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Potential differences in receptor-mediated G-protein activation in postmortem human hippocampal membranes prepared from healthy controls and suicide victims
No full textAim: Postmortem brain studies offer enormous opportunities to study molecular mechanisms associated with suicide. In the present study, conventional [35S]GTPγS binding assay and its version-up method ([35S]GTPγS binding/immunoprecipitation assay) were applied to postmortem human hippocampal membranes prepared from suicide victims and control subjects.Methods: By using conventional [35S]GTPγS binding assay, functional activations of Gi/o proteins coupled with multiple GPCRs (5-HT1A receptor, α2A-adrenoceptor, M2/M4 mAChRs, adenosine A1 receptor, histamine H3 receptor, group II mGlu, GABAB receptor, μ-opioid receptor, δ-opioid receptor, and NOP receptor) were detected by using 15 different agonists. Furthermore, 5-HT2A receptor- and M1 mAChR-mediated Gαq/11 activation and adenosine A1 receptor-mediated Gαi-3 activation were detectable by means of [35S]GTPγS binding/immunoprecipitation assay.Results: No significant differences in pharmacological parameters of all concentration-response curves investigated were found between suicide victims and control subjects. Significant correlations were obtained for the maximal percent increases between some distinct signaling pathways.Conclusion: Although only preliminary and auxiliary results were obtained as to the potential differences between suicide victims and control subjects because of the limited number of subjects as well as unmatched age and postmortem delay, adenosine A1 receptor-mediated Gαi/o activation and 5-HT2A receptor-mediated Gαq/11 activation appear worth focusing on in the future investigations. This study also indicates the possibility that some distinct signaling pathways are interrelated with each other, for example, functional activations of Gi/o proteins coupled to M2/M4 mAChR and 5-HT1A receptor, NOP receptor, and GABAB receptor, and NOP receptor and δ-opioid receptor.</p
Analysis of cell type-specific adverse effects in ovaries
No full textThe global rise in infertility, coupled with an increasing reliance on assisted reproductive technologies (ART), highlights the growing concern over environmental exposures that are associated with negative impacts on fertility. As the ovary's finite pool of oocytes determines female fertility, it is crucial to study the effects of chemicals on ovarian function.The overarching aim of this thesis was to identify key molecular targets and processes affected by chemical exposure in human ovaries, with the ultimate goal of generating novel information that could improve future risk assessment. This aim was approached through two complementary strategies: experimental studies and the development of AOPs. DEHP was chosen as a model compound. In the experimental studies, its primary metabolite, MEHP, was used to expose human ovarian tissue and cell cultures in vitro, followed by transcriptomic analyses to identify effects in an unbiased manner. For AOP development, the focus was on DEHP's hypothesized mode of action, androgen signaling, mapping its role in ovarian follicle biology through semi-systematic literature reviews.Paper I aimed to identify the effects of MEHP exposure on human ovarian tissue and ovarian cells in culture, utilizing RNA-sequencing and histological assessments. The findings highlighted significant effects on follicle survival and growth in vitro in human ovarian cortical cultures, along with gene expression changes related to cytoskeleton, adherens junctions and Hippo pathway in ovarian cells.Paper IV focused on cell type-specific responses, employing single-cell RNA sequencing to investigate the molecular effects of MEHP exposure on different ovarian cell types. This approach confirmed the disruption of cytoskeleton genes observed in Paper I in more cell types, and identified disruption of oxidative phosphorylation genes. Additionally, it revealed reduced cell interactions upon exposure and identified glial cells as the most sensitive cell types to the exposure.Paper II aimed to develop a Key Event Relationship (KER) that links androgen receptor antagonism to reduced granulosa cell proliferation, as part of an AOP on androgen antagonism leading to reduced female fertility (AOP345). The KER was developed through a systematic literature review, as it was non-canonical knowledge, and studies were collected and assessed for their quality.Paper III was built on Paper II, by continuing the development of the AOP, populating the information of all the included Key Events (KEs) and KERs. A similar systematic approach was followed to connect granulosa proliferation with ovarian cycle irregularities. A weight-of-evidence approach was followed, to inform the confidence in the complete AOP.Overall, this thesis provides new insights into the impact of chemical exposures on ovarian function, advancing understanding through both experimental and AOP-based approaches. In Papers I and IV processes including cytoskeleton organization