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Contributions of the mTOR pathway to striatal alterations in environmental and genetic models of autism
No full textAutism Spectrum Disorders (ASDs) are amongst the most prevalent neurodevelopmental disorders, and are characterized by repetitive behaviours, social impairment and cognitive inflexibility. Neuropathological studies in both ASDs patients and animal models recurrently reveal morphological and functional alterations in several brain regions, notably the striatum (STR). As the main input nucleus of the basal ganglia, the striatum plays a central role in motor control, cognitive flexibility, social behaviour, and learning. The STR is predominantly composed of Spiny Projection Neurons (SPNs), whose activity is modulated by dopamine (DA).ASDs are polygenic disorders arising from the interplay between susceptibility genes, epigenetic regulation, and environmental factors. Many genetic and environmental models of ASDs converge on dysregulated mammalian Target Of Rapamycin (mTOR) signalling. mTORC1, a key complex in this pathway, regulates autophagy and protein synthesis. Hyperactivation of mTORC1 leads to impaired autophagic flux and enhanced translation, both of which have been implicated in ASDs pathophysiology; yet their relative contributions remain poorly understood. This thesis investigates the impact of dysregulated mTORC1-signalling over striatal development and function in environmental and genetic models of ASDs.In Paper I we characterized the roles of neuronal autophagy in the development of the STR during the postnatal period. Along this time window, mTORC1 activity steadily increases, which results in a gradual reduction in autophagic activity in SPNs. Furthermore, we provide evidence that in utero exposure to valproic acid leads to mTORC1 hyperactivity at late developmental stages, which inhibits autophagic flux. This mTORC1 overactivation results in impaired sociability and decreased glutamatergic synaptic transmission.In Paper II we investigated the consequences of translational dysregulation over striatal dopaminergic modulation. Increased translation leads to altered dopaminergic transmission, due to impaired nicotinic receptors on DA terminals. This defective cholinergic modulation of DA terminals, in turn, underlies cognitive inflexibility.Altogether, the work presented in this thesis sheds new light onto the consequences of mTOR signalling dysregulation over striatal function in environmental and genetic models of ASDs.List of scientific papersI. mTOR supresses macroautophagy during striatal postnatal development and is hyperactive in mouse models of autism spectrum disordershttps://doi.org/10.3389/fncel.2020.00070II. Dysregulated acetylcholine-mediated dopamine neurotransmission in the eIF4E Tg mouse model of autism spectrum disordershttps://doi.org/10.1016/j.celrep.2024.114997</p
Skeletal muscle and adipose tissue omics and phenotypes : a comparison of highly trained and untrained males and females
No full textRegular exercise training or physical activity improves athletic performance and reduces the risk of developing noncommunicable diseases such as diabetes, hypertension and cancer. This thesis aimed to examine the physiological and molecular adaptations to long-term exercise training, with a particular focus on the role of sex differences. To do so, three groups of males and females with distinctly different exercise training backgrounds were recruited, 1) highly endurance-trained individuals with at least 15 years of experience of regular endurance exercise, 2) highly strength-trained individuals with at least 15 years of experience of regular resistance exercise, and 3) a healthy untrained age- matched group of individuals not participating in regular exercise training.Whole-body magnetic resonance imaging revealed that both endurance- and strength-trained individuals, compared with untrained, displayed a greater relative whole-body muscle mass, larger muscle cross-sectional areas, lower adipose tissue volumes and lower fatty infiltration in skeletal muscles of both the upper and lower body. However, this finding was more pronounced in males than in females.To better understand the underlying molecular mechanisms of the skeletal muscle phenotypic differences, skeletal muscle transcriptomic and proteomic analyses were performed. The transcriptomic analysis showed large differences in the resting baseline muscle transcriptome between endurance trained and untrained individuals. The proteomic analyses further unraveled the molecular adaptations to exercise and largely confirmed the skeletal muscle phenotype observed in the transcriptomic analysis. Over 650 proteins were identified as differentially expressed in both endurance-trained men and women compared with their respective control groups. Of these, a striking 92% were mitochondrial proteins. While large differences were detected between the endurance-trained and untrained, few transcriptomic or proteomic differences were identified in the strength-trained group compared with the untrained, despite the distinct phenotypic and physiological performance differences between these groups. Furthermore, prominent sex differences between male and female controls were identified on the transcriptomic level, however these differences were reduced in the endurance trained state. The proteomic analysis confirmed the transcriptomic data suggesting the skeletal muscle sex differences identified in the untrained individuals are almost completely diminished in endurance-trained.Next, exercise training influences whole-body metabolism and thus, other peripheral tissues than skeletal muscle are affected. As such, the adipose tissue has recently gained interest in the field of exercise physiology, however, it is less well studied in an exercise physiology context than skeletal muscle. So, subcutaneous abdominal white adipose tissue (scWAT) showed distinct gene expression patterns related to aerobic metabolism and immune response, which were notably influenced by the training status of the individuals. The study also identified significant sex differences in the transcriptomic profiles of long-term trained and untrained individuals, with female scWAT