 grams and agreed to consent. The women were randomly approached for an interview following a randomisation factor depending on the number of women to be discharged. Linear regression with fixed effects and cluster robust standard errors were conducted to assess the associations between presence of labour companionship and respectful treatment using a factor-weighted respectful treatment score and its sub-scores: maintained respect and dignity, privacy and confidentiality and no physical and verbal abuse. The model was adjusted for age, parity, educational level, mode of birth and the ALERT intervention.Study III was a qualitative study aiming to understand the interactions between maternity care providers, women and their relatives in two hospitals in Southern Tanzania. Eleven natural group discussions were conducted with 21 maternity care providers in the labour and postnatal wards at the two hospitals. Data were analysed following Braun and Clarke's reflexive thematic analysis. Three themes were developed with both semantic and latent content.Study IV was a scalability assessment to understand the factors influencing the scalability of the QUALI-DEC intervention in Argentina, Burkina Faso, Thailand and Viet Nam. Data from the formative research conducted in the four countries were deductively analysed using the framework analysis. The analysis was guided by the scalability assessment tool developed by Zamboni and colleagues and incorporated into the QUALI-DEC intervention. The tool included 34 items covering four dimensions on the attributes of innovation, implementers, adopting organizations and socio-political context.Results suggested that having a companion during labour or birth was associated with reduced levels of physical and verbal abuse in Benin, Malawi, Tanzania and Uganda. However, the coverage of labour companionship across these countries was low (around 39%) and women reported poor experiences of care during labour and birth.Three distinct levels of implementation were identified across the eight countries: pre-implementation (Benin, Thailand and Viet Nam); early implementation (Burkina Faso, Malawi, Tanzania and Uganda); and institutionalisation (Argentina). These implementation levels were shaped by different processes and factors at the meso and macro-levels.At the meso-level, several processes facilitated implementation and scalability, including adaptations to the intervention to better align it with users' needs. These adaptations included reviewing the educational material, introducing structural changes to the maternity ward and addressing physical constraints that limited the inclusion of companions. Organizational norms also shaped care processes, influencing the integration of labour companionship. In Tanzania, gatekeeping practices by maternity ward providers further influenced the integration of labour companions, reflecting a provider-centred model of care that may conflict with women's autonomy.At the macro-level, the broader policy landscape generally portrayed labour companionship as a luxurious add-on rather than a woman's right (except in Argentina). Strict legal frameworks and the absence of clear financing mechanisms limited integration into existing maternity care systems, particularly in Thailand and Viet Nam.These findings underscore that labour companionship is a dynamic process that needs to be negotiated at the macro, meso and micro-levels of the maternity care system. Its integration is shaped by institutional and systemic factors, including policy landscape, availability of adequate physical structures and an aligned organisational culture.List of scientific papersI. El-Halabi S, Pembe AB, Dumont A, Betrán AP, Kaboré C, Chipeta EK, Carroli G, Alvesson HM, Kidanto H, Dossou JP, Annerstedt KS., Beňová L, Gross MM, Waiswa P, Lumbiganon P, Mac QNH, Bohren MA, Hanson C. Towards a universal implementation of labor companionship: A synthesis of the policy and facility environment of eight low-and-middle income countries. Frontiers in health services. 2025 Jul 23;5:1550473. https://doi.org/10.3389/frhs.2025.1550473II. El-Halabi S, Annerstedt KS, Agossou C, Al-Beity FMA, Pembe AB, Wanduru P, Kandeya B, Dossou JP, Molsted-Alvesson H, Bohren MA, Hanson C. Labour companionship and respectful treatment of women during childbirth: a cross-sectional study across 16 hospitals in Benin, Malawi, Tanzania and Uganda. BMJ Public Health. 2025 May 12;3(1):e002462. PMID: 40391249; PMCID: PMC12086892. https://doi.org/10.1136/bmjph-2024-002462III. El-Halabi S, Al-Beity FMA, Hanson C, Pembe AB, Alvesson HM. "Labour under lock: providers' perspectives on relatives' access to two maternity wards in Southern Tanzania". [Manuscript]IV. El-Halabi S, Hanson C, Dumont A, Cleeve A, Alvesson HM, Kaboré C, Carroli G, Lumbiganon P, Mac QNH, Betran AP, Annerstedt KS, Bohren MA, Zamboni K. Planning for scale: analysis of adaptations and contextual factors influencing scale-up of the QUALI-DEC intervention to optimize caesarean section use. Implement Sci Commun. 2025 May 21;6(1):61. PMID: 40400046; PMCID: PMC12093684. https://doi.org/10.1186/s43058-025-00737-6</p
Understanding implementation mechanisms : a cluster-randomized controlled trial in schools