and oxidative phosphorylation are identified through MEHP results, as potential targets for female fertility endpoints. Furthermore, the future perspective of expanding the assessment of chemical effects beyond follicles and onto ovarian cell types like glial cells is presented. Through Papers II and III a pragmatic way of developing KEs, KERs and whole AOPs is demonstrated. AOP345 highlights the importance of follicle counts as a key endpoint in toxicological assessments, an approach currently considered optional in test guidelines. However, as follicle counting is both subjective and time-consuming, these papers identify the possibility of developing granulosa cell proliferation as an alternative.List of scientific papersI. E.M. Panagiotou, A. Damdimopoulos, T. Li, E. Moussaud-Lamodière, M. Pedersen, F. Lebre, K. Pettersson, C. Arnelo, K. Papaikonomou, E. Alfaro-Moreno, C. Lindskog, T. Svingen, P. Damdimopoulou. Exposure to the phthalate metabolite MEHP impacts survival and growth of human ovarian follicles in vitro. Toxicology. 505 (2024). https://doi.org/10.1016/j.tox.2024.153815II. E.M. Panagiotou, M.K. Draskau, T. Li, A. Hirschberg, T. Svingen, P. Damdimopoulou. AOP key event relationship report: Linking decreased androgen receptor activation with decreased granulosa cell proliferation of gonadotropin-independent follicles. Reproductive Toxicology. 112 (2022) 136-147. https://doi.org/10.1016/j.reprotox.2022.07.004III. E.M. Panagiotou, H.K.L. Johansson, J. Zilliacus, A. Beronius, T. Svingen, P. Damdimopoulou. AOP report: Androgen receptor (AR) antagonism leading to decreased fertility in females via reduced granulosa cell proliferation. [Submitted]IV. E.M. Panagiotou*, L. Méar*, E. Moussaud-Lamodière, A. Kamposiora, N. Throumpari, C. Arnelo, K. Pettersson, K. Papaikonomou, C. Lindskog, P. Damdimopoulou. Single-cell insights on the effects of the phthalate metabolite MEHP on the human ovary in vitro. [Manuscript]* Equal contribution authors</p
Implementing Precision Cancer Medicine: organisational challenges, innovations, and the role of Molecular Tumour Boards
No full textPrecision Cancer Medicine (PCM) has been heralded as a transformative approach in oncology, tailoring treatments to the molecular profiles of individual patients. While PCM offers the potential for improved outcomes and reduced side effects, its implementation in clinical practice remains fraught with challenges. This thesis investigates the organisational dimensions of PCM, aiming to uncover barriers and facilitators to its integration into healthcare. By examining the emerging organisational and translational research efforts surrounding PCM, it illustrates how precision and targeted treatments can be equitably delivered across diverse healthcare settings.The thesis consists of three empirical studies. The first study describes the development of a clinical decision-support system for molecular profiling used in the Molecular Tumour Boards (MTBs) of a multicentre clinical trial designed to follow a PCM framework. This project provided insights into the practical and organisational conditions for implementing PCM within a translational research environment. The second study explores systemic and organisational hurdles to implementing PCM in the Stockholm healthcare system. Through qualitative interviews with healthcare professionals, researchers, and policymakers the study identified fragmented funding models, siloed organisational structures, and difficulties integrating genomic data into patient care as significant obstacles. The third study aimed to investigate how MTBs function as an emerging organisational form for PCM implementation. The study analysed MTBs at five comprehensive cancer centres and demonstrated their role in facilitating case discussions, educating clinicians about actionable molecular biomarkers, and fostering interdisciplinary collaboration.The findings show that while PCM has the potential to revolutionise cancer care, its success is contingent on addressing systemic challenges, such as creating sustainable funding mechanisms, improving cross-disciplinary collaboration, and ensuring that omics technologies are integrated into clinical workflows. The thesis highlights MTBs as a pivotal organisational innovation that bridges translational research and clinical practice. By addressing these challenges, this thesis contributes actionable insights for policymakers, researchers, and clinicians, advocating for a more inclusive and adaptive approach to PCM implementation.List of scientific papersI. Tamborero, D., Dienstmann, R., Haj Rachid, M., Boekel, J., Lopez- Fernandez, A., Jonsson, M., Razzak, A., Braña, I., De Petris, L., Yachnin, J., Baird, R.D., Loriot, Y., Massard, C., Martin-Romano, P., Opdam, F., Schlenk, R.F., Vernieri, C., Masucci, M., Villalobos, X., Chavarria, E., Cancer Core Europe consortium, Balmaña, J., Apolone, G., Caldas, C., Bergh, J., Ernberg, I., Fröhling, S., Garralda, E., Karlsson, C., Tabernero, J., Voest, E., Rodon, J., Lehtio, J. The Molecular Tumor Board Portal supports clinical decisions and automated reporting for precision oncology. Nat Cancer 3, 251-261 (2022). https://doi.org/10.1038/s43018-022-00332-xII. Masucci, M., Del Villar Pérez, J., Mazzocato, P., Ernberg, I., Brommels, M. Implementing Personalized Cancer Medicine: Insights from a Qualitative Interview Study. J. Pers. Med., 2025, 15, 150. https://doi.org/10.3390/jpm15040150III. Masucci, M., Pérez, J., Mazzocato, P., Ekman, S., Brommels, M., Ernberg, I. Molecular Tumour Boards in Precision Oncology: An Exploratory Case Study of Five Comprehensive Cancer Centres. (2025). [Manuscript]</p
