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Oral graft versus host disease : salivary gland pathology and tissue modelling
No full textChronic Graft versus Host Disease (cGVHD) is a consequence of dysregulated immune reconstitution after allogenic hematopoietic cell transplantation (alloHCT), a treatment for high-risk blood malignancies or immune disorders. cGVHD is a donor T-cell-driven rejection of the host tissues associated with high morbidity and mortality. It may affect nearly any site or organ of the body, and manifestations in the oral cavity are common. These manifestations may involve both oral mucosa and salivary glands. There are no clinical diagnostic criteria for salivary gland cGVHD (sg-cGVHD), which primarily presents as sicca syndrome with xerostomia or salivary dysfunction, similar to Sjögren’s syndrome.Gold standard for diagnosing sg-cGVHD is through biopsy and histopathological diagnosis. There are defined diagnostic features for histopathological diagnosis of sg-cGVHD but only vague guidelines for the assessment of severity.Standard of care can be improved by semi-quantitative grading schemes for histopathology, but also through image analysis with artificial intelligence and machine learning. Digital pathology produces large and intricate images that are not easily analysed through traditional machine learning techniques. Although modern algorithms called convolutional neural networks have improved object detection and segmentation in histopathology images, they struggle with analysis of complex context. Alternative tissue representation and machine learning adapted for analysis of relationships are emerging and may be further explored in the context of oral diseases. Using graph theory, tissue may be represented as an interconnected network of its constituents. By modelling tissue as a graph, we can also apply deep learning with graph neural networks for representation of more advanced properties of the tissue.In Study I, we validated histopathological features in grading schemes for both diagnosis and severity rating of sg-cGVHD. We devised a semi-quantitative grading scheme based on pathology criteria defined by the National Institutes of Health’s Consensus Project and compared it to a proposed grading scheme by Imanguli et al. that was inspired by the assessment of Sjögren’s syndrome. In addition to the histopathological grading, we assessed the characteristics of the inflammatory infiltrate by quantitative immunohistochemistry through the software CellProfiler. The formalised NIH sg-cGVHD grading scheme ranged from Grade 0 to Grade IV (G0-GIV), with histopathological verification of diagnostic cutoffs for possible cGVHD at GII and Likely cGVHD at GIII. Acinar and periductal inflammation were closely correlated, and the inflammation was often widespread. The quantification of inflammatory infiltrate confirmed CD8+ T-cells to be the predominant cell type, followed by CD4+ T cells. Macrophages (CD68+) were also detected at increased levels, whereas B-cells (CD19+, CD20+) were not detected.In Study II, we presented a model for tissue representation of oral mucosa that is based on graph theory. We proposed a 2-stage pipeline for modelling of the tissue in the form of a cellgraph with labelled edges for identification of the basement membrane. It encompassed modules for predicting cells with convolutional neural networks and constructing cell-graphs, as well as for basement membrane localisation with the deep learning algorithm GraphSAGE trained on the concept of basement membranes as crossing edges in a cell-graph. This representation yielded a model that fitted low-level features like cellular properties, their relationships in the tissue, and the high-level features of the basement membrane. The model performed with a reliable accuracy of 0.88 in predicting the basement membrane interface.In conclusion, sg-cGVHD can be diagnosed and assessed for severity using the formalised NIH sg-cGVHD grading scheme for histopathology, and histopathological severity is associated with increased levels of CD8+ and CD4+ T cells, as well as macrophages (CD68+). Grading schemes can benefit from artificial intelligence, but this requires fair image representation. Oral tissues can be represented as graphs, and complex features like the basement membrane inferred by graph learning.List of scientific papersI. Tollemar, V., Arvidsson, H., Häbel, H., Tudzarovski, N., Legert, K. G., Le Blanc, K., Gunnar Warfvinge & Sugars, R. V. (2023). Grading of minor salivary gland immuno-histopathology post-allogenic hematopoietic cell transplantation. Heliyon, 9(4), e15517. https://doi.org/10.1016/j.heliyon.2023.e15517 II. Nair, A.*, Arvidsson, H.*, Gatica V, J. E., Tudzarovski, N., Meinke, K., & Sugars, R. V. (2022). A graph neural network framework for mapping histological topology in oral mucosal tissue. BMC bioinformatics. 23(1), 506. *contributed equally https://doi.org/10.1186/s12859-022-05063-5 </p
Skeletal muscle responses to physical activity in health and metabolic disease
No full textSedentary lifestyles, characterised by a lack of physical activity and prolonged periods of sitting, have been linked to reductions in whole-body metabolic flexibility and the increased risk of metabolic diseases, including type 2 diabetes. This can be attributed, at least partially, to the direct negative effects of physical inactivity on skeletal muscle insulin sensitivity, oxidative capacity, and overall metabolic health. In addition, sedentary behaviour can lead to anabolic resistance, resulting in losses of skeletal muscle mass and strength, which can further contribute to conditions like sarcopenic obesity, impairing physical performance and overall quality of life. Conversely, physical activity plays a crucial role in maintaining and improving skeletal muscle health. Exercise is associated with various adaptations in skeletal muscle that enhance tissue oxidative capacity, substrate handling, insulin sensitivity, as well as skeletal muscle mass and strength. These positive changes in skeletal muscle contribute to improvements in systemic metabolic wellbeing. The molecular mechanisms underlying skeletal muscle adaptation to the perturbations caused by physical activity are complex and involve intrinsic processes within the muscle fibre itself, as well as communication between different cell populations in composite skeletal muscle tissue. However, our understanding of the intricate details of these mechanisms remains incomplete. Gaining deeper insights into the regulation of skeletal muscle adaptation could not only facilitate personalised exercise recommendations but also uncover novel opportunities for drug discovery, ultimately leading to improvements in human health.Despite the well-known benefits of exercise, physical activity guidelines are often not met by the general population. Therefore, there is a pressing need for low-level entry paradigms that can promote physical activity and reduce sedentary behaviour for the betterment of individual and public health. One such approach is the incorporation of frequent activity breaks or ‘exercise snacks’ into daily routines, which involves short-duration physical activity breaks throughout the day to disrupt prolonged periods of sitting. These interventions have demonstrated efficacy for cardiometabolic health in controlled settings, such as laboratory-based clinical trials. However, it is essential to evaluate the benefits of breaking sedentary time using strategies that better mimic real-world scenarios to inform practical public health guidelines.In this thesis, the following objectives were pursued: (1) To assess the translational efficacy of interrupting sedentary time in improving cardiometabolic health. (2) To investigate the skeletal muscle transcriptome following exercise or physical inactivity in the context of health and metabolic diseases. (3) To determine the metabolic effects of physical activity- responsive transcription factors and signalling molecules in skeletal muscle.Study I revealed that even a minor addition of ≈750 steps dispersed throughout the day, equivalent to ≈10 minutes of extra walking time, improved dynamic glucose control in individuals with obesity and insulin resistance. Notably, those