No full textBackground: Within the working population, mental health problems are common. To address this challenge, policies and regulations recommend that workplaces systematically assess the psychosocial work environment and act on identified risks. Evidence-based guidelines exist to support this process, but they are seldom used without implementation strategies. However, knowledge about the effectiveness of implementation strategies is inconsistent, with strategies showing mixed effects across studies and settings. This has led to calls for research on the mechanisms through which implementation strategies produce outcomes.Aim: This thesis aims to understand the effectiveness of implementation strategies and their mechanisms of change. It compares the effectiveness of a multifaceted implementation strategy with a discrete strategy for implementing the Swedish Guideline for the Prevention of Mental Ill-Health at the Workplace. Study 1 focuses on comparing the effectiveness of the multifaceted vs. the discrete strategy on intervention outcomes. Study 2 focuses on comparing the effectiveness of the implementation strategies on guideline fidelity from the implementers' and recipients' perspectives. Study 3 tests whether the multifaceted strategy leads to larger improvements in capability, opportunity, and motivation, and whether these components mediated the effects of the strategy on fidelity. Study 4 used qualitative data to investigate the processes implementers went through with the discrete strategies to achieve fidelity as a downstream outcome.Methods: The thesis is based on two cluster-randomized waiting-list controlled trials conducted in Swedish schools. The first trial (2017-2019, 19 schools) tested a multifaceted strategy including an educational meeting, implementation teams, ongoing training, and small cyclical tests of change. In the second trial (2021-2023, 55 schools), the multifaceted strategy was refined and therefore included an additional strategy in the form of implementation facilitation. In both trials, the multifaceted strategy was compared to an educational meeting, but in contrast to the first trial, the implementation team strategy was also part of the discrete strategy in the second trial. Study 1, conducted within the first trial, used a quantitative design in which data were collected on fidelity, psychosocial work environment, and health at baseline, 6 months, and 12 months using questionnaires. Linear Mixed Models (LMM) were used to compare the effectiveness of the strategies on intervention outcomes, as well as to examine associations between guideline fidelity and intervention outcomes. Study 2, conducted in the second trial, used a mixed-method design. Fidelity from school staff (recipients) was assessed at baseline and 12 months using questionnaires, which were analyzed with LMMs. Fidelity from school management (implementers) was measured via a checklist at 12 months, which was analyzed qualitatively. Study 3 used a quantitative design in which data on fidelity were collected at baseline and 12 months. Data on capability, opportunity, and motivation were collected after the educational meeting, and at 3 and 9 months. LMMs compared the effectiveness of the strategies on fidelity, and mediation analyses were conducted using the PROCESS macro. In Study 4, process tracing with comparative case studies was used to analyze qualitative data, including interviews and documents, from a subsample of 16 implementers. The analysis followed four steps: (1) constructing a process theory of change, (2) gathering data, (3) coding data through qualitative content analysis, and (4) conducting causal analysis to identify mechanisms and contextual conditions.Findings: Study 1 found that the discrete strategy was more effective than the multifaceted strategy in improving the primary outcome of exhaustion and some secondary intervention outcomes at 12 months. Higher guideline fidelity was associated with positive outcomes in the work environment and health-related outcomes. Study 2 found that the multifaceted strategy was more effective than the discrete strategy in improving guideline fidelity from baseline to 12 months. This was supported by the implementer's perspective, showing a larger proportion of schools that achieved fidelity. Study 3 showed that the multifaceted strategy led to larger improvements in capability, opportunity, and motivation. The effect of the multifaceted strategy on fidelity was partially mediated by nine of ten tested mediators, with the largest effects explained by skills and behavioral regulation. Study 4 extended these findings by identifying five strategy-specific pathways that illuminated the more proximal outcomes, or strategy-specific milestones, that preceded the more downstream outcome of fidelity.Conclusion: Schools benefit from using a multifaceted strategy that can engage multiple mechanisms, promote coordination across organizational levels, and support ongoing implementation when implementing this guideline. This thesis contributes knowledge on the effectiveness of multifaceted, multi-level strategies in workplace settings, as well as provides guidance for both researchers and practitioners on how to design, adapt, and evaluate such implementation.List of scientific papersI. A. Toropova†, A. Rödlund†1, C. Björklund, L. Schäfer Elinder, I. Jensen and L. Kwak. The effectiveness of implementing the Guideline for the Prevention of Mental Ill-health Problems at the Workplace on health-outcomes, organizational and social risk factors: a cluster-randomized controlled trial in Swedish schools. Scandinavian Journal of Work, Environment & Health. 2023;49(6):428-38. https://doi.org/10.5271/sjweh.4108II. A. Rödlund, A. Toropova, R. Lengnick-Hall, B.J. Powell, L. Schäfer Elinder, C. Björklund and L. Kwak. A cluster-randomized controlled trial assessing the effectiveness of a multifaceted versus a discrete implementation strategy on fidelity to an Occupational Guideline for the Prevention of Mental Health Problems at the Workplace: a dual perspective from Swedish schools. [Manuscript]III. A. Rödlund, A. Toropova, R. Lengnick-Hall, B.J. Powell, L. Schäfer Elinder, C. Björklund and L. Kwak. Mechanisms of change of a multifaceted implementation strategy on fidelity to a guideline for the prevention of mental health problems at the workplace: a mechanism analysis within a cluster-randomized controlled trial. Imple- mentation Science. 2025;20(1):25. https://doi.org/10.1186/s13012-025-01437-4IV. A. Rödlund, A. Toropova, R. Lengnick-Hall, B.J. Powell, L. Schäfer Elinder, C. Björklund and L. Kwak. The Roads Towards Fidelity: A mixed-method study of mechanisms within a multifaceted implementation strategy. [Manuscript]1 †= These authors have contributed equally to this work and should be given equal credit as first authors</p