Molecular phenotyping of Parkinson's disease
No full textParkinson's disease is among the most prevalent neurodegenerative diseases, clinically characterized by motor symptoms resulting from progressive degeneration of dopaminergic neurons in the ventral midbrain. Pathologically, Parkinson's disease is defined by the accumulation of alpha-synuclein aggregates, forming Lewy bodies and Lewy neurites. However, the disease exhibits heterogeneous symptoms. Motor symptoms can vary substantially between patients, and non-motor symptoms often precede and accompany disease progression. Furthermore, Lewy pathology extends beyond the ventral midbrain to other brain regions, underscoring systematic cellular dysregulation in Parkinson's disease.Current standard treatment with levodopa, a dopamine precursor, can provide symptomatic relief. However, its effectiveness varies between patients and diminishes over time, and it fails to reverse disease progression. Cell replacement therapy has since emerged as a promising alternative, supported by the pioneering transplantation of human fetal tissue showing long-term benefits in some patients. However, the variability in outcomes and ethical concerns have since shifted the research focus toward human pluripotent stem cells (hPSCs)- based strategies, requiring robust differentiation protocols for generating authentic midbrain dopaminergic neurons.This thesis broadens our understanding of ventral midbrain cellular heterogeneity through (1) Developing an improved hPSCs differentiation protocol by recapitulating key spatiotemporal developmental signals and introducing a single- cell RNA-seq-based quality assessment framework (Paper I); (2) Characterizing ventral midbrain cellular diversity in both human development and adult brain (Paper Il and III); and (3) Multiomic profiling of Parkinson's disease midbrain and nearby regions, revealing disease-associated transcriptional dysregulation (Preliminary work).This thesis begins with a literature review on ventral midbrain development, Parkinson's disease, and single-cell genomics to contextualize the work. Subsequent chapters then present and discuss the results from the studies included in this thesis.List of scientific papersI. Single-cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation.Nishimura, K., Yang, S., Lee, K.W., Ásgrímsdóttir, E.S., Nikouei, K., Paslawski, W., Gnodde, S., Lyu, G., Hu, L., Salto, C., Svenningsson, P., Hjerling-Leffler, J., Linnarsson, S. and Arenas. E.Stem Cell Reports, 2022, 18(1), pp.337-353. https://doi.org/10.1016/j.stemcr.2022.10.016II. Comprehensive cell atlas of the first-trimester developing human brain.Braun, E., Danan-Gotthold, M., Borm, L.E., LEE, K.W., Vinsland, E., Lönnerberg, P., Hu, L., Li, X., He, X., Andrusivová, Ž., Lundeberg, J., Barker, R.A., Arenas, E., Sundström, E. and Linnarsson. S.Science, 2023, 382(6667):eadf1226. https://doi.org/10.1126/science.adf1226III. Transcriptomic diversity of cell types across the adult human brain.Siletti, K., Hodge, R.D., Albiach, A.M., LEE, K.W., Ding, S.L., Hu, L., Lönnerberg, P., Bakken, T.E., Casper, T., Clark, M., Dee, N., Gloe, J., Hirschstein, D., Shapovalova, N.V., Keene, C.D., Nyhus, J., Tung, H., Yanny, A.M., Arenas, E., Lein, E.S. and Linnarsson, S.Science, 2023, 382(6667):eadd7046. https://doi.org/10.1126/science.add7046</p
Medical abortion : improving access and care from very early pregnancy to the second trimester
No full textINTRODUCTION: Medical abortion with mifepristone and misoprostol is a safe and effective treatment. Removing unnecessary barriers to treatment increases access and improves health and wellbeing. Since the introduction of medical abortion, treatment has been simplified, allowing women to self-manage the treatment at home in the first trimester. Telemedicine provision can further increase access to safe abortion, but randomized trials from high income settings are lacking. Additionally, there is insufficient evidence on efficacy of medical abortion in very early gestation, before a pregnancy can be visualized on ultrasound. This leads to inconsistencies in treatment recommendations, and for some women abortion is delayed. For women seeking abortion in the second trimester, it is recommended that the procedure is performed in a health care facility, due to the increased risk of complications. This can create a barrier to treatment. Optimizing the protocol to include administration of misoprostol at home before hospital admission, could shorten the time spent in hospital. This can improve access by increasing number of patients