related to enhanced aerobic respiration while the male scWAT was enriched for inflammatory pathways. However, the inflammatory profile of male scWAT was significantly attenuated in endurance-trained vs untrained males. These findings underscore the importance of regular exercise training for improving or maintaining metabolic health and reducing inflammation, particularly in males, where endurance training attenuated the inflammatory profile seen in untrained individuals.To further contextualize the data, the current data were cross-referenced to publicly available data from patients with various metabolic disorders, such as the metabolic syndrome, type 2 diabetes or obesity. Endurance-trained individuals display many genes (muscle and scWAT) and proteins (muscle) with an opposite expression compared to patients with various metabolic disorders. A majority of these were mitochondria-related, with many being directly involved in energy generating processes or in mitochondrial structure. After 6 to 12 months of exercise training in patients with the metabolic syndrome or type 2 diabetes, the number of oppositely regulated genes was reduced. In male patients with type 2 diabetes, a substantial number of genes became reciprocally expressed relative to endurance-trained males.Taken together, based on a highly controlled study design comparing three groups with distinctly different training backgrounds, this thesis addressed tissue- specific differences in skeletal muscle and adipose tissue transcriptome and proteome. The results showed large transcriptomic and proteomic differences between the endurance trained group and the other two groups as well as distinct sex differences. These results provide a deepened understanding of how humans respond to training, which may help to impact future optimization of training regimens for performance and health.List of scientific papersI. Emanuelsson EB, Berry DB, Reitzner SM, Arif M, Mardinoglu A, Gustafsson T, Ward SR, Sundberg CJ, Chapman MA. MRI characterization of skeletal muscle size and fatty infiltration in long- term trained and untrained individuals. Physiological reports 2022 10;14 e15398-https://doi.org/10.14814/phy2.15398II. Chapman MA, Arif M, Emanuelsson EB, Reitzner SM, Lindholm ME, Mardinoglu A, Sundberg CJ. Skeletal Muscle Transcriptomic Comparison between Long-Term Trained and Untrained Men and Women. Cell reports 2020 31;12 107808-https://doi.org/10.1016/j.celrep.2020.107808III. Emanuelsson EB, Arif M, Reitzner SM, Perez S, Lindholm ME, Mardinoglu A, Daub C, Sundberg CJ, Chapman MA. Remodeling of the human skeletal muscle proteome found after long-term endurance training but not after strength training. iScience 2024 27;1108638-https://doi.org/10.1016/j.isci.2023.108638IV. Eric B. Emanuelsson*, Stefan M. Reitzner*, Allister Quizon, Joshua Burrows, Merve Elmastas, Jutta Jalkanen, Sean Perez, Hampus Gilljam, Malene E Lindholm, Jessica Norrbom, Bernhard O. Palsson, Daniel Zielinski, Mikael Rydén, Mark A. Chapman, Carl Johan Sundberg. Transcriptomic profiling of adipose tissue in highly trained and untrained individuals - a cross-sectional analysis. [Manuscript]* indicates equal contribution</p
Molecular methods for the detection of nucleic acids applied to infection and cancer diagnostics
No full textThere is now an array of molecular methods to detect, quantify and elucidate nucleic acids sequences (DNA & RNA) with the field in a constant state of developmental flux. Molecular approaches are now the standard for clinical viral diagnostics with the qPCR the cornerstone of most modern hospitals. However as shown during the Covid-19 pandemic, inherent complexity, costs and the need for advanced read-out thermocyclers impede its wide scale deployment across settings. There is an ongoing movement to develop novel diagnostic approaches which are cheaper and simpler to facilitate sensitive viral detection at scale. Such developments can enable the democratization of diagnostics, bringing us closer to POC and help ensure better preparedness for future pandemics. For most bacterial infections phenotypic based antimicrobial susceptible tests (AST) are used with some molecular assays which complement them. However, the majority of these clinical tests require a number of days to provide actionable results. Such delays impact patient outcomes and promote imprecise antibiotic use. With the growing threat of antimicrobial resistance (AMR) there is an obvious need for novel rapid tests which accelerate clinical decisions and promote antibiotic stewardship. Sequencing based approaches have also recently become more accessible to clinical and research-based microbiology laboratories. They can provide insights into mechanisms of antimicrobial resistance and help track variant evolution. This revolution in high throughput sequencing technologies has also seen the concurrent development of numerous novel methods applied to human health and disease. These methods are now increasingly important to help identify high risk patients and inform cancer treatment. In this thesis the role of molecular diagnostics in microbial detection are reviewed along with an overview of phenotypic - antimicrobial susceptibility tests (AST) and their recent advances. In addition, I present an overview of sequencing technology and its application to the detection of rare DNA variants.In Paper I, we developed a method to detect SARS-CoV-2 in a reverse transcription loop mediated isothermal amplification reaction (RT-LAMP) using in-house produced (non-commercial) enzymes directly on heat inactivated (non-extracted) samples. This work was developed during the Covid-19 pandemic as a potential alternative for viral diagnostics. We aimed to complement the gold standard qPCR which was unable in certain cases to fulfill the testing needs. We focused on developing an isothermal approach using in house produced enzymes without the need for ribonucleic acid (RNA) extraction or heat cycling devices to enable a wider potential usage. We produced and isolated reverse transcriptase's and strand displacing DNA polymerases, tested and optimized their performance and subsequently benchmarked our approach against commercial alternative showing comparable results. We then validated