New plasma and platelet products : evaluation of hemostatic and functional properties in experimental studies
No full textBlood transfusion plays a vital role in managing major bleeding, preventing mortality, and maintaining hemodynamic stability. Strong evidence indicates that early transfusion significantly improves survival rates. However, access to blood can be challenging in remote hospitals with constrained inventories, during mass casualty events, at the site of injury, or in military operations where proper storage conditions are lacking. Developing new strategies to overcome these challenges is essential to meet transfusion needs. One such strategy involves alternative methods for storing blood components, which can help improve the availability and resilience of the blood supply. Cryopreserved platelets, for example, can be stored frozen for several years, unlike conventional platelets which have a limited shelf life of 5-7 days and are often in short supply. They are intended for use in patients with active bleeding, as they exhibit a pro-coagulant phenotype. Another promising component is dried plasma, which has gained increased attention in the context of emergency preparedness. Although traditionally restricted to military settings, dried plasma is now becoming more widely available, thereby expanding its use in both pre-hospital and in-hospital care. Dried plasma offers logistical advantages, as it does not require a cold chain and remains stable for at least two years at room temperature. With the introduction of new blood components into clinical practice, studies are needed to evaluate not only their individual quality but also their combined hemostatic effects. To address these challenges, this thesis evaluated the hemostatic and functional properties of cryopreserved platelets and dried plasma through a series of experimental studies.In Paper I, we investigated the interplay between platelets and plasma components. Various combinations were tested, including conventional platelets with either fresh frozen plasma or pathogen-inactivated plasma, and cryopreserved platelets with the same two plasma types. Additionally, the quality of each plasma and platelet type was assessed individually. The study revealed that both pathogen-inactivated plasma and cryopreserved platelets demonstrated altered characteristics relative to their respective counterparts. However, only the groups containing cryopreserved platelets showed reduced clot strength in ROTEM, suggesting that platelet quality, rather than differences in coagulation factor levels, plays a more significant role in in vitro clot formation.In Paper II, we explored the possibility of cryopreserving platelets without using the toxic cryoprotectant DMSO, which is the current standard. Interestingly, DMSO-free freezing resulted in a higher platelet count, although the platelets showed slightly more damage compared to those preserved with DMSO. We further tested controlled-rate freezing, which improved the viability of platelets frozen in the absence of DMSO. These findings suggest that DMSO-free cryopreservation, particularly when combined with controlled-rate freezing, may be a promising alternative that avoids the risk of toxicity.In Paper III, we shifted focus to plasma, comparing three differently produced dried plasma products with fresh frozen plasma of respective origin, as controls. The comparison included user evaluations by healthcare professionals from various fields, as well as laboratory tests assessing coagulation factor content and hemostatic function. All dried plasma units could be reconstituted within 10 minutes, with even faster times among experienced users. An advantage deemed important was the ability to deliver the final product in a plastic bag, facilitating transfusion under pressure. Laboratory testing revealed some variation in coagulation factor levels among the plasma types, although all remained within reference ranges. No impairment in clot formation through TEG was observed. Overall, the dried plasma products were feasible for use and supported hemostasis in vitro, highlighting their potential role in emergency preparedness.In Study IV, we assessed the use of dried plasma as a reconstitution medium for cryopreserved platelets, in comparison to regular fresh frozen plasma. The study found that dried plasma was comparable to fresh frozen plasma in this application. Interestingly, platelet concentrations were higher when dried plasma was used. However, phenotypic and functional testing of the final component revealed no significant differences between the two groups. Dried plasma thus appears to be a promising alternative reconstitution medium, particularly due to its fast preparation time and logistical advantages, including room temperature storage. These benefits make it especially suitable for emergency preparedness, including use in remote hospitals, prehospital care settings, and military operations.In summary, this thesis provides valuable insights into the evaluation of alternative platelet and plasma components that can improve transfusion logistics and availability in resource-limited settings. It also highlights the potential implications of combining these components during transfusions, an important consideration for clinical practice. Overall, this research supports efforts to make blood components more accessible in emergencies where transfusions are crucial, yet access is often limited.List of scientific papersI. Sandgren P, Ehn K, Larsson L, Uhlin M, Wikman A. Cryopreserved platelets and amotosalen-treated plasma in an experimental clot formation set-up. Blood Transfus. 2023;21(2):137-145. https://doi.org/10.2450/2022.0279-21II. Ehn K, Wikman A, Uhlin M, Sandgren P. Cryopreserved Platelets in a Non-Toxic DMSO-Free Solution Maintain Hemostatic Function In Vitro. Int J Mol Sci. 2023;24(17):13097. https://doi.org/10.3390/ijms241713097III. Ehn K, Skallsjö G, Romlin B, Sandström G, Sandgren P, Wikman A. An experimental comparison and user evaluation of three different dried plasma products. Vox Sang. Published online 2025 Jan 27. https://doi.org/10.1111/vox.13798IV. Ehn K, Sandgren P, Asplund Högelin K, Wikman A. Improved Platelet Recovery in Cryopreserved Platelets Reconstituted in Freeze-Dried Plasma. Submitted for publication, 2025-09-10. [Manuscript]</p