managed with a day-care procedure.AIM: The overall aim of this thesis is to improve access to medical abortion by finding evidence for further de-medicalizing of treatment in first and second trimester and expanding access to treatment in very early gestation.METHOD: We conducted 3 randomized trials and 1 observational, cross-sectional study. In study I patients seeking abortion before 6 weeks of gestation with an unconfirmed intra-uterine pregnancy (IUP) was randomized to immediate or standard delayed care after 7-14 days (at confirmed IUP). In study III women seeking abortion up to 9 weeks of gestation, were randomized at the telephone booking to an online consultation or standard in-clinic provision of abortion. In study IV patients seeking abortion in the second trimester were randomized to home initiation of misoprostol before hospital admission or hospital administration. In study II we analyzed requests for telemedicine abortion (using the same online consultation from Study III) in Italy before and during the pandemic.RESULTS: We could show a non-inferiority of efficacy compared to standard care for both very early medical abortion (95.2% vs 95.3%) (study I) and after telemedicine consultation (98.2% vs 98.1%) (study III). We could also show that home initiation of misoprostol in second trimester abortion resulted in more patients being completed as day-care patients (71% vs 46%)(study IV). All interventions had similar safety outcomes as standard care. In study II we could show a 12% increase in requests for telemedicine abortion during the pandemic and that reasons for seeking an abortion were mainly related to need for privacy and due to the pandemic.CONCLUSION: Offering treatment in very early gestation before confirmed IUP is effective and safe. Telemedicine abortion is effective and safe, and there is a need for alternative provision also in settings with legal abortion. Home initiation of misoprostol in seconds trimester abortion is safe and increases number of patients being treated with a day-care procedure.List of scientific papersI. Randomized Trial of Very Early Medication Abortion. Brandell K, Jar-Allah T, Reynolds-Wright J, Kopp Kallner H, Hognert H, Gyllenberg F, Kaislasuo J, Tamang A, Tuladhar H, Boerma C, Schimanski K, Gibson G, Løkeland M, Teleman P, Bixo M, Mandrup Kjaer M, Kallfa E, Bring J, Heikinheimo O, Cameron S, Gemzell- Danielsson K; VEMA (Very Early Medication Abortion) Study Group. New England Journal of Medicine. 2024 Nov 7;391(18):1685-1695. https://doi.org/10.1056/nejmoa2401646II. Telemedicine as an alternative way to access abortion in Italy and characteristics of requests during the COVID-19 pandemic. Brandell K, Vanbenschoten H, Parachini M, Gomperts R, Gemzell- Danielsson K. BMJ Sexual & Reproductive Health. 2022 Oct;48(4):252-258. https://doi.org/10.1136/bmjsrh-2021-201281III. A Randomized Trial on Efficacy of Telemedicine Abortion. Isabella Bizjak*, Karin Brandell*, Ninni Mannerberg, Anette Aronsson, Amanda Cleeve & Kristina Gemzell Danielsson. [Manuscript]IV. First dose of misoprostol administration at home or in hospital for medical abortion between 12-22 gestational weeks in Sweden (PRIMA): a multicentre, open-label, randomised controlled trial. Rydelius J, Hognert H, Kopp-Kallner H, Brandell K, Romell J, Zetterström K, Teleman P, Gemzell-Danielsson K. Lancet. 2024 Aug; 404(10455):864-873. https://doi.org/10.1016/s0140-6736(24)01079-1*Shared first author</p
Mechanisms of activation and cytotoxicity in γδ T cells and natural killer cells
No full textγδ T cells and natural killer (NK) cells have been shown to play crucial roles in the immune response. While the activation mechanisms of these two cell types differ, with γδ T cells being activated by small metabolites from the isoprenoid synthesis pathway and NK cells through balancing signals between activating and inhibitory receptors, their effector functions were believed to resemble each other. Both cell types are well known for their quick release of IFN-γ and TNF-α and for their cytotoxic responses to infected and malignant cells. Consequently, both γδ T cells and NK cells are regarded as promising candidates for immunotherapies. However, the outcomes of clinical trials conducted thus far remain inconsistent, suggesting the presence of a lack of fundamental knowledge. To enhance our understanding of these multifaceted immune cells, this thesis aimed to address several key research questions. We explored the cytotoxic pathways employed by γδ T cells and NK cells, trying to identify the factors that limit their effectiveness. We further examined how activation affects their potency and employed advanced imaging techniques to investigate NK cell infiltration in tumour spheroids. To achieve this, we employed a range of single-cell microscopy-based approaches to elucidate the heterogeneity of γδ T cell and NK cell populations.We found that following a three-day period of stimulation, γδ T cells exhibited an upregulation of activating receptors, with a particularly pronounced increase in granzyme B expression. However, after the proliferation and