who engaged in higher levels of physical activity while interrupting sedentary time experienced greater benefits, indicating that more breaks from sedentary behaviour lead to better metabolic health outcomes. Nevertheless, adherence to the intervention, which involved 3-min activity bouts every 30 min between 08:00-18:00, was lower than anticipated. This raises questions about the long- term feasibility of such approaches when considered in isolation from other modifiable lifestyle factors, including changes in dietary habits or structured exercise routines.Study II employed a comprehensive meta-analytical approach to compare the skeletal muscle transcriptomic response to acute aerobic or resistance exercise, exercise training, and physical inactivity. This analysis revealed distinct gene signatures in skeletal muscle after a single bout of exercise in the naïve state, which differed from those observed after training of the same exercise modality. Interestingly, there was greater overlap in the skeletal muscle transcriptome between acute aerobic and resistance exercise than there was between acute exercise and exercise training. These findings highlight the refinement of the adaptive response in skeletal muscle over time through dedicated training to a specific exercise modality. Study II identified the transcription factor nuclear receptor subfamily 4 group A member 3 (NR4A3) as a gene that is upregulated in skeletal muscle after exercise but downregulated in response to physical inactivity. Study III delved deeper into the role of NR4A3 in the context of physical inactivity and revealed its regulatory role in translation within skeletal muscle. Depletion of NR4A3 resulted in skeletal muscle atrophy and compromised glucose oxidation, instead favouring increased lactate production. Therefore, decreased levels of NR4A3 during physical inactivity may directly contribute to muscle disuse atrophy and impaired skeletal muscle metabolism.Furthermore, study II identified that individuals with obesity and/or type 2 diabetes exhibit an altered skeletal muscle transcriptional response to exercise training compared to healthy individuals. Study IV uncovered a heightened inflammatory response during the recovery period after exercise in individuals with type 2 diabetes. This response was attributed to an increased influx of immune cells into skeletal muscle tissue, potentially facilitating crosstalk between different cell types within the skeletal muscle interstitial space. Notably, the cytokine stromal cell-derived factor 1 (CXCL12/SDF-1) was found to be expressed by macrophages or endothelial cells in response to factors released by skeletal muscle fibres or hypoxia, respectively. CXCL12 activation, in turn, promoted the proliferation of skeletal muscle satellite cells, which could be integral for adaptive remodelling following exercise.In conclusion, the research presented in this thesis emphasises the central role of physical activity in improving human health, with a specific focus on the ability of exercise to induce adaptations in skeletal muscle. The findings herein shed light on the intricate molecular mechanisms underlying skeletal muscle responses to physical activity, which contribute to the metabolic fitness of this tissue and of the human body as a whole.List of scientific papersI. Smith JAB, Savikj M, Sethi P, Platt S, Gabriel BM, Hawley JA, Dunstan D, Krook A, Zierath JR, Näslund E. Three weeks of interrupting sitting lowers fasting glucose and glycemic variability, but not glucose tolerance, in free-living women and men with obesity. Am J Physiol Endocrinol Metab. 2021;321:2, E203-E216. https://doi.org/10.1152/ajpendo.00599.2020 II. Pillon NJ, Gabriel BM, Dollet L, Smith JAB, Sardón Puig L, Botella J, Bishop DJ, Krook A, Zierath JR. Transcriptomic profiling of skeletal muscle adaptations to exercise and inactivity. Nat Commun. 2020;11:470. https://doi.org/10.1038/s41467-019-13869-w III. Smith JAB, Gabriel BM, Krook A, Zierath JR, Pillon NJ. Downregulation of NR4A3 During Inactivity Alters Glucose Metabolism and Impairs Translation in Human Skeletal Muscle. [Manuscript]IV. Pillon NJ, Smith JAB, Alm PS, Chibalin AV, Alhusen J, Arner E, Carninci P, Fritz T, Otten J, Olsson T, van Doorslaer de Ten Ryen S, Deldicque L, Caidahl K, Wallberg-Henriksson H, Krook A, Zierath JR. Distinctive exercise-induced inflammatory response and exerkine induction in skeletal muscle of people with type 2 diabetes. Sci Adv. 2022;8:36, eabo3192. https://doi.org/10.1126/sciadv.abo3192 </p
Immediate skin-to-skin contact after a very preterm birth : supporting the parent-infant relationship
No full textBackground: Preterm birth poses challenges that may negatively affect parental mental health and infant development. A well-functioning parent-infant relationship can mitigate the risk of preterm birth on infant development, and early interventions that foster close parent-infant contact and support parenthood from birth are therefore warranted. Skin-to-skin contact between parents and infants has been found to decrease distress in parents and improve parent-infant interaction behaviors, yet little is known regarding its benefits when initiated immediately after birth for more vulnerable infants. Further, there is a lack of knowledge regarding the difference in effects of SSC provided during the first postnatal hours and later in the neonatal period. The overall aim of this thesis was to gain knowledge about, and a deeper understanding of, the impact of skin-to-skin contact between parents and very preterm infants when practiced immediately after birth, as compared to later in the neonatal period, on parents’ mental health and the parent-infant relationship within the first year of life.Methods: The four studies in this thesis derive from two multicenter studies: a prospective longitudinal study, “The 2nd International Closeness Survey” (study I) and a randomized clinical trial, “The Immediate Parent-Infant Skin-to-Skin Study” (IPISTOSS) (study II-IV). Study I involved mothers (n=684) and fathers (n=574) to preterm infants born less than 35 gestational weeks of age from 23 neonatal units in 15 countries. In study I, associations between the amount of proximity between parents and infants in the neonatal unit, including time spent in skin-to-skin contact, and parents’ symptoms of depression (assessed with Edinburgh Postnatal Depression Scale, EPDS) at discharge and at 4 months were investigated. Study II-IV derived from the randomized controlled trial IPISTOSS that compared care in skin-to-skin contact immediately after birth with standard incubator care for very preterm infants (28–33 gestational weeks of age) in three neonatal units in Sweden and Norway. Study II included 73 parent couples to 91 infants and investigated the effect on parents’ symptoms of depression (EPDS) and anxiety (assessed with Spielberger State-Trait Anxiety Inventory, STAI) within the infants’ first year of life. Study III included 71 infants and their 56 mothers and investigated the effect on mother-infant interaction quality (measured with the Parent-Child Early Relational Assessment, PCERA) at 4 months. In study IV, 12 parents participated in individual interviews at the time of discharge to home to explore their experiences of immediate skin-to-skin contact and the care and support from healthcare staff.Results: Study I found no association between the duration of parent-infant proximity in the neonatal unit and symptoms of depression in parents at discharge and at 4 months. Study II found that immediate skin-to-skin contact after a very preterm birth decreased EPDS scores in mothers (mean [SD] 9.8 [6.0] vs 12.3 [5.9] in the control group, p Conclusion: The main findings in this thesis suggest that the practice of skin-to-skin contact in the immediate postpartum period has an impact on the early parent-infant relationship following a very preterm birth, which is supported by the positive influence of immediate skin-to-skin contact on parents’ mental health and mother-infant interaction quality as well as by parents’ experiences. Beyond the immediate postpartum period, parent-infant proximity and skin-to-skin contact in the NICU need to be continued to be supported, along with other elements within infant- and family-centered developmental care that may further contribute to parental mental health after a preterm birthList of scientific papersI. Lehtonen L, Lilliesköld S, De Coen K, Toome L, Gimeno A, Caballero S, Tameliene R, Laroche S, Retpap J, Grundt H, Van Hoestenberghe MR, Skene C, Pape B, Axelin A, Separation, Closeness Experiences in Neonatal Environment (SCENE) research group. Parent-infant closeness after preterm birth and depressive symptoms: A longitudinal study. Frontiers in Psychology. 