Clinical and genetic studies of papillary thyroid carcinoma : with focus on telomere maintenance and the microRNA machinery
No full textPapillary thyroid carcinoma (PTC) is generally indolent, but a subset exhibits aggressive behavior driven by molecular alterations. Identifying reliable biomarkers and molecular mechanisms to predict and manage this aggressive subset remains an important unmet clinical need, as current risk stratification tools are insufficient to guide personalized therapy. Reactivation of telomerase reverse transcriptase (TERT), most often via promoter mutations, is a key feature of tumor aggressiveness. Follicular-patterned tumors, including follicular thyroid carcinoma (FTC), pose diagnostic challenges. While epigenetic alterations such as global loss of 5-hydroxymethylcytosine (5hmC) have been proposed as surrogate markers for TERT promoter mutations, their predictive value is limited. Moreover, the regulation of TERT through transcriptional and epigenetic mechanisms, and its interaction with other oncogenic pathways in PTC, remain incompletely understood. In parallel, dysregulation of microRNAs (miRNAs) has been implicated in tumor progression, invasion, and metastasis in multiple cancers, including thyroid carcinoma, highlighting their potential as biomarkers and therapeutic targets.In Paper I, immunohistochemical (IHC) detection of 5hmC was assessed as a potential predictive marker for TERT promoter mutations in FTC. Using two antibody clones across 29 tumors with known TERT status, 5hmC IHC showed low sensitivity for predicting TERT promoter mutations, despite prior associations in PTC. These results indicate that global 5hmC loss is not a reliable predictor in follicular thyroid tumors, highlighting the need for more direct molecular assessments of TERT activation.Paper II identified the transcription factor, ETS homologous factor (EHF), as a critical mediator linking BRAFV600E signaling to TERT transcription. In analyses of PTC cohorts from public databases and Karolinska, high EHF expression was associated with BRAFV600E mutation, TERT promoter mutations, worse clinical behaviors, and shorter survival. Functional assays in vitro demonstrated that EHF overexpression increased TERT expression in cells with BRAFV600E and TERT promoter mutations, whereas BRAFV600E knockdown reduced both EHF and TERT expression levels. Chromatin immunoprecipitation suggested direct EHF binding to the mutant TERT promoter, providing mechanistic insight into transcriptional TERT activation in PTC.Paper III explored alternative mechanisms of TERT activation, including promoter hypermethylation and copy number gains. Hypermethylation at promoter specific CpG sites and copy number gains were associated with elevated TERT expression, advanced disease, and poor survival, even in tumors lacking TERT promoter mutations. Integrative analyses indicate that promoter mutations, promoter hypermethylation, and copy number gains represent complementary mechanisms that all are associated with TERT activation, contributing to PTC aggressiveness.In Paper IV, post-transcriptional regulation by miRNAs was examined in relation to PTC aggressiveness. DICER1 expression was downregulated in PTC, particularly in advanced stages, and associated with widespread miRNA dysregulation. Among these, miR-1179, miR-126-5p, and miR-139-5p were consistently associated with aggressive clinical features and poor survivals in two PTC cohorts. Notably, reduced miR-139-5p expression retained prognostic value in TERT promoter wild- type tumors, suggesting its potential as an independent prognostic biomarker and therapeutic target. Functional enrichment analyses of predicted targets implicated these miRNAs in key pathways regulating proliferation, survival, and cell motility.Collectively, these studies provide a comprehensive view of TERT regulation in PTC, highlighting the multifaceted mechanisms driving tumor aggressiveness. While global 5hmC loss is not a reliable marker for TERT promoter mutations in follicular-patterned tumors, transcriptional regulation via the ETS factor EHF, TERT promoter hypermethylation, and copy number gains represent complementary pathways contributing to TERT activation and poor clinical outcomes in PTC. Furthermore, dysregulation of specific miRNAs offers insight into post- transcriptional control of PTC progression and identifies potential prognostic biomarkers. Together, these findings advance our understanding of molecular determinants of aggressiveness in thyroid cancer and underscore the importance of integrated molecular and epigenetic profiling for risk stratification and precision treatment.List of scientific papersI. Martin Hysek#, Samuel L Hellgren, Vincenzo Condello, YIYI XU , Catharina Larsson, Jan Zedenius, C Christofer Juhlin. 5hmC immunohistochemistry: A predictor of TERT promoter mutational status in follicular thyroid carcinoma? J Histochem Cytochem. 2023 Aug;71(8):451-458. https://doi.org/10.1369/00221554231190437II. YIYI XU, Jiwei Gao, Na Wang, Jan Zedenius, Inga-Lena Nilsson, Weng- Onn Lui, Dawei Xu, C Christofer Juhlin, Catharina Larsson", Ninni Mu#. BRAF-induced EHF expression affects TERT in aggressive papillary thyroid cancer. J Clin Endocrinol Metab. 2025 Feb 18;110(3):693-705. https://doi.org/10.1210/clinem/dgae589III. YIYI XU#, Jiwei Gao, Vincenzo Condello, Weng-Onn Lui, C Christofer Juhlin, Ninni Mu, Catharina Larsson#. Epigenetic and genetic mechanisms of TERT activation in papillary thyroid carcinoma. [Manuscript]IV. YIYI XU#, Ninni Mu, Vincenzo Condello, C Christofer Juhlin, Weng- Onn Lui, Catharina Larsson#. Identification of clinically relevant microRNAs in papillary thyroid carcinoma. [Manuscript]# Corresponding author</p