expansion of the γδ T cells, there was a decline in granzyme B expression levels. Investigation of the cytotoxic mechanisms employed by γδ T cells revealed that they predominantly utilise degranulation as their primary mode of action, with the engagement of death receptors playing a comparatively minor role. Further, early activated γδ T cells with high levels of granzyme B used degranulation more often than the late expanded γδ T cells. However, the substantial quantities of granzyme B did not facilitate the serial killing of multiple target cells, as γδ T cells only expressed low levels of perforin.In addition, we established that γδ T cells were efficiently activated by monocytes through the sensing of phosphoantigens as well as cytokines. These cells responded by releasing IFN-γ and TNF-α within hours of these interactions and formed close contacts with monocytes for extended periods. The investigation of these close contacts revealed that calcium responses in γδ T cells reached a peak at approximately 10 hours, which coincided with the maturation and potential accumulation of IPP in monocytes.NK cells are known to be effective killers, capable of eliminating numerous target cells in succession. Here we investigated the factors that disable NK cells from continuing to execute their function. We revealed that even after killing of several target cells NK cells retained a reserve of lytic granules. Instead of experiencing a depletion of their lytic cargo, these cells appeared to be receiving an insufficient level of signalling. However, upon stimulation with novel stimuli, even those NK cells that had ceased to kill were able to resume their function.Finally, to enhance our understanding of infiltrating NK cells, an expansion microscopy approach was utilised to enhance imaging of NK cells in tumour spheroids. This technique has the potential to be employed in future studies to combine functional live cell assays with high-resolution imaging of infiltrating immune cell.In summary, this thesis contributes to a better understanding of the fundamental biology in γδ T cells and NK cells, particularly their cytotoxic functions. These findings are of critical importance in the development of more effective immune therapies.List of scientific papersI. P. A. Sandoz*, K. Kuhnigk*, E. K. Szabo, S. Thunberg, E. Erikson, N. Sandström, Q. Verron, A. Brech, C. Watzl, A. K. Wagner, E. Alici, K.J. Malmberg, M. Uhlin and B. Önfelt. Modulation of lytic molecules restrain serial killing in γδ T lymphocytes. Nature Communications. 14, 6035 (2023). https://doi.org/10.1038/s41467-023-41634-7II. K. Kuhnigk, H. Zhang, A. Stikvoort, P. A. Sandoz* and B. Önfelt *. Activation dynamics of γδ T cells in response to monocyte interaction. [Manuscript]III. H. van Ooijen, Q. Verron, K. Kuhnigk, P. A. Sandoz, N. Sandström and B. Önfelt. Loss of signaling limits NK cell serial killing. [Submitted]IV. S. J. Edwards, V. Carannante, K. Kuhnigk, H. Ring, T. Tararuk, F. Hallbook, H. Blom, B. Önfelt and H. Brismar. High-Resolution Imaging of Tumor Spheroids and Organoids Enabled by Expansion Microscopy. Frontiers in Molecular Biosciences. 7:208 (2020). https://doi.org/10.3389/fmolb.2020.00208*Authors contributed equally</p
Supporting maternal health and wellbeing in the perinatal period : the impact of midwifery care
No full textThe perinatal period involves significant psychological and physical changes that impact women's health and wellbeing. Despite improvements in maternal health, key gaps remain in understanding and supporting women during this period. This thesis addresses these gaps by exploring physical and mental health challenges, the need for continuity and support, and the impact of intimate partner violence (IPV).STUDY I describes new mothers' and midwives' experiences and perceptions of a coordinated postnatal care intervention. The study found that mothers highlighted continuity and accessibility as empowering factors, while midwives emphasized the care model in facilitating care tailored to individual needs.STUDY II investigates the prevalence of IPV among pregnant women and its association with sociodemographic and health factors. The study found 2.1% of 3371 women experienced IPV, primarily psychological violence, with significant associations with living situation, depressive symptoms, education, and employment status.STUDY III investigates new mothers' health problems, reasons for emergency department visits, and support from midwifery clinics. A lot of symptoms and problems were reported and 16% of 580 mothers sought emergency care, with higher age and poorer health increasing the likelihood. Mothers who did not receive the supported they wanted expressed a need for earlier contact and better accessibility.STUDY IV explored changes in IPV exposure before and during pregnancy, and the frequency of screening and disclosure. IPV decreased from 6.2% pre-pregnancy to 2.1% during pregnancy, with 64% out of 3371 exposed women asked about violence, and diverse reasons for disclosure or non-disclosure.Conclusion: Both pregnant women and new mothers face a range of challenges, including