our method in heat inactivated Covid-19 patient samples which had been diagnosed using qPCR.In Paper II, we applied the RT-LAMP reaction into an integrated standalone centrifugal and heating platform with POC diagnostics in mind. Here, we incorporated discs packed with n-benzyl-n-methylethanolamine (NBNM) modified agarose beads. These beads enabled the removal of primer dimers and allowed an enhanced end-point fluorescent detection of the sample via a smartphone at room temperature. We subsequently validated the approach in a range of Covid-19 clinical samples, demonstrating a detection limit of 100 RNA copies within one hour on non-extracted samples. Thus providing an alternative approach to enable cheap mass scale testing in resource-limited environments or closer to POC diagnostics.In Paper III, we developed a protocol to produce DNA Nanoball structures during an RT-LAMP via the addition of compaction oligos. These structures are produced in solution of approximately one micron in size without the need for any nucleation agents e.g. microbeads. We were then able to quantify these nanoballs individually in an impedance based microfluidic chamber without the need for any labelling (e.g. colorimetric or fluorescence). We initially developed our protocol using synthetic SARS-CoV-2 RNA. We then validated our ability to produce DNA Nanoballs from heat inactivated Covid-19 clinical samples. We thereafter produced Nanoballs from different target sequences and detected them with impedance. These included for HIV, influenza, mycobacterium and the AMR gene beta-lactamase highlighting the flexibility of our approach.In Paper IV we developed a novel approach to produce double stranded (ds) unique molecular identifiers (dsUMIs) within a standard short read sequencing library preparation workflow. This enables the accurate detection of low frequency variants in DNA molecules which are relevant in many settings including cancer (e.g. minimal residual disease). In standard workflows the detection of such rare variants is complicated by errors which can occur at a similar frequency. One solution to address this is to label both strands of a DNA molecule with a barcode (dsUMI). This can then allow for the identification and removal of a number of low frequency errors and resolution of boundary collisions. However, to develop such dsUMI barcodes is laborious and expensive. Here our approach called one pot double stranded UMI sequencing (OPUSeq) produces a dsUMI DNA molecule within a modified PCR. We then show its ability to identify rare variants in an experiment which mimics minimal residual disease (MRD). Our research also revealed that the fragmentase mix produces low frequency errors on both strands across the molecule.In our Preliminary data, we developed a rapid one pot multiplex reaction. Here we use molecular inversion probes coupled to sequencing based readout to detect bacterial and fungal targets, as well as AMR genes. We optimized the method using ZymoBiomics microbiome cell (Bacterial and Fungi) standards and synthetic fungal ITS and AMR spike-ins. Currently we are validating our approach to detect infections from blood samples taken from acute myeloid leukemia (AML) patients.List of scientific papersI. Direct detection of SARS-CoV-2 using non-commerical RT- LAMP reagents on heat-inactivated samples. Alisa Alekseenko*, Donal Barrett*, Yerma Pareja-Sánchez*, Rebecca J Howard, Emilia Strandback, Henry Ampah-Korsah, Urška Rovšnik, Silvia Zuniga-Veliz, Alexander Klenov, Jayshna Malloo, Shenglong Ye, Xiyang Liu, Björn Reinius, Simon J Elsässer, Tomas Nyman, Gustaf Sandh, Xiushan Yin, Vicent Pelechano. Scientific Reports 2021, Jan 19;11(1):1820. https://doi.org/10.1038/s41598-020-80352-8 *Authors contributed equally to this study II. Sample-to-answer COVID-19 nucleic acid testing using low- cost centrifugal microfluidic platform with bead-based signal enhancement and smartphone read-out. Ruben R G Soares, Ahmad S Akhtar, Ines F Pinto, Noa Lapins, Donal Barrett, Gustaf Sandh, Xiushan Yin, Vicent. Pelechano, Aman Russom. Lab Chip, 2021, 21, 2932. https://doi.org/10.1039/D1LC00266JIII. Digital Assay for rapid electronic quantification of clinical pathogens using DNA Nanoballs. Muhammad Tayyab*, Donal Barrett*, Gijs van Riel, Shujing Liu, Björn Reinius, Curt Scharfe, Peter Griffin, Lars M. Steinmetz, Mehdi Javanmard, Vicent Pelechano. Sci. Adv.9,eadi4997(2023). https://doi.org/10.1126/sciadv.adi4997*Authors contributed equally to this studyIV. OPUSeq simplifies detection of low frequency DNA variants and uncovers fragmentase-associated artifacts. Alisa Alekseenko, Jingwen Wang, Donal Barrett, Vicent Pelechano. NAR Genomics and Bioinformatics 2022, Jun 27;4(2):lqac048. https://doi.org/10.1093/nargab/lqac048</p
Unveiling the cellular heterogeneity of cancer metastasis
No full textTechnologies within single-cell omics have revolutionized our ability to explore cellular heterogeneity with remarkable precision. Encompassing a range of methods that can analyze the molecular characteristics of individual cells, single- cell omics promise to provide valuable insights into the DNA, RNA, and protein levels at a single-cell resolution. In particular, single-cell RNA-sequencing (scRNA- seq) and spatial-omics stand out as pivotal tools providing insights into gene expression profiles, transcriptional states, and the spatial organization of cancer cells thereby providing leveraged to reveal the cellular heterogeneity of cancer cells and the tumour microenvironment (TME). Especially in the context of cancer metastasis, which remains a therapeutic enigma, the use of scRNA-seq and spatial-omics can be used to delineate the complex cellular landscapes of tumours and identify the interactions within the TME, providing crucial information for developing targeted therapies and improving patient outcomes.In Paper I we investigate clear cell renal cell carcinoma (ccRCC) using scRNA-seq to analyze primary tumours and their metastatic counterparts. A distinct transcriptional signature was identified in tumour cells that predict metastatic potential and patient survival and highlights the role of the TME, particularly vascular remodelling and an immunosuppressive microenvironment in promoting metastasis. Finally, we