Biodistribution of extracellular vesicles in physiological and pathological conditions
No full textEVs are membrane-enclosed nanostructures released by various cell types and serve as critical mediators of intercellular communication. Their ability to transport proteins, nucleic acids, and lipids has positioned EVs as promising biomarkers and delivery vehicles in diagnostics and therapeutics. However, a comprehensive understanding of their in vivo biodistribution, clearance, and immunomodulatory potential remains limited, hindering translational progress. The work described in this thesis summarizes recent advances in EV engineering, labeling, biodistribution analysis, and their roles under physiological and pathological conditions.To address limitations in EV analysis, luciferase-based endogenous labeling systems were optimized for sensitive and specific EV detection. Among several luciferases tested, NanoLuc and ThermoLuc demonstrated superior luminescence, pH stability, and suitability for in vivo applications. Fusion of these luciferases to tetraspanins enabled efficient EV incorporation without altering vesicle morphology, size, or producer cell viability. Importantly, CD63-NanoLuc provided high signal intensity and sensitivity. In contrast, CD63-ThermoLuc exhibited stronger EV specificity with minimal soluble background signal, thus offering advantages in unpurified systems. Luciferase-labelled EVs enabled quantification of vesicle secretion kinetics under different media conditions and facilitated assessment of cellular uptake in various recipient cell lines.In vivo tracking of luciferase-labelled EVs revealed rapid EV clearance from circulation following intravenous administration, with a half-life of less than 2 minutes in plasma. The liver and spleen represented major uptake sites, with over 90% of EVs accumulating in these organs within minutes. Systemic biodistribution was route-dependent; intravenous and intraperitoneal administration effectively delivered EVs to peripheral organs, while subcutaneous and oral routes showed limited systemic dispersion.To further investigate the fate of injected EVs, we employed high-resolution imaging flow cytometry methods and discovered that EV clearance is mediated, in part, by transient binding to platelets and red blood cells. These interactions peaked within 5 minutes post-injection and coincided with a drop in circulating blood cell counts, suggesting clearance via phagocytic uptake in the liver and spleen. Co-localization of EVs with platelets and red blood cells in macrophages supports a hitchhiking-based mechanism for EV sequestration. We hypothesized that by changing the affinity of EVs to different blood components, their clearance will be drastically altered. To test this, EVs were engineered to display albumin- binding domains. This modification significantly reduced their interaction with blood components, thereby extending circulation time and offering a strategy to enhance delivery efficiency.Having established a foundational understanding of EV pharmacokinetics in physiological conditions, we wanted to determine how inflammatory conditions impacted these properties. In LPS-primed mice, circulating EV levels remained elevated up to 24 hours post-injection, a stark contrast to non-inflammatory conditions. Enhanced accumulation was observed in immune-rich organs, including the liver, spleen, lungs, kidneys, and brain. Flow cytometric analysis demonstrated increased EV association with myeloid cells, particularly with macrophages and neutrophils, and adaptive immune cells under inflammatory conditions. However, this increased uptake did not translate to improved cytosolic delivery of functional protein cargo, suggesting that endosomal escape remains a major bottleneck in EV-mediated delivery.Given the clear differences in the fate of injected EVs between physiological and inflammatory conditions, we were interested in studying the functional properties of EVs derived from pathological sources. Tumor-derived EVs (tEVs) from melanoma cells play a key role in hematopoietic dysregulation and immune suppression. These tEVs contain angiogenesis-related factors like VEGF and immunomodulatory chemokines. tEV injection in mice induced splenomegaly, extramedullary hematopoiesis, and expansion of myeloid-derived suppressor cells (MDSCs) and erythroid progenitors, mimicking tumor-bearing profiles. Heat inactivation or VEGF pathway blockade prevented these effects, highlighting VEGF as a critical driver of immunosuppressive remodeling.These findings emphasize the complexity of EV biodistribution, the influence of inflammatory and tumor microenvironments, and the utility of engineered luciferase-EVs in quantitative and mechanistic studies. The dual challenges of rapid clearance and limited functional cargo release highlight the need for continued development of EV modifications to enhance stability, targeting, and delivery efficiency. Understanding and manipulating EV interactions with the immune system and blood components is essential for their successful application in therapeutic settings.List of scientific papersI. Gupta, Dhanu, Xiuming Liang, Svetlana Pavlova, Oscar P. B. Wiklander, Giulia Corso, Ying Zhao, Osama Saher, Jeremy Bost, Antje M. Zickler, Andras Piffko, Cecile L. Maire, Franz L. Ricklefs, Oskar Gustafsson, Virginia Castilla Llorente, Manuela O. Gustafsson, R. Beklem Bostancioglu, Doste R. Mamand, Daniel W. Hagey, André Görgens, Joel Z. Nordin, and Samir EL Andaloussi. 