IPV, that impact their health and perception of care. This thesis highlights the importance of high-quality maternity care that meets women's individual needs, emphasizing midwives' role in preventive care and addressing the complexities of violence during pregnancy and postpartum health challenges. Keywords: Support, Continuity of care, Transition to motherhood, Intimate partner violence, Pregnancy, Postnatal care, Emergency department visits, Midwifery, Screening.List of scientific papersThis thesis is based on the following papers, referred to in the text by their Roman numerals I - IV.I. Eikemo, R., Vikström, A., Nyman, V., Jonas, W., & Barimani, M. (2023). Support during the postnatal period: Evaluating new mothers' and midwives' experiences of a new, coordinated postnatal care model in a midwifery clinic in Sweden. Scandinavian Journal of Caring Sciences, 37(1), 260-270. https://doi.org/10.1111/scs.13103II. Eikemo, R., Barimani, M., Elvin-Nowak, Y., Eriksson, J., Vikström, A., Nyman, V., Backman-Enelius, M & Jonas, W. (2023). Intimate partner violence during pregnancy-Prevalence and associations with women's health: A cross-sectional study. Sexual & Reproductive Healthcare, 36, 100843. https://doi.org/10.1016/j.srhc.2023.100843III. Eikemo, R., Barimani, M., Nyman, V., Jonas, W., & Vikström, A. (2024). Health challenges and midwifery support for new mothers after childbirth: A cross-sectional study in Sweden. Midwifery, 134, 104020. https://doi.org/10.1016/j.midw.2024.104020IV. Eikemo, R., Elvin-Nowak, Y., Åhlund, S., Vikström, A., Nyman, V., Jonas, W., Barimani, M. (2024) I had to tell to survive"- a cross-sectional study on exposure to intimate partner violence in pregnant women and the importance of screening. Sexual & Reproductive Healthcare, 42, 101045. https://doi.org/10.1016/j.srhc.2024.101045</p
RNA in motion : NMR insights into ribosomal RNA dynamics
No full textRNAs perform many different tasks in biology. The ribosome is an RNA-protein complex which synthesises proteins based on the message from DNA. It consists of two subunits which associate during translation initiation and form non- covalent interactions, known as intersubunit bridges. rRNA helix 44 (h44) participates in several of the intersubunit interactions, some of which are purely rRNA-mediated.The ribosome is a common antibiotic target. The rise of antibiotic resistance, along with side effects of existing antibiotics, highlights the need for research to explore alternative structural layers within rRNA as targets, and to compare them across species. During the COVID-19 pandemic, multiple ways to target the virus were studied, with the viral genome being one of them.The first chapter introduces RNA structure and conformational dynamics, as well as the ribosome, which is the system under study in Paper I. The theory of NMR spectroscopy and its application to studying conformational dynamics of RNA is also introduced. The SARS-CoV-2 (SCoV2) viral RNA genome is introduced to contextualise Papers III and IV.In Paper I, the work to characterise the dynamics at the intersubunit bridge B3 of the human cytosolic 18S rRNA h44 is described. We show a transient protonation of an adenine, as a result of a structurally altered pKa, and a guanine nucleobase flip about the glycosidic bond. As B3 is located at the pivot point of intersubunit rotation during translation and is important for subunit association, protonation at B3 may play a role in translation initiation. In Paper II, one of the workflows employed within Paper I is described. Paper II also provides a Python script pipeline to extract and visualise chemical shifts from different base pairs to aid in inferring excited-state structures from chemical shifts. In Paper III, we determine the secondary structure of conserved stem-loop elements of the SCoV2 genome by NMR spectroscopy and show the validity of studying RNA stem-loops in isolation by NMR. In Paper IV, we provide near-complete NMR resonance assignments of stem-loop 5a of the 5'-UTR, as a basis for further structure elucidation.List of scientific papersI. Riad, M., Marušič, M., Albers, M., Halbeisen, M., Montserrat-Canals, M., Steinmetzger, C. & Petzold, K. Visualizing a transiently protonated RNA state at intersubunit bridge B3 of human ribosomal RNA helix 44 by relaxation dispersion NMR spectroscopy. [Manuscript]II. Riad, M., Hopkins, N., Baronti, L., Karlsson, H., Schlagnitweit, J. & Petzold, K. Mutate-and-chemical-shift-fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy. Nat Protoc. 16, 5146-5170 (2021) https://doi.org/10.1038/s41596-021-00606-1III. Wacker, A., Weigand, J. E., Akabayov, S. R., Altincekic, N., Bains, J. K., Banijamali, E., Binas, O., Castillo-Martinez, J., Cetiner, E., Ceylan, B., Chiu, L .- Y., Davila-Calderon, J., Dhamotharan, K., Duchardt-Ferner, E., Ferner, J., Frydman, L., Fürtig, B., Gallego, J., Grün, J. T., Hacker, C., Haddad, C., Hähnke, M., Hengesbach, M., Hiller, F., Hohmann, K. F., Hymon, D., de Jesus, V., Jonker, H., Keller, H., Knezic, B., Landgraf, T., Löhr, F., Luo, L., Mertinkus, K. R., Muhs, C., Novakovic, M., Oxenfarth, A., Palomino-Schätzlein, M., Petzold, K., Peter, S. A., Pyper, D. J., Qureshi, N. S., Riad, M., Richter, C., Saxena, K., Schamber, T., Scherf, T., Schlagnitweit, J., Schlundt, A., Schnieders, R., Schwalbe, H., Simba- Lahuasi, A., Sreeramulu, S., Stirnal, E., Sudakov, A., Tants, J .