highlight potential therapeutic targets, such as the CXCL9/CXCL10-CXCR3 and CD70-CD27 axes, as potential targets for treatments.In Paper II, building on the insights of Paper I, we use scRNA-seq to analyze the TME of primary and bone metastatic ccRCC with a unique comparison to tumour involved-, benign- and healthy- bone marrow. Our findings highlight a population of tumour-associated mesenchymal stromal cells promoting epithelial-to- mesenchymal transition and bone remodelling as well as an immunosuppressive microenvironment with exhausted CD8+ T cells and tumour-associated macrophages that aids the metastatic process to bone. Finally, we show that tumour-associated mesenchymal cells exert their effects on bone remodelling via the RANK/RANKL/OPG signalling pathway, crucial for regulating bone resorption and remodelling in metastatic sites and suggest the pathway as a therapeutic target.In Paper III, we investigated spatially resolved chromosomal aberrations in high- risk neuroblastoma (NB) patient samples and cell lines using spatial omics. We describe the tumour clonal landscape in patient samples and the evolutionary trajectories of cells in response to chemotherapy in NB cell lines. We reveal that targeting both proliferation and DNA damage response pathways via a combination treatment with doxorubicin, gemcitabine, and an ATM inhibitor, can effectively reduce tumour growth and inhibit regrowth both in vitro and in vivo, highlighting the importance of addressing genetic and phenotypic heterogeneity in NB.Overall, this thesis provides novel insights into the intricate cellular landscape of primary and metastatic tumours as well as their surrounding microenvironments, revealing potential culprits of the metastatic process. Using high-resolution single-cell omics technologies, we enable the identification of key therapeutic targets and the development of more effective, personalized treatment strategies to combat cancer metastasis.List of scientific papersI. Adele M. Alchahin*, Shenglin Mei*, Ioanna Tsea, Taghreed Hirz, Youmna Kfoury, Douglas Dahl, Chin-Lee Wu, Alexander O. Subtelny, Shulin Wu, David T. Scadden, John H. Shin, Philip J. Saylor, David B. Sykes, Peter V. Kharchenko & Ninib Baryawno. A transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma. Nat Commun 13, 5747 (2022). https://doi.org/10.1038/s41467-022-33375-wII. Shenglin Mei*, Adele M. Alchahin*, Ioanna Tsea*, Youmna Kfoury, Taghreed Hirz, Nathan Elias Jeffries, Ting Zhao, Yanxin Xu, Hanyu Zhang, Hirak Sarkar, Shulin Wu, Alexander O. Subtelny, John Inge Johnsen, Yida Zhang, Keyan Salari, Chin-Lee Wu, Mark A. Randolph, David T. Scadden, Douglas M. Dahl, John Shin, Peter V. Kharchenko, Philip J. Saylor, David B. Sykes & Ninib Baryawno. Single-cell analysis of immune and stroma cell remodelling in renal cell carcinoma primary tumours and bone metastatic lesions. Genome Med 16, 1 (2024). https://doi.org/10.1186/s13073-023-01272-6III. Ioanna Tsea*, Xiaoze Li Wang*, Yuwei Lin, Sen Li, Agnes Luise Sorteberg, Maja Lundström, Alexandra Johannesson, Pranauti Panshikar, Eleonor O'Brien, Kristina Ihrmark Lundberg, Ingrid Lilienthal, Quentin Verron, Nikolas Herold, Susanne Fransson, Tommy Martinsson, Per Kogner, Jacob Stenman, Malin Wickström, John Inge Johnsen, Ninib Baryawno, Charlotte Stadler, Magda Bienko & Shahrzad Shirazi Fard. Spatially resolved chromosomal aberrations are sensitive towards targeted therapy against proliferation and DNA damage response. [Manuscript] *Equal contribution</p
Exploring the human oligodendrocyte lineage during development and adulthood through the lens of single-cell methods
No full textEven though much is known about oligodendroglia based on animal models, human oligodendroglia biology knowledge is still lacking. In this thesis, we attempt to characterize human oligodendroglia molecular profiles using single-cell technologies. In papers I and II, we explored human oligodendroglia in the context of development. We identified oligodendrogenesis already occurring as early as 8 postconceptional weeks and revealed the molecular changes in OPC specification with single-cell RNA-seq. We also inspected the epigenetic landscape using single-cell ATAC-seq, revealing which transcription factors are active in OPC specification. Using spatial transcriptomics, we managed to map the OPC to ventral regions, suggesting that the first wave of OPCs also arises from ventral regions as in the mouse. Following the same principles, we investigated the second trimester of gestation in paper II. We characterized the molecular profile of the second-trimester oligodendroglia using single-nuclei RNA-seq and single-nuclei ATAC-seq. We investigated the molecular changes in OPC specification and compared it with the first trimester, showing some overlap, but several unique genes, indicating that in the second trimester OPC specification follows a different pathway. Next, we investigated the human oligodendroglia in adulthood in papers III and IV. Using single-nuclei RNA-seq we characterized the oligodendroglia from different regions-cortex, cerebellum, and spinal cord-and identified unique oligodendroglia populations. We also investigated the changes in oligodendroglia transcriptomic by age and sex. While we observed that aged individuals have reduced expression of genes related to myelination-indicating lower myelination capacity-, sex differences were more subtle. Finally, we investigated the epigenomic landscape of adult human oligodendroglia in paper IV. We used a combination of single-nuclei ATAC-seq and single-nuclei CUT&Tag to investigate the regulatory mechanism in human oligodendroglia. We identified a new candidate SOX10 enhancer in oligodendrocytes and observed the epigenetic priming of the HOX gene family in spinal cord oligodendrocytes. Together, we provided a deep molecular characterization of human oligodendroglia both in development and adulthood.List of scientific papersI. Developmental landscape of human forebrain at a single-cell level identifies early waves of oligodendrogenesis. David van Bruggen, Fabio Pohl, Christoffer Mattsson Langseth, Petra Kukanja, Hower Lee, Alejandro Mossi Albiach, Mukund Kabbe, Mandy Meijer, Sten Linnarsson, Markus M. Hilscher, Mats Nilsson, Erik