2022; 22;13:906531. https://doi.org/10.3389/fpsyg.2022.906531 II. Lilliesköld S, Lode-Kolz K, Rettedal S, Westrup B, Bergman N, Sorjonen K, Ådén U, Mörelius E, Jonas W. Skin-to-skin contact at birth for very preterm infants and symptoms of depression and anxiety in parents during the first year: A secondary outcome of a randomized clinical trial. [Manuscript]III. Lilliesköld S, Lode-Kolz K, Rettedal S, Lindstedt J, Linnér A, Markhus Pike H, Ahlqvist-Björkroth S, Ådén U, Jonas W. Skin-toskin contact at birth for very preterm infants and mother-infant interaction quality at 4 months: A secondary analysis of the IPISTOSS randomized clinical trial. JAMA Network Open. 2023; 6(11): e2344469. https://doi.org/10.1001/jamanetworkopen.2023.44469 IV. Lilliesköld S, Zwedberg S, Linnér A, Jonas W. Parents' experiences of immediate skin-to-skin contact after the birth of their very preterm neonates. Journal of Obstetric, Gynecological & Neonatal Nursing. 2022; 51(1): 53-64. https://doi.org/10.1016/j.jogn.2021.10.002 </p
Hepatic diseases at the molecular level : identifying novel targets and biomarkers through transcriptomics
No full textIn the last few decades, the prevalence of obesity and metabolic diseases have dramatically increased and pose a major threat to global healthcare systems. The chronic excess of carbohydrates and fat is a risk factor for many diseases, including cardiovascular disease, several types of cancer, and metabolic dysfunction-associated steatotic liver disease (MASLD). Concomitant with the obesity pandemic, MASLD has become the most prevalent liver disease and affects more than a third of the global population. Early MASLD stages are characterized by hepatic lipid droplet accumulation that are considered asymptomatic, however, more severe stages include liver inflammation, scarring, liver cirrhosis and even hepatocellular carcinoma. Apart from recently approved drugs like Wegovy, weight loss is the only effective treatment, hence creating a demand for new therapeutic targets for MASLD as well as reliable diagnostic biomarkers.In the work presented in this doctoral thesis, we explored novel potential therapeutic targets and diagnostic biomarkers in MASLD and hepatocellular carcinoma by combining molecular biology techniques, high-throughput sequencing-based methods such as transcriptomics, and computational analyses.In paper 1, we took advantage of the observation that women of fertile age are less frequently diagnosed with MASLD than women after menopause or men, and investigated the protective effects of estrogen signaling in MASLD. We found that only male mice fed a high-fat diet (HFD) developed intrahepatic lipid droplets, and treatment with different estrogen-like molecules ameliorated this phenotype. Using transcriptomics, we dissected the altered hepatic gene and pathway landscape upon activated estrogen signaling and determined affected cell types by integrating publicly available single cell RNA-seq atlases. Since transcriptomic changes can occur via modulation of the epigenomic landscape, we profiled the histone modifications histone 3 lysine 27 acetylation (H3K27ac) and histone 3 lysine 4 trimethylation (H3K4me3) using chromatin immunoprecipitation followed by sequencing (ChIP-seq). We found that estrogen treatment also partly reversed diet-induced changes of these histone modifications. Additionally, we identified enhancer gene pairs that were sensitive to diet and estrogen signaling. In a publicly available RNA-seq dataset comprising 206 patients with MASLD, we confirmed that many genes altered in mice followed similar gene expression trends in human disease. Lastly, we found that the TEA associated domain 1 (TEAD1) gene, encoding an important transcription factor of the Hippo pathway, was among the identified candidate genes. TEAD1 inhibition in a mouse cell line and primary human hepatocyte-derived spheroids demonstrated that TEAD1 regulates hepatic steatosis and could be a promising therapeutic target.In paper 2, we uncovered novel serological diagnostic biomarkers for MASLD using a transcriptomics approach. We re-analyzed two RNA-seq datasets comprising more than 300 patients with MASLD, and identified genes that encoded for secreted proteins and were upregulated in disease compared to healthy individuals. We found that the protein levels of our top-ranked candidate, Sulfatase 2 (SULF2), were significantly increased in the serum of 31 patients with MASLD compared to 11 healthy individuals. These results suggest that SULF2 is a promising candidate to non-invasively diagnose MASLD.In paper 3, we investigated the roles of RNA binding proteins (RBPs) in hepatocellular carcinoma (HCC) since this class of proteins has been implicated to regulate central cell functions. After shortlisting candidate RBPs that were associated with low patient survival, we depleted the mRNAs of ten RBPs in HCC cell models and characterized the effect on cell survival and the transcriptome. A noncanonical RBP, i.e., RBP without harboring known RNA-binding domains (RBDs), called T-complex protein 1 subunit gamma (CCT3) exhibited the strongest effect on proliferation and surprisingly deregulated many noncoding (nc)RNAs. One long noncoding (lnc)RNA called LINC00326 was upregulated upon CCT3 depletion, and we hypothesized that this link could be responsible for cancer cell death. We mimicked the LINC00326 upregulation by using a CRISPR activation (CRISPRa) approach that endogenously increased LINC00326 RNA levels and found a strong detrimental effect on HCC cell survival. We found evidence that CCT3 and LINC00326 physically interact. Moreover, we determined that CCT3 and LINC00326 were functionally dependent. The simultaneous knockdown of CCT3 and LINC00326 partially rescued the phenotype on cell survival, indicating that CCT3 killed HCC cells by upregulation of LINC00326. Lastly, we investigated through which mechanism the CCT3-LINC00326 axis functioned and uncovered higher lipid peroxidation levels and reduced intracellular storage lipids upon CCT3 knockdown or LINC00326 overexpression, suggesting that this favored HCC cell death.In paper 4, we developed a cost-effective and easy-to-apply method to overcome low transfection efficiencies in experiments dependent on large plasmids. We found out that the addition of small plasmids to large CRISPR-Cas9 plasmids increased transfection efficiency and reduced cell death. We uncovered that the method elevated transfection efficiency in all tested cell lines and types, including several hard-to-transfect cell lines and primary cells. The observed benefit was independent of gene content on the small plasmid.List of scientific papersI. Estrogen receptor activation remodels TEAD1 gene expression to alleviate hepatic steatosis. Christian Sommerauer*, Carlos J Gallardo-Dodd*, Christina Savva, Linnea Hases, Madeleine Birgersson, Rajitha Indukuri, Joanne X Shen, Pablo Carravilla, Keyi Geng, Jonas Nørskov Søndergaard, Clàudia Ferrer-Aumatell, Grégoire Mercier, Erdinc Sezgin, Marion Korach-André, Carl Petersson, Hannes Hagström, Volker M Lauschke, Amena Archer, Cecilia Williams, Claudia Kutter. Molecular Systems Biology. 2024, 20:374-402. *Equal contribution. https://doi.org/10.1038/s44320-024-00024-x II. Sulfatase 2 mRNA and serum levels correlate with metabolic dysfunction and increase in early stages of MASLD. Christian Sommerauer, Rasmus M Sandsdal, Carlos J Gallardo-Dodd, Signe Sørensen Torekov, Hannes Hagström, Claudia Kutter. [Manuscript]III. CCT3-LINC00326 axis regulates hepatocarcinogenic lipid metabolism. Jonas Nørskov Søndergaard, Christian Sommerauer, Ionut Atanasoai, Laura C Hinte, Keyi Geng, Giulia Guiducci, Lars Bräutigam, Myriam Aouadi, Lovorka Stojic, Isabel Barragan, Claudia Kutter. Gut. 2022, 71:2081-2092. https://doi.org/10.1136/gutjnl-2021-325109 IV. Successful delivery of large-size CRISPR/Cas9 vectors in hard-to-transfect human cells using small plasmids. Jonas Nørskov Søndergaard, Keyi Geng*, Christian Sommerauer*, Ionut Atanasoai, Xiushan Yin, Claudia Kutter. Communications Biology. 2020, 3:319. *Equal contribution. https://doi.org/10.1038/s42003-020-1045-7 </p