- N., Tolbert, B. S., Vögele, J., Weiß, L., Wirmer-Bartoschek, J., Wirtz Martin, M. A., Wöhnert, J. & Zetzsche, H. Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy. Nucleic Acids Res. 48, 12415-12435 (2020) https://doi.org/10.1093/nar/gkaa1013IV. Schnieders, R., Peter, S. A., Banijamali, E., Riad, M., Altincekic, N., Bains, J. K., Ceylan, B., Fürtig, B., Grün, J. T., Hengesbach, M., Hohmann, K. F., Hymon, D., Knezic, B., Oxenfarth, A., Petzold, K., Qureshi, N. S., Richter, C., Schlagnitweit, J., Schlundt, A., Schwalbe, H., Stirnal, E., Sudakov, A., Vögele, J., Wacker, A., Weigand, J. E., Wirmer-Bartoschek, J. & Wöhnert, J. 1H, 13C and 15N chemical shift assignment of the stem- loop 5a from the 5'-UTR of SARS-COV-2. Biomol Nmr Assigm. 15, 203-211 (2021) https://doi.org/10.1007/s12104-021-10007-w</p
Cardiovascular drug use after myocardial infarction : adherence, kidney dysfunction, and real-world outcomes
No full textPatients with acute coronary syndromes (ACS) face a substantial risk of recurrent events and mortality, particularly at higher age or when chronic kidney disease (CKD) is present. Several factors may contribute to this elevated risk, including suboptimal prescription, initiation, and adherence to guideline-recommended secondary prevention therapies. This thesis utilized merged real-world data from national and regional sources, including the nationwide Swedish Web-system for Enhancement and Development of Evidence-based care in Heart disease (SWEDEHEART) registry, the Stockholm Creatinine Measurements (SCREAM) database, and the National Prescribed Drug Register. Its main objective was to examine secondary prevention drug use and long-term outcomes after ACS or acute myocardial infarction (AMI) in various contexts, with a specific focus on patients with CKD.Study I assessed the relationship between kidney function and use of key secondary prevention drugs among ACS survivors. All patients admitted with a first ACS enrolled in SWEDEHEART between 2005-2010 were identified. After excluding those who died in-hospital or lacked serum creatinine data, 75,129 patients remained to analyze. Following adjustment for absolute or relative contraindications, patients with moderately reduced kidney function (eGFR 30-59 mL/min/1.73 m2) less frequently received acetylsalicylic acid, statins, and ß-blockers compared with those with normal-mildly reduced kidney function (eGFR ≥60 mL/min/1.73 m2). They were also more likely to discontinue acetylsalicylic acid (hazard ratio [HR] 1.49, 95% CI 1.42-1.56), statins (HR 1.35, 95% CI 1.29-1.41), renin-angiotensin system inhibitors (HR 1.37, 95% CI 1.31-1.43), and ß-blockers (HR 1.22, 95% CI 1.18-1.27) during the first year post-event. Patients with severely reduced kidney function (eGFR 2) exhibited similar patterns in both prescription and discontinuation.Study Il investigated the association between adherence to ß-blocker therapy after a first AMI and long-term risk of death or readmission due to heart failure (HF). Among 38,608 patients admitted for a first AMI between 2005-2010 enrolled in SWEDEHEART, those who died within the first year, had prior HF, or unknown ejection fraction (EF) were excluded. Adherence was defined as a proportion of days covered 280% at one year. Patients with reduced EF, regardless of in-hospital signs of HF, were more likely to stay adherent to ß-blocker therapy than those with normal EF and no HF. Socioeconomic and clinical factors were associated with better adherence. The whole group of patients with reduced kidney function showed a non-significant trend toward non-adherence, while dialysis was clearly associated with non-adherence. In adjusted analyses, adherence was associated with reduced all-cause mortality (HR 0.77, 95% CI 0.71-0.84) over four years, though this benefit was not statistically significant in patients with normal EF irrespectively of heart failure status. Adherence was also associated with reduced risk of the composite outcome of HF readmission/death in all patients (HR 0.83, 95% CI 0.78-0.89), but the association was uncertain in subgroups with normal EF with and without heart failure signs.Study III examined the association between statin intensity and long-term outcomes, focusing on patients with CKD. Among 112,727 patients admitted for a first AMI enrolled in SWEDEHEART between 2005-2016, those who died in-hospital, lacked creatinine data, or did not initiate statin therapy within 90 days were excluded. High-intensity statins were initiated in 38.7% of patients, while 61.3% received low-moderate intensity. Among one-year survivors, statin discontinuation in CKD patients was 25%, with similar rates across intensity groups. After further exclusions for death, therapy switch, or non-adherence at one year, 84,705 patients remained for on-treatment analysis. In patients