Sundström, Gonçalo Castelo-Branco. Developmental Cell 2022. https://doi.org/10.1016/j.devcel.2022.04.016II. Specification of oligodendrocyte progenitor cells and their precursors during diferent stages of human neural development. Fabio Pohl, Luise A. Seeker, Mukund Kabbe, Eneritz Agirre, Neemat Mahmud, Carmen Abaurre, David Van Bruggen, Nadine Bestard- Cuche, Zahra Moslehi, Gioele La Manno, Steve Goldman, Anna Williams, Gonçalo Castelo-Branco. [Manuscript]III. Brain matters: unveiling the distinct contributions of region, age, and sex to glia diversity and CNS function. Luise A. Seeker, Nadine Bestard-Cuche, Sarah Jäkel, Nina-Lydia Kazakou, Sunniva M. K. Bøstrand, Laura J. Wagstaff, Justyna Cholewa-Waclaw, Alastair M. Kilpatrick, David Van Bruggen, Mukund Kabbe, Fabio Baldivia Pohl, Zahra Moslehi, Neil C. Henderson, Catalina A. Vallejos, Gioele La Manno, Goncalo Castelo-Branco, Anna Williams. Acta Neuropathologica Communications 2023. https://doi.org/10.1186/s40478-023-01568-zIV. Single-nuclei histone modification profiling of the adult human central nervous system unveils epigenetic memory of developmental programs. Mukund Kabbe, Eneritz Agirre, Karl E. Carlström, Fabio Baldivia Pohl, Nicolas Ruffin, David van Bruggen, Mandy Meijer, Luise A. Seeker, Nadine Bestard-Cuche, Alex R. Lederer, Jilin Zhang, Virpi Ahola, Steven A. Goldman, Marek Bartosovic, Maja Jagodic, Anna Williams, Gonçalo Castelo-Branco. BioRxiv 2024. https://doi.org/10.1101/2024.04.15.589512 [Manuscript Preprint]</p
Molecular mechanisms of WNT signaling and receptor-transducer coupling
No full textWingless and int-1 (WNT) signaling is a group of evolutionarily conserved signal transduction pathways that are vital for fundamental cellular processes. The signals are transduced via receptors at the cell membrane, Frizzleds (FZDs), which belong to class F of GPCRs. In this thesis, the focus is on WNT signaling, the binding of WNT-3A to FZDs, and their coupling to their primary intracellular transducer, Dishevelled (DVL), constitutively, i.e. in the absence of ligands.WNT-3A has been extensively studied for its role in beta-catenin dependent signaling. In this work, eGFP-WNT-3A was used in combination with HiBiT-tagged FZD1-10 to determine the binding kinetics in a live cell nanoBRET assay. The kinetic parameters were determined from both kinetic and saturation binding curves. Among all FZDs, except for FZD3 and FZD9 for which binding could not be detected, the determined affinities range from single to double digit nanomolar concentrations. Additionally, the utility of this assay for other experimental modes was shown, including competition binding with commercial preparations of WNT- 3A, WNT-5A, WNT-5B, WNT-10B, WNT-11 and WNT-16B.In addition to ligand induced signaling, constitutive activity, which is defined as coupling of the receptor to its transducer in the absence of stimulation by a ligand can have important functional consequences. To this end, G protein tri-cistronic activity sensors (G-CASE) were designed and in addition to measuring ligand induced G protein coupling, were validated specifically for determining constitutive coupling in GPCRs.FZDs couple constitutively to DVL. To this end, a FZD6-DVL3 complex was formed and stabilized for cryogenic electron microscopy (cryoEM) experiments, which enabled the determination of the structure at an overall resolution of 3.6 Å. The structure consists of FZD6 and residues 409-495 of DVL3 (DEP domain). The receptor exhibits a typical GPCR-like organization with seven transmembrane helices. The interaction of FZD6 with DEP is characterized by two sites: 1) a lipophilic cavity formed by residues L3935.69, V3965.72, V3995.75 and I4005.76 on FZD6 and residues L434 and I436 on DVL3 and 2) a polar site formed by residues R22612.49, K4126.28, R4166.32 and K4988.49 on FZD6 and K435 on DVL3, surrounding an unassigned density. Single, alanine point mutations in R22612.49, R4166.32, K4988.49, as well as the molecular switch W4937.55 in the vicinity, reduce the relative affinity of FZD6 to DVL3 (amino acids 243-496). The moiety in this unassigned density may play a central role in coordinating the polar residues surrounding this site.Overall, the work presented in this thesis adds to our knowledge on WNT signaling and receptor-transducer coupling methodologically with new approaches to study WNT-FZD kinetics and constitutive GPCR coupling, as well as mechanistically with novel information on the molecular interaction of FZD6-DVL3, with the first structure of a FZD-DVL complex reconstituted in a lipid environment.List of scientific papersI. Quantitative Profiling of WNT-3A Binding to All Human Frizzled Paralogues in HEK293 Cells by NanoBIT/BRET Assessments. Paweł Kozielewicz, Rawan Shekhani, Stefanie Moser, Carl-Fredrik Bowin, Janine Wesslowski, Gary Davidson, Gunnar Schulte. ACS Pharmacol Transl Sci. 2021 May 11;4(3):1235-1245.https://doi.org/10.1021/acsptsci.1c00084II. Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors. Hannes Schihada, Rawan Shekhani, Gunnar Schulte. Sci Signal. 2021 Sep 7;14(699):eabf1653. https://doi.org/10.1126/scisignal.abf1653III. Structural insight into the functional interaction between Frizzled 6 and the transducer protein Dishevelled. Rawan Shekhani, Magdalena M Scharf, Jan Hendrik Voss, Jia Yu Ho, Fanny Peysson, Shi Min Tan, Allan H. Pang, Jitender Kumar, Vítězslav Bryja, Konstantinos Tripsianes, Sébastien Granier, Rémy Sounier, Yong Zi Tan, Gunnar Schulte. [Manuscript]</p
Various aspects on indwelling urinary catheter treatment and its relation to asymptomatic bacteriuria and urinary tract