Exploring allergic and irritant contact dermatitis : biomarkers, mechanisms, and allergen uptake
No full textContact dermatitis (CD) is a common disease resulting from direct skin contact with allergens or irritants and greatly affects the life quality of suffering individuals. It is categorized into two main forms, namely allergic CD (ACD) and irritant CD (ICD), depending on the triggering agent. Despite having distinct underlying mechanisms, the clinical presentation of ACD and ICD can provoke resembling clinical manifestations, particularly in chronic cases, which hampers the clinical discrimination and subsequent disease management strategies. Furthermore, while the pathogenesis of ACD and ICD is better understood, effective therapeutics remain elusive. Additionally, the ongoing introduction of novel materials in industrial and consumer products, including metal nanoparticles, underscores the need for continuous evaluation of their skin absorption and potential to induce ACD. The overall aim of this thesis was to elucidate pathomechanisms of CD, offering new insights into its pathology, identifying disease classifiers, and comprehensively characterizing the absorption and distribution patterns of metal-based allergens in human skin.Transcriptomic profiling of human skin biopsies obtained from positive patch test reactions to allergens and irritants was conducted in Paper I-III. In Paper I, integrative transcriptomic analysis and machine-learning approaches were utilized to identify disease-specific signatures for robust disease classification. By analyzing the transcriptomic profiles of positive patch test reactions to four allergens and two irritants with distinct properties, 28 disease-classifying gene sets with high prediction accuracy were identified. Notably, the proposed biomarker gene sets were validated in an independent patient group (n=31), and their potential clinical applicability was confirmed through performance testing in external datasets. In Paper II, time-dependent changes in the transcriptomic signature of ACD induced by nickel were investigated, revealing leukocyte compositional alterations in the late phases (48 and 96h) of nickel exposure. Specifically, deconvolution algorithms and enrichment analyses suggested infiltration and activation of various innate immune cells including natural killer (NK) cells, mast cells, and inflammatory macrophages. Notably, the transcriptomic profiling results from both Paper I and II indicated that NK cell activation might distinguish ACD from ICD. In Paper III, dysregulation of the short non-coding microRNA (miRNA), and its regulatory impact on the post-transcriptional expression in ACD and ICD was explored. The results revealed miRNA-mRNA pairs enriched for processes relevant to CD, identifying miR-155-5p, miR-142-3/5p and miR-497-5p as potential key regulators of the inflammatory process of ACD. Paper IV and V assessed the hazards of skin exposure to the metal nanoparticles, specifically CoNPs, in individuals with cobalt contact allergy, identifying CoNPs as a hazardous material capable of inducing ACD. Furthermore, the uptake and distribution of CoNPs in the skin were evaluated in Paper V, showing minimal uptake of CoNP compared to the standard test substance of cobalt contact allergy.Overall, the findings from these studies provide novel insights into the molecular pathways involved in CD, encompassing the delineation of key cellular players and associated pathways, regulatory mechanisms, and proposal of robust disease classifiers of ACD and ICD. Additionally, the results underscore the potential hazards of allergens in nanoparticulate form as a potent inducers of elicitation responses in contact allergic individuals.List of scientific papersI. Fortino V*, Wisgrill L*, Werner P, Suomela S, Linder N, Jalonen E, Suomalainen A, Marwah V, Kero M, Pesonen M, Lundin J, Lauerma A, Aalto-Korte K, Greco D, Alenius H, Fyhrquist N. Machine-learning-driven biomarker discovery for the discrimination between allergic and irritant contact dermatitis. Proc Natl Acad Sci U S A. 2020 Dec 29;117(52):33474- 33485. Epub 2020 Dec 14. *These authors contributed equally. https://doi.org/10.1073/pnas.2009192117 II. Wisgrill L, Werner P, Jalonen E, Berger A, Lauerma A, Alenius H, Fyhrquist N. Integrative transcriptome analysis deciphers mechanisms of nickel contact dermatitis. Allergy. 2021 Mar;76(3):804-815. Epub 2020 Aug 12. https://doi.org/10.1111/all.14519 III. Werner P, Wisgrill L, Riskumäki M, Jalonen E, Vendelin J, Suomela S, Lauerma A, Alenius H, Fyhrquist N. Identification of novel miRNA-mRNA regulatory networks in contact dermatitis by integrated microarray analysis. Allergy. 2021 Apr;76(4):1257-1261. Epub 2020 Sep 20. https://doi.org/10.1111/all.14578 IV. Midander K*, Werner P*, Isaksson M, Wisgrill L, Lidén C, Fyhrquist N, Julander A. Cobalt nanoparticles cause allergic contact dermatitis in humans. Br J Dermatol. 2022 Oct 28:ljac043. Epub ahead of print. *These authors contributed equally. https://doi.org/10.1093/bjd/ljac043 V. Werner P, Schaier M, Braun G, Julander A, Midander K, Isaksson M, Köllensperger G*, Wisgrill L*, Fyhrquist N. Integrative transcriptomic analysis and assessment of skin permeation of cobalt nanoparticles and cobalt salts in human skin by RNAseq and LA-ICP-TOFMS. 2024. *These authors contributed equally. [Manuscript]</p
Prostate cancer : investigating diagnosis, treatment and outcomes
No full textBackground: Prostate cancer is difficult to study. The prostate is located so that it is hard to palpate and because of the dense pelvic bones it is hard to visualize using radiology such as computed tomography. The prostate increases in size with age and it can be irregular in shape and consistency without the presence of cancer. Prostate cancer can be aggressive and deadly on one hand, while on the other hand, it can also be clinically insignificant, never causing any issues for the patient. Even if ignoring the most benign prostate cancers, the natural course of most prostate cancers is still slow. This means that researchers need long follow up times, up to 20 years, to observe cancer specific mortality. The efficiency of any diagnostic test or treatment often needs to be evaluated with surrogate endpoints because of the indolent nature of the disease. Prostate cancer should ideally be diagnosed before symptoms arise, since prostate cancer, in general, does not cause symptoms until it has become locally advanced or developed into metastatic disease. These are some of the many challenges for clinicians and researchers working with prostate cancer patients. Several blood-based tests have been suggested to enhance prostate cancer testing. In a large screening study, the Stockholm3 test has been shown to reduce the number of men undergoing prostate biopsies and to lower the detection rate of low-grade cancers, while still maintaining the detection rate of ISUP Gleason Group 2 to 5 cancers. The Stockholm3 test was trained and validated on prostate biopsies, and it is unknown how well the test performs, compared to PSA and PSA-density (PSA/prostate volume), when the whole prostate is available for pathological analysis. There are studies indicating that patients with prostate cancer could benefit from the use of existing drugs and vaccines not intended for prostate cancer so called drug repurposing. Recent findings suggest that the oral cholera vaccine could be effective in treating inflammatory diseases and certain prevalent cancers. A Swedish register study showed that administering the cholera vaccine following a prostate cancer diagnosis led to a nearly 50% reduction in prostate cancer-specific mortality rates. The ideal scenario after a prostatectomy for prostate cancer is to have undetectable levels of PSA, usually defined as less than 0.1 ng/mL. Nonetheless, some patients exhibit measurable