with moderately reduced kidney function, high-intensity statins were associated with a reduced risk of death, reinfarction, or stroke in both intention-to-treat (HR 0.93, 95% CI 0.87-0.99) and on-treatment analyses (HR 0.90, 95% CI 0.83-0.99), with no evidence of heterogeneity across kidney function groups (P = 0.46).Study IV compared patient characteristics, clinical management, and long-term outcomes between AMI patients enrolled in SWEDEHEART and those not enrolled. Using a regional database (SCREAM), 47,342 AMI hospitalizations (40,935 incident cases) were identified between 2006-2021 in the region of Stockholm. Enrolment was defined as having any record in the Register of Information and Knowledge About Swedish Heart Intensive Care Admissions (RIKS-HIA), a sub register of SWEDEHEART, during the index hospitalization or within one day prior to admission, to account for emergency department registrations. Non-enrolled patients (N = 6,113; 13%) were older, more often had CKD, and had a higher comorbidity burden and frailty risk. They were less likely to undergo invasive procedures and initiate acetylsalicylic acid (HR 0.88, 95% CI 0.84-0.91), ß-blockers (HR 0.87, 95% CI 0.84-0.90), renin-angiotensin system inhibitors (HR 0.73, 95% CI 0.69-0.76), or statins (HR 0.59, 95% CI 0.56-0.61). Adherence during the first year was also lower, partly due to their comorbidity profile. Non-enrolled patients experienced higher in-hospital and long-term mortality (HR 1.15, 95% CI 1.09-1.21) and more reinfarction or stroke (HR 1.16, 95% Cl 1.08-1.26) compared with enrolled patients.Conclusions: Patients with ACS and CKD were less frequently prescribed secondary prevention drugs at discharge and, when initiated, more likely to discontinue within the first year. Adherence to ß-blockers after AMI was associated with improved long-term outcomes, though this benefit may be limited to patients with reduced ejection fraction. High-intensity statin therapy in CKD patients was associated with better long-term outcomes after AMI. Most patients with CKD that started on high-intensity statins remained on this regimen after one year, with overall persistence to statin treatment equal to those that started on low-moderate intensity therapy. Patients not enrolled in SWEDEHEART were less likely to receive guideline-recommended treatments and had worse short- and long-term outcomes. Although overall improvements in adherence to guideline-recommendations have been observed over the years, these findings highlight persistent gaps in cardiovascular care that warrant further studies and interventions.List of scientific papersI. Khedri M, Szummer K, Carrero JJ, Jernberg T, Evans M, Jacobson SH, Spaak J. Systematic underutilisation of secondary preventive drugs in patients with acute coronary syndrome and reduced renal function. Eur J Prev Cardiol. 2017 May;24(7):724-734. https://doi.org/10.1177/2047487317693950II. Desta L*, Khedri M*, Jernberg T, Andell P, Mohammad MA, Hofman-Bang C, Erlinge D, Spaak J§, Persson H§. Adherence to beta-blockers and long-term risk of heart failure and mortality after a myocardial infarction. ESC Heart Fail. 2021 Feb;8(1):344-355. https://doi.org/10.1002/ehf2.13079III. Khedri M, Szummer K, Lundman P, Jernberg T, Desta L, Lindahl B, Erlinge D, Jacobson SH, Spaak J. Statin Treatment Intensity, Discontinuation, and Long-Term Outcome in Patients With Acute Myocardial Infarction and Impaired Kidney Function. J Cardiovasc Pharmacol. 2023 Jun 1;81(6):400-410. https://doi.org/10.1097/FJC.0000000000001402IV. Khedri M, Szummer K, Jacobson SH, Hjemdahl P, Spaak J§. Carrero JJ§. Characteristics, clinical management and outcomes of patients with acute myocardial infarction enrolled or not enrolled in a quality registry. [Submitted]*Contributed equally to the work.§Share senior authorship of the work.</p
The regulatory code of injury-responsive enhancers enables precision cell-state targeting in the CNS
No full textEnhancer elements direct cell-type-specific gene expression programs. After injury, cells change their transcriptional state to adapt to stress and initiate repair. Here we investigate how injury-induced transcriptional programs are encoded within enhancers in the mammalian CNS. Leveraging single-nucleus transcriptomics and chromatin accessibility profiling, we identify thousands of injury-induced, cell-type-specific enhancers in the mouse spinal cord after a contusion injury. These are abundant in glial cells and retain cell-type specificity, even when regulating shared wound response genes. By modeling glial injury-responsive enhancers using deep learning, we reveal that their architecture encodes cell-type specificity by integrating generic stimulus response elements with cell identity programs. Finally, through in vivo enhancer screening, we demonstrate that injury-responsive enhancers can selectively target reactive astrocytes across the CNS using therapeutically relevant gene delivery vectors. Our decoding of the principles of injury-responsive enhancers enables the design of sequences that can be programmed to target disease-associated cell states.</p