No full textCatheter-associated urinary tract infection (CAUTI) is one of the most common kinds of patient harm. One way of introducing bacteria into the urinary tract is by inserting a contaminated indwelling urinary catheter (IUC) due to non-aseptic handling during insertion or during maintenance of the IUC and/or the attached urine collection bag. Another way is to leave the IUC in place. The longer the dwell time the greater the risk of bacteriuria. In Sweden, the recommendations from The National Board of Health and Welfare on IUC-insertion procedure has shifted gradually from requiring sterile catheter to accepting non-sterile catheter over the years. In parallel, various denominations has been introduced for IUC-insertion, one of them called clean/nonsterile technique. In 2012 the European Association for Urology Nurses (EAUN) published a guideline on urethral catheterisation requiring sterile IUC and aseptic technique during procedure. The Swedish national Handbook for Healthcare was inspired by it and revised its recommendations. Though, different hospitals had differing requirements on sterility during IUC-insertion procedure. The overall aim with this thesis was to gain increased knowledge in various aspects on indwelling urinary catheter treatment and its relation to asymptomatic bacteriuria and urinary tract infection.In Study I, a cross-sectional survey, we used a structured questionnaire to explore how registered nurses and assistant nurses from three departments at hospital A described their IUC-insertion procedure in relation to what they called their insertion technique, and whether they followed the hospital guideline for urethral catheterisation. Of 492 included in the study analyses at hospital A, 58% (n=287) said that they followed the hospital guideline. Irrespective of the origin of the denomination used, "clean/non-sterile technique" was used significantly more often by the registered nurses (244/308 vs 104/179, PIn Study II the same questionnaire was used in a cross-sectional survey to compare how registered nurses and assistant nurses from three departments at hospital A and hospital B described their IUC-insertion procedure in relation to the denominations they used for the procedure. A total of 819 participants, 492 from hospital A and 327 from hospital B, were included in the study analyses. Most participants called their IUC-insertion technique "clean/non-sterile". The participants' conformity with all the sterility precautions in the EAUN-guidelines were associated with working at departments of Cardiology and Surgery (OR 2.35, 95% CI 1.69-3.27), use of sterile catheterisation set (OR 2.06, 95% CI 1.42- 2.97), use of sterile drapes for dressing on insertion area (OR 1.91, 95% CI 1.24- 2.96) and using the term "sterile technique" (OR 1.64, 95% CI 1.11-2.43).In Study III, an observational study on asymptomatic bacteriuria (ABU) and first urinary tract infection (UTI) event during the remaining inpatient care, 196 asymptomatic geriatric inpatients from two hospitals were included in the analyses. Significantly more ABU was found in patients with a history of catheterisation compared to those without (38/104, 36,5% vs 19/92, 20,7%, P=0.018). History of catheterisation was significantly associated with ABU after adjustment for confounders (OR 2.79, 95% CI 1.31-5.91). Of 124 patients possible to follow up at one of the study sites, five patients received antibiotic treatment for UTI during the remaining hospital stay. All UTI-cases were women, all had had ABU and four of five had had an IUC on admission. Three of five women had diabetes mellitus.In Study IV same geriatric inpatients and same questionnaire was used. The overall proportion of uropathogens >103 CFU/mL was significantly higher in patients with a history of catheterisation than without (P=0.006). The proportion of secondary uropathogens was also significantly higher in patients with a history of catheterisation compared to those without (P=0.028). Female sex (OR 3.58, 95% CI 1.76-7.29), history of catheterisation (OR 3.03, 95% CI 1.52-6.04) and diabetes mellitus (OR 2.23, 95% CI 1.11-4.47) were significantly associated with the overall detection of uropathogens. The most common uropathogen was E. coli. Overall, 34% of the E.coli isolates were antibiotic resistant to at least one antibiotic group assessed. Among the E. coli isolates assessed for biofilm formation, five were biofilm formers, of which all came from women with ABU detected in prior study urine samples. Four isolates originated from women with a history of catheterisation. Culture results of IUC-urine samples and voided urine samples from the same 73 patients with an IUC on admission were compared. The results were incongruent in 21% of the related urine samples.Conclusions: Clean/non-sterile insertion technique counteracts asepsis and jeopardizes patient safety and efforts to prevent CAUTI. Harmonised and implemented national guidelines and skill training on a regular basis is a great concern for patient safety. Significant bacteriuria is increased in asymptomatic patients with a history of catheterisation up to four weeks prior to the IUC- removal. The proportion of E. coli resistant to cefadroxil and ciprofloxacin in geriatric inpatients is worrying. Biofilm forming E. coli is not common in inpatients catheterised for less than two weeks. To minimise unnecessary antibiotic treatment against secondary uropathogens, voided urine sampling is preferable to IUC-urine sampling, when possible, in patients who have been catheterised for less than two weeks.List of scientific papersI. Kulbay A, Tammelin A. Clean or sterile technique when inserting indwelling urinary catheter: An evaluation of nurses and assistant nurses' interpretations of a guideline at an acute-care hospital in Sweden. Nordic Journal of Nursing Research. 2019;39(2):92-97. https://doi.org/10.1177/2057158518800261II. Kulbay A, Joelsson-Alm E, Tammelin A. The impact of guidelines on sterility precautions during indwelling urethral catheterization at two acute-care hospitals in Sweden - a descriptive survey. BMC Nursing. 2021;20(1):99. https://doi.org/10.1186/s12912-021-00619-xIII. Kulbay A, Joelsson-Alm E, Amilon K, Tammelin A. Asymptomatic bacteriuria and urinary tract infection in geriatric inpatients after indwelling urinary catheter removal: a descriptive two-centre study. Infect Prev Pract. 2024;6(4):100411. https://doi.org/10.1016/j.infpip.2024.100411IV. Kulbay A, Amilon K, White J, Joelsson-Alm E, Tammelin A. Uropathogens, antibiotic resistance and biofilm producing Escherichia coli among inpatients with and without a history of urethral catheterization. [Manuscript]</p
Revise and remember : managing hip resurfacing implant recall