PSA levels above 0.1 ng/mL at their first follow-up post-radical prostatectomy, a condition known as PSA persistence. This condition suggests possible metastatic disease, benign prostate remnants, or incomplete surgical removal. For those whose PSA persistence results from non-radical surgery, adjuvant radiotherapy targeting the prostate area might be beneficial.Aims: Study 1. We aimed to validate the diagnostic performance of the Stockholm3 test in an independent, clinical practice cohort. Study 2. We aimed to investigate if oral cholera vaccine had an effect on overall mortality for men diagnosed with prostate cancer. Study 3. We aimed to evaluate and compare the effectiveness of PSA and PSAdensity against the Stockholm3 test in predicting the occurrence of clinically significant prostate cancer following prostatectomy. Study 4. We aimed to investigate the long-term prognosis of patients with PSA persistence, which risk factors that predict PSA persistence. As a secondary objective we aimed to assess the impact of adjuvant radiotherapy for men with PSA persistence.Methods: Study 1. The study involved men, aged 45–75 who had never been diagnosed with prostate cancer and were scheduled for prostate biopsies at one of three clinics in Norway and Sweden. Before undergoing systematic prostate biopsies, these men had their blood drawn for Stockholm3 testing. Clinically significant prostate cancer was defined as ISUP Grade Group (GG) 2 or above. We compared the predictive performance for prostate cancer of the Stockholm3 test, PSA and PSA-density by modelling receiver operating characteristics curves and calculating the area under the curves. Study 2. We identified men diagnosed with prostate cancer using the Stockholm PSA and Biopsy Register, which is a population-based register of men from Stockholm, Sweden, with detailed social and prostate cancer data. Using the Swedish Prescribed Drug Register and combining it with the register of a large private vaccine clinic we could identify men who were exposed to vaccine after their prostate cancer diagnosis. To assess the effect of oral cholera vaccine on overall mortality we used a marginal structural Cox regression model. We made the same analysis for several other travel vaccines and malaria prophylaxis to use as a comparison. Study 3. The initial STHLM3 study included 58,818 men who were included in a screening by invitation of which 755 men underwent prostatectomy. These were included in the study. We defined clinically significant prostate cancer after prostatectomy as final pathology ISUP grade 2 or more, pT-stage 3 or more or tumor volume more than 0.5 cm3. We calculated the area under the curve (AUC) for predicting clinically significant prostate cancer at whole prostate specimen pathology for Stockholm3, PSA and PSA-density. Study 4. Using the Stockholm PSA and Biopsy Register, used in Study 2, we identified all men in Stockholm, Sweden who underwent a prostatectomy from 2004 to 2018. PSA persistence was defined as a PSA of >0.1 ng/mL at first blood test after 4 weeks or more postoperatively. Adjuvant radiotherapy was defined as radiotherapy within the first year after prostatectomy. Our endpoints were all cause mortality, prostate cancer specific mortality and disease progression, defined as hormonal treatment. Statistics used were chi-square test, Mann- Whitney U test, multivariate Cox regression and competing hazards regression.Results: Study 1. Out of 533 participants, 263 (49%) were diagnosed with prostate cancer, with 162 of these men exhibiting ISUP Gleason Group (GG) ≥ 2 cancer. The Stockholm3 test showed better diagnostic effectiveness in identifying GG ≥ 2 prostate cancer compared to PSA and PSA-density. The predictive accuracy (area under the curve or AUC) for high-grade tumors was 64.2 for PSA alone, 74.8 for PSA density, and 85.9 for the Stockholm3 test. By applying a 10% risk threshold on the Stockholm3 test for when to biopsy, 38% of biopsies could be avoided, albeit with a delayed diagnosis in 6% (10 individuals) of cases with GG ≥ 2 cancer. Conversely, a biopsy threshold set at a PSA-density of 0.1 saved 35% of biopsies but resulted in a delayed diagnosis in 16% (26 individuals) of the men with GG ≥ 2 cancer. Study 2. We included 22,738 men with prostate cancer in the study, of which 4,248 (19%) died during follow-up. The median follow-up time was 5 years. 717 men received oral cholera vaccine after their prostate cancer diagnosis of which 36 died during follow-up. Receiving oral cholera vaccine after prostate cancer diagnosis was associated with a 54% reduced risk of dying (HR 0.46, p-value 0.0001). However, the same effect, or better, was seen for several of the other travel vaccines and for malaria prophylaxis. Study 3. We included 755 patients from the initial STHLM3 study that underwent prostatectomy. Postoperatively 93% had clinically significant prostate cancer on final pathology. For the whole study cohort, the AUC for clinically significant prostate cancer on whole prostate specimen was 77.3 (71.3-82.7) for Stockholm3, 70.5 (63.7-77.2) for PSA and 74.3 (68.3-80.3) for PSA-density. For men with diagnostic ISUP GG1 the AUC for clinically significant prostate cancer was 78.0 (68.1 -88.0) for Stockholm3, 65.3 (53.4-77.2) for PSA and 70.3 (59.3- 81.3) for PSA-density. Study 4. We included 8,905 men who underwent prostatectomy, among whom 496 (6%) exhibited PSA persistence post-surgery. Significant predictors of PSA persistence included the Gleason score, clinical T-status, and elevated preoperative PSA levels. Men experiencing PSA persistence showed increased rates of all-cause mortality (HR 2.23, 95% CI 1.60-3.12), cancer-specific mortality (HR 5.32, 95% CI 2.73-10.37), and an elevated risk of cancer progression (HR 6.44, 95% CI 5.40-7.68). Of these, 95 men with PSA persistence underwent adjuvant radiotherapy. While adjuvant radiotherapy was linked to a reduced risk of disease progression, the association was not statistically significant (SHR 0.53, 95% CI 0.14-1.96).Conclusion: Study 1. The Stockholm3 test was independently validated in a clinical practice cohort and reduces the number of unnecessary biopsies while delaying diagnosis for a limited number of men. Study 2. We conclude that the effect of oral cholera vaccine seen on overall mortality is likely due to a healthy traveler bias and caused by residual confounding. Study 3. Stockholm3 and PSA-density are better predictors for clinically significant prostate cancer after prostatectomy compared to PSA. This validates the Stockholm3 test even further. Stockholm3 has the potential to be used for selecting men for active monitoring or treatment, but further studies are needed. Study 4. Men with PSA persistence following a prostatectomy demonstrated worse prognosis and more adverse tumor characteristics than those with undetectable PSA levels at first control after surgery. Additionally, adjuvant radiotherapy did not significantly impact disease progression, cancer-specific mortality, or overall mortality in these patients.List of scientific papersI. A. Möller, H. Olsson, H. Grönberg, M. Eklund, M. Aly, T. Nordström. The Stockholm3 blood-test predicts clinically significant cancer on biopsy: Independent validation in a multi-center community cohort. Prostate Cancer and Prostatic Diseases. 2019, 22, 137-142. https://doi.org/10.1038/s41391-018-0082-5 II. A. Möller, K. Schwamborn, A. Spillmann, J. Hoogstraate, R. Szulkin, O. Akre, L. Egevad, M. Clements, M. Aly. Travel vaccines are strongly associated to reduced mortality in prostate cancer patients - a real effect or residual confounding? Vaccine. 2022, 40, 3797-3801. https://doi.org/10.1016/j.vaccine.2022.05.028 III. A. Möller, T. Palsdottir, M. Eklund, T. Nordström, L. Egevad, Markus Aly. The performance of Stockholm3, PSA-density and PSA for predicting clinically significant prostate cancer after prostatectomy. [Submitted]IV. A. Möller, O. Akre, T. Nordström, M. Eklund, L. Egevad, M. Aly. PSA persistence after prostatectomy – predictors, prognosis and the effect of adjuvant radiotherapy. [Manuscript]</p
Prediction of mortality in hip fracture patients