2020. "Quantification of Extracellular Vesicles in Vitro and in Vivo Using Sensitive Bioluminescence Imaging." Journal of Extracellular Vesicles 9(1). https://doi.org/10.1080/20013078.2020.1800222II. Andre Görgens*, Svetlana Pavlova*, Doste R. Mamand, Daniel W. Hagey, Xiuming Liang, Yesid Estupiñan Velasquez, Wenyi Zheng, Guannan Zhou, Risul Amin, Antje M. Zickler, Scott Bonner-Harris, Miina Ojansivu, Manuela O. Gustafsson, Oliver G. Hayes, Oscar P. B. Wiklander, Manuchehr Abedi-Valugerdi, Samantha Roudi, Molly M. Stevens, and Samir EL Andaloussi. Injected Extracellular Vesicles and other nanoparticles hitchhike on Erythrocytes and Platelets from circulation towards organ clearance. [Manuscript]* shared first authorshipIII. Svetlana Pavlova, Doste R. Mamand, André Görgens, Antje M.Zickler, Wenyi Zheng, Xiuming Liang, Oscar Wiklander, Manuchehr Abedi- Valugerdi, Elien Van Wonterghem, Junhua Xie, Zheyu Niu, Guannan Zhou, Roosmarjin E. Vanderbroucke, Dhanu Gupta, Samir El Andaloussi Systemic Inflammation Modulates Clearance and drives Extra-Hepatic Distribution of Extracellular Vesicles. [Manuscript]IV. Doste R. Mamand, Safa Bazaz, Dara K. Mohammad, Xiuming Liang, Svetlana Pavlova, Carsten Mim, Susanne Gabrielsson, Joel Z. Nordin, Oscar P. B. Wiklander, Manuchehr Abedi-Valugerdi, Samir El- Andaloussi. Extracellular vesicles originating from melanoma cells promote dysregulation in haematopoiesis as a component of cancer immunoediting. J Extracell Vesicles. 2024 Jul;13(7):e12471. https://doi.org/10.1002/jev2.12471</p
Reaching cancer survivors where they are : effects and experiences of live-remote exercise
No full textThe number of cancer survivors is steadily increasing, yet many experience impaired health outcomes and reduced health-related quality of life. Exercise has been shown to provide important benefits for cancer survivors, including improvements in health-related quality of life. Despite this evidence, physical activity levels remain low, partly due to individual barriers such as treatment- related side effects, lack of time, and distance, as well as limited availability of suitable services. This highlights the need to develop accessible exercise programmes. Supervised, virtually delivered exercise has the potential to overcome such barriers by extending reach while maintaining professional support. However, evidence regarding the efficacy of this delivery format, and survivors' experiences of exercising in this context, remains limited. The overall aim of this thesis was to evaluate the effects of a live-remote exercise intervention for survivors of breast, prostate, and colorectal cancer, and to explore participants' experiences of exercising in this format, in order to inform the development of accessible exercise programmes that support healthy survivorship.Study I evaluated the effects of a live-remote exercise intervention in individuals who had completed curative treatment for breast, prostate, or colorectal cancer. In this randomised controlled trial, 200 participants were allocated to either usual care or a 12-week live-remote exercise intervention. In small groups of up to eight, participants exercised twice weekly for 60 minutes under the virtual supervision of an upskilled personal trainer. The results showed no significant effect on overall health-related quality of life. However, significant improvements were observed in physical activity levels, physical functioning, cardiorespiratory fitness, and upper body strength post-intervention, although most effects were not maintained at the six month follow-up.Study II explored which subgroups of cancer survivors benefited most from the intervention by conducting an exploratory subgroup analysis of the trial data from Study I. Demographic and clinical characteristics, including age, sex, body mass index, cancer type, chemotherapy, and endocrine therapy, were examined as potential moderators of intervention effects, alongside baseline health status. The results suggested that sex and endocrine therapy moderated intervention effects, with women and participants receiving endocrine therapy experiencing greater improvements in health-related quality of life, fatigue, sleep disturbances, and strength. In addition, participants with poorer baseline health, including lower health-related quality of life, lower physical fitness, or higher fatigue, appeared to derive greater benefit.Study III explored cancer survivors' experiences of engaging in exercise within a live-remote format. In this qualitative study, 22 participants from the 12-week intervention took part in four online focus groups. Discussions were analysed using reflexive thematic analysis with an abductive approach, whereby themes were identified inductively and then mapped onto the Capability, Opportunity, Motivation (COM-B) model to help understand factors influencing exercise engagement. Nine themes were identified, reflecting exercise readiness following treatment, accessibility and professional support, peer support, life circumstances, the role of exercise in recovery and daily routines, caring for self and others, positive experiences exceeding expectations, and the development of exercise habits. From the participants' perspectives, live-remote exercise was seen to overcome barriers to participation and to provide both psychosocial and exercise support, thereby facilitating engagement and integration of exercise into daily life.In conclusion, this thesis demonstrates that a 12-week, live-remote supervised exercise intervention led to significant improvements in physical activity, physical function, fitness, and strength among cancer survivors. Although no significant effect was observed for overall health-related quality of life, meaningful gains were seen in the physical functioning domain. Most improvements observed post-intervention were not maintained at six months. Participants with lower health-related quality of life, reduced physical function, or higher fatigue at baseline appeared to derive greater benefit, as did female participants and those receiving endocrine therapy. Qualitative findings highlighted factors that facilitated exercise engagement, including professional supervision, accessibility, and the group format.Looking ahead, greater attention is needed on how virtually delivered exercise can be optimised to achieve benefits comparable to on-site interventions. Furthermore, strategies to help cancer survivors sustain exercise beyond the end of structured support will be essential to maintain long-term health gains. Taken together, these results suggest that live-remote, supervised exercise can support meaningful short-term health benefits, enhance engagement, and improve access to exercise for cancer survivors, thereby contributing to healthy survivorship.List of scientific papersI. Kotte M, Bolam KA, Altena R, Cormie P, Wengström Y, Mijwel S. Effects of live-remote exercise on quality of life and other health- related outcomes in cancer survivors: a randomised controlled trial. Journal of Cancer Survivorship. 