No full textBackgroundTotal hip arthroplasty (THA) with metal-on-polyethylene articulation provides excellent clinical outcomes but remains associated with complications such as polyethylene wear and joint dislocations. For younger and more active patients, metal-on-metal (MoM) implants were initially introduced as a promising alternative. However, MoM bearings have subsequently demonstrated higher complication rates, including severe adverse events such as periprosthetic fractures and soft tissue reactions in the joint. One such implant with particularly high complication rates was the Articular Surface Replacement (ASR) by DePuy/Johnson&Johnson. As a result, the use of MoM implants declined in Sweden by 2015, and the Swedish Hip and Knee Society officially ceased recommending hip resurfacing arthroplasty in 2018. This decision started a debate in the media between pro and con groups for HRA. Adding further complexity, certain European countries have continued to perform the procedure, with follow-up challenges affecting the Swedish healthcare system. Investigating the implications of MoM implants on patients' outcomes and healthcare systems is essential for future clinical guidelines and policy decisions.MethodsPaper I and III studied patients' experiences with the ASR hip resurfacing arthroplasty (HRA). Paper I focused on those living with the recalled implant, while Paper III examined the narratives of patients undergoing revision surgery due to complications.Paper Il compared long-term revision rates, clinical outcomes, and changes in blood metal ion levels between patients with ASR THA and those with ASR HRA.Paper IV investigated potential socioeconomic differences in patients receiving MoM HRA versus uncemented THA in a 1:1 matched ratio from 1999 to 2014.ResultsPatients living with asymptomatic ASR HRA implants without pain reported that they rarely thought about the implant. Many had actively sought referrals to centers performing HRA procedures. Annual hospital check-ups after surgery were highly valued by these patients, and if the patients were taken care of and informed, they continued to express their trust in the healthcare system.Among patients who underwent revision surgery, some were satisfied with their new implants, while others continued to experience pain and further complications. Despite the recall, since the patients were well taken care of, trust in the healthcare system remained intact.In a cohort of 38 patients, 11-year outcomes revealed no revisions among ASR HRA recipients, but 32% of ASR XL THA recipients required revision surgery. The ASR XL THA group also exhibited significantly elevated cobalt ion levels, even in well-functioning implants.Patients with lower education and income levels were more likely to receive uncemented THA than MoM HRA.ConclusionWhen an implant is recalled due to high complication rates, robust support systems, including annual follow-ups, are crucial. Patients emphasized the importance of transparent communication regarding implant risks. ASR XL THA demonstrated poorer outcomes compared to ASR HRA, with rising cobalt ion levels in asymptomatic patients, necessitating regular monitoring. Socioeconomic differences in implant selection challenge the principle of equitable healthcare, warranting further attention.List of scientific papersI. Living with a recalled implant: a qualitative study of patients' experiences with ASR hip resurfacing arthroplasty. Bitar C, Krupic F, Felländer-Tsai L, Crnalic S, Wretenberg P. Patient Saf Surg. 2021;15(1):2. https://doi.org/10.1186/s13037-020-00278-yII. 11-Year outcomes in patients with metal-on-metal ASR hip arthroplasty. Bitar C, Moberg I, Krupic F, Wretenberg P, Otten V, Crnalic S. J Orthop. 2022;32:98-103. https://doi.org/10.1016/j.jor.2022.05.015III. Beyond the procedure of a recalled implant: Patients' experiences of ASR hip implant revision. Bitar C, Wretenberg P, Felländer-Tsai L, Crnalic S, Krupic F. [Submitted]IV. Socioeconomic disparities in the utilization of metal-on-metal hip resurfacing compared to uncemented total hip arthroplasty: A population-based case-control study in Sweden. Oxblom A, Bitar C, Rolfson O, Hedlund H, Qureshi AR, Brismar H, Wretenberg P, Palme M, Adami J, and Felländer-Tsai L. [Submitted]</p
Advanced techniques in ERCP
No full textEndoscopic Retrograde Cholangiopancreatography (ERCP), first described over 50 years ago, has fundamentally remained unchanged. Despite the potential for technical and clinical innovations in the x-ray field, their implementation has progressed at a slow pace. ERCP is a common procedure that is both cost- and resource-intensive but there is a notable absence of tools to assist with scheduling, and the topic of ERCP duration remains largely unexplored. The technique of implanting multiple plastic stents side by side in the pancreatic duct has been well known for nearly 20 years, yet its role remains ambiguously defined. The 4 projects described in this thesis investigated clinically relevant yet scientifically insufficiently explored topics in ERCP with the goal of advancing the technique and improving clinical care.In Studies I and II, we aimed to adapt the use of advanced imaging techniques for ERCP. Study I assessed radiation doses during ERCP with cone beam computed tomography (CBCT) and discussed lessons learned from early clinical experiences with this technique. We showed that, although CBCT requires more radiation, doses were moderate, and highlighted the importance of appropriate case selection. Study II is the first-ever description of image fusion technique used during ERCP which proved feasible and helpful in most cases.In Study III, we created and validated the first specific tool for estimating ERCP duration using large datasets from a national database. A simple addition score achieved estimation of the expected ERCP duration with a mean absolute error of 17 min.Study IV focused on treating pain in chronic pancreatitis with multiple plastic stents in the pancreatic duct. We demonstrated that ERCP with multiple plastic stents is safe and effective for pain relief in patients with co-morbidities in which the risk of standard treatment with surgical resection of the pancreatic head is high. However, our results also highlighted that pain relapse is common in the long- term.Our studies demonstrate that an open-minded approach to established techniques might lead to new knowledge