No full textBackground: Hip fractures are associated with elevated mortality and require extensive health care resources. As populations grow increasingly older the total amount of hip fractures has also escalated. An increasing amount of people live to be more than 100 years old and this group is expected to reach more than 50 times current estimated levels during the course of this century. As a result, the age span of geriatric hip fracture patients is now almost 50 years, and as the defining characteristic of these patients aside from their age is merely the anatomical region of the fracture, it is a very heterogenous group. Our main aim was to develop and externally validate machine learning models for prediction of mortality after hip fracture to identify which patients are the most at risk. In preparation for this we wanted to explore the value of routine blood samples for prediction of mortality after hip fracture. Furthermore, we wanted to investigate temporal trends of incidence and mortality in the emerging group of centenarian hip fracture patients, and finally train machine learning models for estimations of mortality 1, 3, 6 and 12 months after hip fracture and externally validate these models so they ultimately could be used clinically.Methods: Studies 1 and 3 of the thesis are based on a database of consecutive hip fracture patients from Bispebjerg University Hospital in Copenhagen, Denmark, queried at different time points for different data. Study 1 consisted of 792 patients and their age, sex and admission blood samples as well as follow up data on mortality. The predictive value of preoperative blood samples was compared using receiver operating characteristics curves (ROC) and univariate and multivariate regression was performed to determine which blood samples were associated with 3- month mortality. Study 2 was a nationwide survey of all hip fractures in Denmark over a 17-year period. A total of 517 centenarian patients had suffered hip fractures and temporal trends of incidence and mortality were analyzed. Furthermore, comparisons to hip fracture patients from the same cohort with the age interval 70 to 99 (n = 124,007) were done concerning incidence, mortality and comorbidities. Study 3 consisted of 1186 patients from the same database as study 1 but from a longer time interval and with a wide range of biochemical and anamnestic data as potential predictors, as well as follow up data on mortality. Three different types of machine learning models, a Random Forest (RF), an extreme gradient boosting (XGB) and a generalized linear model (GLM) were developed for 1-, 3-, 6-, and 12- month mortality using the 10 to 13 most important features selected by the Boruta algorithm. The data was partitioned so that 70% was used for training and 30% was used as a holdout test set, results were compared using the area under the curve (AUC) for receiver operating characteristics (ROC) curves, calibration slope and intercept and decision curve analysis (DCA). Study 4 was based on 5055 hip fracture patients from Karolinska Solna and Huddinge from a 10-year period, data was collected from RIKSHÖFT and from Karolinska Data (KARDA). The models developed in study 3 were deployed and comparisons using AUC and calibration curves and DCA were performed. The best performing models were recalibrated as event rates were lower in the external validation data than in the development data.Results: Elevated creatinine on admission blood samples was associated with an almost threefold increased 3-month mortality and had an AUC of 0.69 (0.64–0.74). The other blood samples under study had lower AUC values and on multivariate regression only age, creatinine, potassium and albumin remained as risk factors. For the centenarian hip fracture patients, incidence had declined slightly but mortality had remained stable during the study period. The centenarians under study had less registered comorbidities as measured by Charleston comorbidity index (CCI) than the comparison group of younger hip fracture patients. 68 % of centenarian patients had a CCI of 0 versus 46 % in the comparison group, but the mortality was higher. The developed machine learning models had AUC values of approximately 0.8 for all timepoints but the XGB model was the overall best performer as it was more calibrated and had better DCA. An online tool based on the XGB models has been developed for evaluation and educational purposes (https://hipfx.shinyapps.io/hipfx/). For the external validation study, mortality was lower in the Swedish data set than in the data that the models were developed on. The AUC values especially for the longer timeframes was acceptable but lower than in the development study and again, it was the XGB model that performed best overall with values of 0.72, 0.74, 0.75 and 0.77 for 1-, 3-, 6-, and 12-month mortality respectively. In line with the observed difference in event rates models were not well calibrated, so the XGB models were recalibrated using bootstrapped isotonic regression with much improved calibration.Conclusions: Of the blood samples under study, creatinine was the best predictor of mortality after hip fracture with an AUC of 0.69 which is similar to results in studies evaluating the use of ASA classification, CCI index and Possum score, so admission blood samples could be an interesting addition to prediction models for mortality after hip fracture. Our findings on centenarian hip fracture patients suggest the compression of morbidity theory proposed in many other studies on subjects older than 100 years which could be an important finding as this age group is expected to increase drastically in the coming century. Based on our results, healthcare needs do not seems to increase proportionally with age for the group under study. Furthermore, as at least some of the known predictors for mortality does not seem to be linearly separable, prediction models for mortality in hip fracture populations might benefit from machine learning techniques to model complex interactions between factors. The XGB models developed in the third study of the thesis had the best results for prediction of 1-, 3-, 6- and 12-months mortality after hip fracture. The models performed acceptably in the external validation, especially for the later timepoints, but needed recalibration as the mortality rates were different in the development data and the external validation. The previously developed online tool will be updated with the recalibrated models so that they can be used clinically.List of scientific papersI. Value of routine blood tests for prediction of mortality risk in hip fracture patients. Mathias Mosfeldt, Ole B Pedersen, Troels Riis, Henning O Worm, Susanne van der Mark, Henrik L Jørgensen, Benn R Duus, Jes B Lauritzen. Acta Orthop. 2012 Feb;83(1):31-5. https://doi.org/10.3109/17453674.2011.652883 II. Centenarian hip fracture patients: a nationwide population-based cohort study of 507 patients. Mathias Mosfeldt , Christian M Madsen, Jes B Lauritzen, Henrik L Jørgensen. Acta Orthop. 2019 Aug;90(4):342-347. https://doi.org/10.1080/17453674.2019.1602386 III. Development and internal validation of a multivariable prediction model for mortality after hip fracture with machine learning techniques. Mathias Mosfeldt; Henrik Løvendahl Jørgensen; Jes Bruun Lauritzen; Karl-Åke Jansson. Calcif Tissue Int. 2024 Apr 16. Online ahead of print. https://doi.org/10.1007/s00223-024-01208-1 IV. External validation of machine learning models for estimation of mortality 1, 3, 6 and 12 months after hip fracture. Mathias Mosfeldt; Henrik Løvendahl Jørgensen; Jes Bruun Lauritzen; Karl-Åke Jansson. [Manuscript]</p
Parkinson's disease and Gaucher disease : focus on the GBA1 link