2025. https://doi.org/10.1007/s11764-025-01845-xII. Kotte M, Mijwel S, Bolam KA, Altena R, Wengström Y. Who benefits most? Subgroup effects in a randomised controlled trial of live- remote exercise for cancer survivors. [Submitted]III. Kotte M, Ringborg CH, Wengström Y. The experience of live-remote exercise-perspectives after cancer treatment. Supportive Care in Cancer. 2024;32(8):526. https://doi.org/10.1007/s00520-024-08736-4</p
Beyond effectiveness : implementation factors influencing uptake of IV iron for anaemia in pregnancy in Nigeria
No full textIntroductionAnaemia in pregnancy (AIP) is a major global health challenge, affecting 37% of women worldwide and up to 56% in Nigeria, leading to increased risk of developing complications such as postpartum haemorrhage, maternal death, preterm birth, low birth weight, and long-term neurodevelopmental problems. Beyond these health risks, AIP also causes fatigue and low productivity, adding financial strain on families, especially in contexts where healthcare expenses are largely out-of- pocket.Iron deficiency anaemia (IDA) is the leading cause, yet oral iron, the standard treatment, often fails due to poor adherence from the daily pill burden, forgetfulness and gastrointestinal side effects. Intravenous (IV) iron is a safe, rapid, and effective alternative, particularly for women who cannot tolerate or do not respond to oral iron. Despite its benefits, the use of IV iron remains limited in Nigeria, with little understanding of how to translate its evidence of effectiveness into practice. For widespread use, IV iron must be acceptable to users and providers, feasible to deliver safely with adherence to protocols, and offer better health outcomes at a good value, particularly in a health system dominated by out-of-pocket payments.Aim To increase the understanding of the implementation factors and how they influence key implementation outcomes related to the uptake of IV iron therapy for anaemia in pregnancy in Nigeria.MethodsThis doctoral research was embedded within the context of an open-label Intravenous versus Oral Iron for Iron Deficiency Anaemia in Pregnant Nigerian Women (IVON) trial, a hybrid type 1 effectiveness-implementation study conducted in Kano and Lagos States, Nigeria. The doctoral research, which focused on the implementation arm of the trial, was structured around four mixed-method studies. Study I was an exploratory qualitative study conducted in the pre-implementation phase to explore the acceptability of IV iron among diverse stakeholders. Among purposively selected and consenting participants, 12 focus group discussions (with 140 participants) and 29 key informant interviews were conducted across 10 healthcare facilities. Transcribed data were analysed deductively using the Theoretical Framework of Acceptability. Studies II, III, and IV were conducted during the implementation phase of the IVON trial in 11 facilities.Study II was a repeated cross-sectional survey of trained healthcare providers (HCPs), with a longitudinal component for a subset, to assess how their perceptions of IV iron's acceptability and feasibility varied by provider characteristics and changed over time. The Acceptability of Intervention Measure (AIM) and Feasibility of Intervention Measure (FIM) survey tools were used at baseline (August 2021) and endline (May 2023) to collect data. Analysis was conducted using descriptive statistics, independent and paired t-tests, and ANOVA. In Study III, a sequential explanatory mixed-method design was employed to assess fidelity. Quantitatively, alternate IV iron administrations were directly observed using a checklist, with adherence compared across facilities and states. Qualitatively, 14 in-depth interviews (IDIs) were conducted with HCPs across nine facilities, with transcribed data analysed using deductive thematic analysis based on the Conceptual Framework of Implementation Fidelity. Both qualitative and quantitative data were analysed in NVivo 12 Plus, while Stata version 17.0 was used for the quantitative studies.In Study IV, a decision tree model was used to assess the cost-effectiveness of IV iron (Ferric Carboxymaltose (FCM)) versus oral iron (Ferrous Sulphate (FS)). Costs, health outcomes, and disability-adjusted life years (DALYs) averted were estimated using the IVON Trial and secondary data sources from a limited societal perspective over a one-year horizon. Incremental cost-utility ratios (ICURs) were compared against an opportunity cost-based threshold, with sensitivity analyses conducted to assess parameter uncertainty.ResultsIn Study I, stakeholders highlighted some key factors that contributed to the acceptability of IV iron. The perception of IV iron benefits includes its single-dose administration, which promotes better adherence and rapid health improvements, as well as its potential to avert complications such as the need for blood transfusions. However, some factors hindered acceptance, including misconceptions surrounding IV iron, high out-of-pocket expenses, limited trained HCPs, space and irregular supply. These insights informed strategies to support the implementation of IV iron during the IVON trial.During the implementation phase in Study II, 53 and 39 trained HCPs were surveyed at baseline and endline, respectively, with 20 HCPs participating at both time points. Mean AIM scores differed by state at baseline (p = 0.001) but not at endline, and no differences were found by cadre or facility level. However, mean AIM scores increased