and could help paving the way for broader clinical use of advanced ERCP techniques in the future.List of scientific papersI. Radiation dose in cone beam CT guided ERCP Alexander Waldthaler, Marcus Reuterwall Hansson, Urban Arnelo, Nils Kadesjö European Journal of Radiology 2020; 123.108789https://doi.org/10.1016/j.ejrad.2019.108789II. Bimodal ERCP, a new way of seeing things Marcus Reuterwall Hansson, Alexander Waldthaler, Jeanne Lubbe, Nils Kadesjö, Raffaella Pozzi Mucelli, Marco Del Chiaro, Johannes-Matthias Löhr, Urban Arnelo Endoscopy International Open 2020; 8: E368-E376https://doi.org/10.1055/a-1070-8749III. Predicting ERCP procedure time - the SWedish Estimation of ERCP Time (SWEET) tool Alexander Waldthaler, Anna Warnqvist, Josefine Waldthaler Miroslav Vujasinovic, Poya Ghorbani, Erik von Seth, Urban Arnelo, Johannes-Matthias Löhr, Annika Bergquist Endoscopy 2025; 57: 31-40https://doi.org/10.1055/a-2371-1367IV. Multiple plastic stents for treatment of obstructive chronic pancreatitis Alexander Waldthaler, Ebba Asplund, Paula Steiner, Laura Vossen Engblom, Erik von Seth, Miroslav Vujasinovic, Urban Arnelo, J .- Matthias Löhr, Annika Bergquist [Manuscript]</p
The Alzheimer disease continuum : findings from monogenic Alzheimer disease
No full textAlzheimer disease (AD), with accumulation of typical deposits of amyloid-beta (AB) and phosphorylated tau, starts to develop in the CNS many years before the onset of early clinical symptoms. The perception of AD has evolved and it is in- creasingly recognized as a biological entity, not just a distinct clinical phenotype. It has been proposed that the presence of a deterministic mutation or the pres- ence of pathological levels of biomarkers that are surrogate measures to detect AD neuropathological change (AB plaques and tau neurofibrillary tangles), will de- fine whether an individual is on the AD continuum. Concordantly, the AD contin- uum spans over both asymptomatic and symptomatic phases. Autosomal domi- nant AD (ADAD) is caused by deterministic genetic variants in the APP, PSEN1 and PSEN2 genes. Such mutations elicit early-onset disease, with the first symptoms observed before the age of 65 years. Studies of ADAD families provide a unique and important model for investigation of the natural progression of AD patho- physiology and symptoms. In Sweden, the Familial Alzheimer Disease (FAD) study has been ongoing at Karolinska Institutet since the 1990's. This longitudinal obser- vational study enrolls relatives with 50% risk of developing AD and the research protocol includes sampling of biofluids, brain MRI, electroencephalography and neurocognitive testing. The objective of this thesis was to continue profiling of ADAD and investigate novel clinical, biofluid and genetic biomarkers for AD in or- der to explore preclinical and clinical phases of AD pathophysiology, monitor dis- ease severity and improve the diagnostic work-up.In Study I, we found that plasma concentrations of glial fibrillary acidic protein (GFAP), tau phosphorylated at position 181 (p-tau181) and neurofilament light chain (NfL) all started to increase in the presymptomatic phase and, thus, are potential biomarkers for monitoring asymptomatic individuals on the AD continuum. Eleva- tions of plasma GFAP concentrations were the earliest to be detected, supporting that this is a sensitive marker in presymptomatic AD.In Study Il we investigated plasma AB concentrations over the AD continuum by mutation type, due to strong variant-specific effects. In the Swedish double mu- tation (APP p.KM670/671NL), AB peptides were more than 3-fold increased in both presymptomatic and symptomatic mutation carriers compared to non-carrier controls at baseline. Thus, measurement in clinical routine would indirectly confer the risk of disclosing mutation status. Plasma Aß did not correlate to CSF AB levels, nor did longitudinal results suggest any mutation-specific effects on AB42/40 ratio with increasing age. Thus, the results did not support the utility of plasma AB measurement in ADAD.Study III included analyses of transient global topographies or maps of summated neuronal potentials, i.e microstates, as recorded by electroencephalography (EEG). The EEG microstate methodology was employed for the first time in ADAD, and the results pointed out selective changes associated with either clinical symptoms or mutation status. Albeit exploratory, the findings shed light on the activity of intrinsic global neuronal networks over the AD continuum and may be hypothesis-generating for future work in the field.Lastly, Study IV reported biomarker results and clinical characteristics from a novel C-terminally located APP (p.I718M) mutation, not previously described, po- tentially causative of both AD and mixed AD-DLB phenotypes.In summary, this thesis integrated studies of fluid and novel EEG biomarkers over the AD continuum. Also, biomarker profiles and clinical characteristics of a novel APP mutation were systematically described. The findings may be crucial for psy- chosocial support and genetic counselling to specific families, advance the gen- eral understanding of pathogenic variants and, thus, may be of importance for the ADAD community worldwide.List of scientific papersI. Johansson C, Thordardottir S, Laffita-Mesa J, Rodriguez-Vieitez E, Zetterberg H, Blennow K, Graff C. Plasma biomarker profiles in autosomal dominant Alzheimer's disease. Brain. 2023 Mar 1;146(3):1132- 1140. https://doi.org/10.1093/brain/awac399II. Johansson C, Thordardottir S, Laffita-Mesa J, Pannee J, Rodriguez- Vieitez E, Zetterberg H, Blennow K, Graff C. Gene-variant specific effects of plasma amyloid-beta levels in Swedish autosomal dominant Alzheimer disease. Alzheimers Res Ther. 2024;16(1):207. https://doi.org/10.1186/s13195-024-01574-wIII. Johansson C, Koenig T, Smailovic U, Hallstrom V, Jelic V*, Graff C *. Selective association of EEG microstates with clinical symptoms and mutation status in monogenic Alzheimer disease. *Shared last authors. [Submitted]IV. Johansson C, Rodriguez-Vieitez E, Bluma M, Nennesmo I, Thonberg H, Ullgren A, Jelic V, Zetterberg H, Blennow K, Nordberg A, Graff C. Phenotypic variability in early-onset dementia segregating with the novel APP p.I718M variant. [Submitted]</p