No full textParkinson’s disease (PD) is the 2nd most common neurodegenerative disorder in the world, characterized by both motor as well as many non-motor features. There are many symptomatic treatments available, that may shift significantly during the course of the disease. The biggest medical need in PD management however is treatments that actively targets disease pathology, thus being able to slow or stop the disease progression. When such therapies become available, another obstacle to administer them as early as possible in the disease progress is the fact that PD diagnosis is based on motor symptoms, that does not appear until late in the neurodegenerative process. Thus, the notion of prodromal PD has gained increased research attention in the search for earlier signs and biomarkers specific for PD.The most important genetic risk marker for PD development is mutations in the GBA1 (Glucocerebrosidase 1) gene. The gene codes for a lysosomal enzyme called Glucocerebrosidase, that normally degrades Glucocerebroside - a glucolipid mainly found in cellular membranes. When the GBA1 gene is altered, the enzyme becomes defective, and Glucocerebroside is pathologically accumulated within cells. In its biallelic state, GBA1 mutations gives rise to Gaucher disease (GD), a systemic disease mainly characterized by visceral, hematological and skeletal manifestations, but in rare cases also shows severe neurological signs and symptoms. There is a particular high endemic incidence of neuronopathic GD in the northern parts of Sweden with a distinct phenotype called “the Norrbottnian GD variant”. The GBA1-PD connection has therapeutic implications, since disease modifying treatments targeting the lysosomal pathway may be a way to find diseasemodifying treatments for PD. Since GBA1 mutations, and subsequent lysosomal dysfunction, most likely are linked to PD pathology, an overall aim of this thesis is to investigate different aspects of this gene in relation to the Swedish PD population.In Study I and II we screened the most common variations in the GBA1 gene in the largest Swedish PD cohort reported. Comparing 1625 PD patients and 2025 healthy controls from 5 different sites throughout Sweden, we found the most significant association with highest effect size with the severe mutation L444P. There was also a markedly skewed geographical distribution of this mutation, with 58% of the mutation carriers coming from northern Sweden, consistent with the high GD prevalence in Norrbotten. The other GBA1 variants N370S and E326K also showed association to PD risk, but not as markedly as the L444P mutation. The relevance for the GBA1-variant T369M on PD risk has been debated, and thus we also analyzed this in our cohort. However, despite a slight overrepresentation of T369Mcarriers in the PD cohort, this difference did not prove significant in our population. In conclusion, alterations in the GBA1 gene results in a significantly greater risk of developing PD in Sweden. However, there are marked differences between which specific genetic alteration is investigated – the mutation matters. There is also a marked geographical accumulation of the severe GBA1 mutation L444P in northern Sweden.Study III assessed polyneuropathy (PNP) in a small number of PD patients and healthy controls. The increased frequency of polyneuropathy we saw in Parkinson’s disease patients can be both a clinical feature of the disease, but also aggravated by the L-dopa treatment and subsequent disturbances in the B-vitamin metabolism, especially Cobalamin. Genetic analyses also revealed an increased PNP risk for PD patients with mutations in the COMT gene, coding for an enzyme implicated in Cobalamin metabolism. However, other investigated mutations in another gene related to B-vitamin metabolism (MTHFR), or to GBA1, did not correlate to PNP risk in PD patients.Study IV followed 12 Norrbottnian GD patients for 10 years, reporting on clinical outcomes. Neurological GD affection was measured with the modified Severity Scoring Tool (mSST), showing slow progressive deterioration over time. The neurological symptoms that mostly contributed to the decline was Ataxia of gait and Cerebellar tremor. Almost all patients had horizontal gaze palsy and 50% had epilepsy at end-of-follow-up. One patient deteriorated rapidly both cognitively and functionally, being diagnosed with Dementia with Lewy bodies. However, for the rest cognition remained mostly stable, although at a slightly impaired level. The main conclusion is that there is an overall slow worsening of neurological signs and symptoms among the Norrbottnian GD patients over time, but many live long and the clinical heterogeneity, despite almost identical genotype, is large.Also, plasma Neurofilament Light (NfL) was assessed in selected GD patients and compared to healthy controls, investigating its possible function as a biomarker for neurological affection in GD. There were no significant differences in NfL levels between GD patients and controls, but a trend for association between NfL levels and neurological affection. This suggests that following NfL over time in larger neurologically affected GD cohorts might reveal interesting associations. However, the clear lack of correlation also raises a warning about being too liberal in using the unspecific marker NfL as a proxy for neurological affection in neuronopathic GD.List of scientific papersI. Ran C, Brodin L, Forsgren L, Westerlund M, Ramezani M, Gellhaar S, Xiang F, Fardell C, Nissbrandt H, Söderkvist P, Puschmann A, Ygland E, Olson L, Willows T, Johansson A, Sydow O, Wirdefeldt K, Galter D, Svenningsson P, Belin AC. Strong association between glucocerebrosidase mutations and Parkinson's disease in Sweden. Neurobiol Aging. 2016 Sep;45:212.e5-212.e11. https://doi.org/10.1016/j.neurobiolaging.2016.04.022 II. Ran C, Brodin L, Gellhaar S, Westerlund M, Fardell C, Nissbrandt H, Söderkvist P, Sydow O, Markaki I, Hertz E, Wirdefeldt K, Svenningsson P. Glucocerebrosidase variant T369M is not a risk factor for Parkinson's disease in Sweden. Neurosci Lett. 2022 Jul 27;784:136767. https://doi.org/10.1016/j.neulet.2022.136767 III. Andréasson M, Brodin L, Laffita-Mesa JM, Svenningsson P. Correlations Between Methionine Cycle Metabolism, COMT Genotype, and Polyneuropathy in L-Dopa Treated Parkinson's Disease: A Preliminary Cross-Sectional Study. J Parkinsons Dis. 2017;7(4):619-628. https://doi.org/10.3233/JPD-171127 IV. Brodin L, Khosousi S, Machaczka M, Kämpe Björkvall C, Svenningsson P. Slow worsening of neurological signs and symptoms in patients with Norrbottnian Gaucher disease type 3b: A 10-year longitudinal follow-up study. [Submitted]</p
Decoding the symphony : illuminating the mammalian mitochondrial RNA binding proteome with a novel interactome approach
No full textMitochondria, colloquially known as the "powerhouses of the cell" are crucial for generating cellular energy through a process known as oxidative phosphorylation (OXPHOS). These minute but mighty organelles are distinct and semi-independent entities, containing their own DNA, which is circular in nature. This DNA has the capacity for replication, transcription, and translation, processes collectively referred to as mitochondrial gene expression. Out of the OXPHOS system's peptides, mitochondria encode for 13, with the remainder being produced in the nucleus and then transported into the mitochondria. The complex system of gene expression within mitochondria relies on an intricate interaction among DNA, RNA, and proteins. However, disruptions in this system have been linked to a variety of diseases, such as diabetes, neurodegenerative disorders, and cancer. RNA-binding proteins (RBPs) are essential in regulating various aspects of RNA's lifecycle, including its synthesis, stability, processing, epitranscriptomic modification, translation, and degradation. Yet, the specifics of how these proteins operate remain partially understood.My doctoral research has been devoted to uncovering new RBPs and elucidating their roles in mitochondrial gene expression. Specifically, in paper 1, I introduced a technique named ‘mt-TRIzol-RBP-Ex’ for the efficient and unbiased enrichment of mitochondrial RBPs from HEK293T cells, leading to the identification and partial characterization of a potential novel RBP, C8orf82, in mitochondrial translation. Subsequent papers focused on other RBPs such as C6orf203 (in paper 2) and GTPBP8 (in paper 3), as well as translation initiation factors (in paper 4), revealing their indispensable roles in mitochondrial post-transcriptional pathways. The depletion of these proteins resulted in significant impacts on translation.This doctoral thesis provides a comprehensive review of current knowledge on mitochondrial RBPs and presents both preliminary and published findings on the roles of specific, previously uncharacterized, or novel RBPs in mitochondrial gene expression.List