significantly over time (p = 0.002). Mean FIM scores did not vary by time, cadre, state, or facility. In Study III, overall fidelity to the protocol was moderate (66%), with the highest rates observed in tertiary facilities (84%) and the lowest in primary facilities (36%). Factors such as working as a team and the readily available charts and protocols facilitated high fidelity. However, individual variations, institutional challenges like heavy workloads and logistical issues such as prolonged patient waiting times contributed to lower fidelity levels. In Study IV, FCM cost more per woman (US104.28), but was also more effective, with lower DALYs incurred (0.0111) compared to that of FS (0.0372). The resulting ICUR was US$1,196.06 per DALY averted, which is below the threshold value, indicating that FCM was a cost-effective intervention.ConclusionThe findings from this doctoral research increase the understanding of the factors influencing the implementation of IV iron therapy for anaemia in pregnancy in Nigeria. While the acceptability of IV iron was high and improved over time, its feasibility and fidelity were influenced by health system constraints. Although there are context-specific factors influencing its use, IV iron (FCM) has proven to be cost-effective and offers clear clinical benefits over oral iron. These findings highlight that with tailored strategies, including the integration of IV iron into health insurance schemes and national guidelines, there is a high potential for the wide uptake and utilisation of IV iron in Nigeria. Consequently, reducing the burden of AIP and improving outcomes for mothers and babies across Nigeria.List of scientific papersI. Akinajo OR, Babah OA, Banke-Thomas A, Beňová L, Sam-Agudu NA, Balogun MR, Adaramoye VO, Galadanci HS, Quao RA, Afolabi BB, Annerstedt KS. Acceptability of IV iron treatment for iron deficiency anaemia in pregnancy in Nigeria: a qualitative study with pregnant women, domestic decision-makers, and health care providers. Reprod Health. 2024;21(1):22. https://doi.org/10.1186/s12978-024-01743-yII. Akinajo OR, Banke-Thomas A, Annerstedt KS, Beňová L, Adelabu YA, Sam-Agudu NA, Afolabi BB. Intravenous iron for iron deficiency anaemia in pregnancy: A quantitative study of acceptability and feasibility of its integration into routine antenatal care practice in Nigeria. [Submitted]III. Akinajo OR, Annerstedt KS, Banke-Thomas A, Obi-Jeff C, Sam- Agudu NA, Babah OA, Balogun MR, Beňová L, Afolabi BB. Implementation fidelity of intravenous ferric carboxymaltose administration for iron deficiency anaemia in pregnancy: a mixed- methods study nested in a clinical trial in Nigeria. Implement Sci Commun. 2024;5(1):81. https://doi.org/10.1186/s43058-024-00609-5IV. Akinajo OR, Annerstedt KS, Santos MT, Afolabi BB, Banke-Thomas A. Economic Evaluation of Ferric Carboxymaltose versus Oral Ferrous Sulphate for Iron Deficiency Anaemia in Pregnancy in Nigeria: A Cost-Utility Analysis. [Submitted]</p
SF3B1-mutant models of RNA mis-splicing uncover UBA1 as a therapeutic target in myelodysplastic neoplasms
No full textAbstract
Myelodysplastic syndromes with somatic mutations in the splicing factor SF3B1 gene (MDS-SF3B1) result in RNA mis-splicing, erythroid dysplasia and ultimately refractory anemia. Precision medicine approaches for MDS-SF3B1 remain challenging due to both the complexity of the mis-splicing landscape and its evaluation in disease-accurate models. To uncover novel RNA mis-splicing events, isogenic SF3B1
K700E and SF3B1
WT iPSC lines from an MDS-SF3B1 patient were differentiated into hematopoietic cells and analyzed via unsupervised splicing event profiling using full-length RNA sequencing. This identified SF3B1
K700E-specific mis-splicing of ubiquitin-like modifier activating enzyme 1 (UBA1), which encodes a key E1 protein at the apex of the ubiquitination cascade. UBA1 mis-splicing (UBA1
ms) introduced protein instability and decreased total UBA1 levels, rendering mutated cells susceptible to the small-molecule UBA1 inhibitor TAK-243. Analysis of CD34+ RNA sequencing data from an MDS patient cohort confirmed unique and ubiquitous UBA1
ms in MDS-SF3B1 patients, absent in other splicing factor-mutated MDS cases or healthy controls. TAK-243 selectively targeted MDS-SF3B1 primary CD34+ cells and reduced mutant cell numbers in colony-forming assays. In contrast, normal hematopoietic progenitor cells were unaffected. Altogether, we here define UBA1
ms as a novel therapeutic vulnerability in SF3B1-mutant cells, introducing UBA1 inhibition as a potential avenue for future MDS-SF3B1 treatments
In vivo composition of the mitochondrial nucleoid in mice.
No full textMitochondrial DNA (mtDNA) is compacted into dynamic structures called mitochondrial nucleoids (mt-nucleoids), with the mitochondrial transcription factor A (TFAM) as the core packaging protein. We generated bacterial artificial chromosome (BAC) transgenic mice expressing FLAG-tagged TFAM protein (Tfam-FLAGBAC mice) to investigate the mt-nucleoid composition in vivo. Importantly, we show that the TFAM-FLAG protein is functional and complements the absence of the wild-type TFAM protein in homozygous Tfam knockout mice. We performed immunoprecipitation experiments from different mouse tissues and identified 12 proteins as core mt-nucleoid components by proteomics analysis. Among these, eight proteins correspond to mtDNA replication and transcription factors, while the other four are involved in the mitoribosome assembly. In addition, we used the Tfam-FLAGBAC mice to identify ten proteins that may stabilize TFAM-FLAG upon depletion of the mitochondrial RNA polymerase despite the absence of mtDNA and induction of the LONP1 protease. Finally, we evaluated the changes in mt-nucleoids caused by very high levels of TFAM unraveling nine interactors that could counteract the high TFAM levels to maintain active mtDNA transcription. Altogether, we demonstrate that the Tfam-FLAGBAC mice are a valuable tool for investigating the mt-nucleoid composition in vivo.</p