of scientific papersI. Shreekara Gopalakrishna, Ilian Atanassov, Aishe Angeletti Sarshad, Daniel Ben Halevy, Shadi Bavafa, Benedikt Beckmann, Joanna Rorbach†. (2024). Enrichment of mammalian mitochondrial RNA-binding proteins using TRIzolEx reveals novel uncharacterised mt-RNA interacting proteins. †Corresponding author. [Manuscript]II. Shreekara Gopalakrishna*, Sarah F Pearce*, Adam M Dinan, Florian A Rosenberger, Miriam Cipullo, Henrik Spåhr, Anas Khawaja, Camilla Maffezzini, Christoph Freyer, Anna Wredenberg, Ilian Atanassov, Andrew E Firth, Joanna Rorbach†. (2019). C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis. Nucleic Acids Res. 2019 Sep 26;47(17):9386-9399. *These authors contributed equally. †Corresponding author. https://doi.org/10.1093/nar/gkz684 III. Miriam Cipullo, Genís Valentín Gesé, Shreekara Gopalakrishna, Annika Krueger, Petra Palenkova, Dmitrii Shiriaev, Yong Liu, Jelena Misic, Yu Cai, Minh Nguyen, Abubakar Abdelbagi, Xinping Li, Michal Minczuk, Ilian Attanasov, Martin Hällberg, Joanna Rorbach†. (2024). GTPBP8 is essential for mitoribosome formation in human mitochondria. †Corresponding author. [Submitted]IV. Cristina Remes, Anas Khawaja, Sarah F Pearce, Adam M Dinan, Shreekara Gopalakrishna, Miriam Cipullo, Vasileios Kyriakidis, Jingdian Zhang, Xaquin Castro Dopico, Olessya Yukhnovets, Ilian Atanassov, Andrew E Firth, Barry Cooperman, Joanna Rorbach†. (2023). Translation initiation of leaderless and polycistronic transcripts in mammalian mitochondria. Nucleic Acids Res. 2023 Jan 25;51(2):891-907. †Corresponding author. https://doi.org/10.1093/nar/gkac1233 </p
Reproductive and lifestyle risk factors for sarcoidosis
No full textSarcoidosis is a systemic inflammatory condition with an unknown origin, characterized by the formation of granulomatous lesions primarily in the lungs and lymphatic system. Despite over a century since it was first described, our understanding of its etiology remains limited. Sarcoidosis incidence peaks in males in mid-thirties (30-50 years old) and in women in mid-fifties (50-60 years old; around the time when menopause starts). This delayed onset in women could be due to exposure to endogenous hormones (i.e. hormones produced within the body), mainly estrogens, suggesting that reproductive and hormonal factors (events associated with changes in levels of estrogens in the blood that occur throughout a woman's life) play a role in disease development. Current research also indicates that lifestyle factors trigger granulomatous inflammation in genetically predisposed people. In this doctoral thesis four individual studies are included. Two of them dealt with reproductive risk factors for sarcoidosis such as age at menarche, pregnancy, menopause (serving as indicators of endogenous hormones) and the use of exogenous hormones (i.e. hormones not produced within the body) such as oral contraceptives and menopausal hormone therapy. The remaining two studies investigated lifestyle risk factors for sarcoidosis, namely cigarette smoking, snus (smokeless tobacco product), obesity, physical activity and alcohol consumption.Study I was a nested case-control study in which we examined whether reproductive and hormonal factors are associated with risk of developing sarcoidosis in women using a population-based cohort in Northern Sweden with questionnaires and health exams completed before sarcoidosis diagnosis. We found that serval indicators of exposure to endogenous hormones (e.g. later age at menopause, earlier age at menarche, earlier age at pregnancy) may contribute to reduction of sarcoidosis risk. On the other hand, exogenous hormones, especially menopausal hormone therapy, were associated with an increased risk.Similarly to Study I, in Study II we conducted a nested case-control study and investigated whether lifestyle risk factors are associated with risk of developing sarcoidosis using prospectively collected data from a population-based cohort in Northern Sweden. We found that current smoking was associated with a 52% lower sarcoidosis risk and former smoking with 33% higher risk. This pattern with current smoking could not be explained away by early signs or symptoms of the disease before diagnosis (so-called preclinical phase) causing symptomatic individuals to stop smoking or report being non-smokers. Snus use, however, was not associated with sarcoidosis. We also showed that there was a 34% increased risk of sarcoidosis associated with being obese but not with being overweight. Moreover, high physically activity was associated with higher risk of sarcoidosis which could be explained by preclinical sarcoidosis, where extensive exercise might worsen the symptoms of undiagnosed sarcoidosis, making it more likely for individuals to seek medical care and receive a diagnosis. No association was found with alcohol consumption.In Study III, using high-quality and prospectively collected data from population-based registers in Sweden, we examined whether menopausal hormone therapy is associated with the risk of developing sarcoidosis in women and whether this risk varied by treatment type, route of administration and duration of use. In this nested case-control study, we found a 25% increased risk of sarcoidosis associated with a history of menopausal hormone therapy use, with women receiving systemic estrogen having the highest risk. We also found a stronger association between short-term use (In Study IV, we performed a systematic review and meta-analysis to summarize the existing literature and estimate the relative risk of sarcoidosis associated with smoking. We found that current smoking was associated with a 39% lower sarcoidosis risk; even when we included studies with data on smoking status collected years before sarcoidosis diagnosis, for which preclinical sarcoidosis is unlikely to explain the pattern. Ever smoking was associated with a small decreased risk of sarcoidosis (13%), while former smoking did not seem to be associated with sarcoidosis.Overall, findings from studies on reproductive risk factors in this thesis suggest that indicators of endogenous hormones, specifically later age at menopause, earlier age at menarche and earlier age at first pregnancy may contribute to a decrease in sarcoidosis risk. However, this beneficial effect does not seem to extend to exogenous hormones such as menopausal hormone therapy. Studies on lifestyle risk factors in this thesis showed that current smoking is associated with a reduced sarcoidosis risk. Obesity and being physically active separately increases the risk of sarcoidosis. Alcohol consumption does not seem to be related to sarcoidosis risk. These findings deepen our understanding of long-suspected (e.g. smoking and obesity) and novel (e.g. hormonal factors, alcohol consumption and physical activity) risk factors for sarcoidosis. However, our quest to examine and detect causal exposures for this enigmatic disease should continue by using actual measurements of levels of estrogen or nicotine, rather than relying on indicators. Future epidemiological studies on risk factors for sarcoidosis should take into account the issue of reverse causation bias due to the long preclinical phase in some sarcoidosis patients. Moreover, since sarcoidosis also has a genetic component, future studies should consider genetics by investigating possible gene-environment interactions.List of scientific papersI. Dehara M, Sachs MC, Kullberg S, Grunewald J, Blomberg A, Arkema EV. Reproductive and hormonal risk factors for sarcoidosis: a nested case–control study. BMC Pulmonary Medicine. 2022;22;43. https://doi.org/10.1186/s12890-022-01834-1 II. Dehara M, Sachs MC, Grunewald J, Blomberg A, Arkema EV. Modifiable lifestyle risk factors for sarcoidosis: a nested case-control study. European Respiratory Journal Open Research. 2023;9(2):00492-2022. https://doi.org/10.1183/23120541.00492-2022 III. Dehara M, Kullberg S, Bixo M, Sachs MC, Grunewald J, Arkema EV. Menopausal hormone therapy and risk of sarcoidosis: a population-based nested case-control study in Sweden. European Journal of Epidemiology. 2024. https://doi.org/10.1007/s10654-023-01084-3 IV. Dehara M, Nguyen NV, Arkema EV. Smoking and the risk of sarcoidosis: a systematic review and meta-analysis. [Manuscript]</p