| 1. |
- Bergström, Gunnar
(författare)
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Microfluidic biosensor systems for cardiotoxicity assaying
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Toxicity screening is an important part of pharmaceutical development and early detection of toxic side effects provide the opportunity for early redesign or termination of unfeasible projects. Today toxicity testing is relying on experiments on animals. Ethical concerns, high costs and problems with interspecies variability in animal experiments have introduced incentives for cell-based toxicity assays. The recent development of stem cell technology have raised the hope for toxicity testing with higher predictivity that can reduce the amount of animals sacrificed, increase the patient safety and reduce the costs in pharmaceutical development.Cell development and behavior is to a large extent dependent on the microenvironment. Microfluidic techniques can be used to build small-sized structures that provide the opportunity to introduce a high degree of control of the cell culture environment with features in cell sizes. In this thesis is demonstrated two different methods for infusing cells into microfluidic cell culture devices using either cells clustered in cardiac bodies during differentiation or cells pre-seeded in microporous carriers prior to infusion.Microfluidic cell culture devices are well suited for optical evaluation. Demonstrated in this thesis is fluorescent staining in combination with confocal microscopy as well as automated imaging with evaluation of beating frequency of cardiomyocyte cell clusters can be used to assess toxicity of cells cultured in microfluidic devices.Biosensors use biological recognition elements to measure the presence of a chemical substance, for example low concentrations of biomarkers secreted by cells in a toxicity assay. Especially capacitive biosensors have shown very low limit of detection. In addition, protein G is demonstrated as an affinity ligand to capture IgG antibodies used as recognition element in a biosensor application or used for antibody screening.
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| 2. |
- Ljustell, Pär, 1977-
(författare)
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Fatigue crack growth experiments and analyses - from small scale to large scale yielding at constant and variable amplitude loading
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is on fatigue crack growth experiments and assessments of fatigue crack growth rates. Both constant and variable amplitude loads in two different materials are considered; a nickel based super-alloy Inconel 718 and a stainless steel 316L. The considered load levels extend from small scale yielding (SSY) to large scale yielding (LSY) for both materials.The effect of different load schemes on the fatigue crack growth rates is investigated on Inconel 718 and compact tension specimens in Paper A. It is concluded that load decreasing schemes give a to high Paris law exponent compared to constant or increasing load amplitude schemes. Inconel 718 is further analyzed in Paper B where growth rates at variable amplitude loading in notched tensile specimens are assessed. The predictions are based on the fatigue crack growth parameters obtained in Paper A. The crack closure levels are taken into consideration and it is concluded that linear elastic fracture mechanics is incapable of predicting the growth rates in notches that experience large plastic cyclic strains. Even if crack closure free fatigue parameters are used and residual stresses due to plasticity are included. It is also concluded that crack closure free and nominal fatigue crack growth data predict the growth rates equally well. However, if the crack closure free parameters are used, then it is possible to make a statement in advance on the prediction in relation to the experimental outcome. This is not possible with nominal fatigue crack growth parameters.The last three papers consider fatigue crack growth in stainless steel 316L. Here the load is defined as the crack tip opening displacement parameter. Paper C constitutes an investigation on the effect of plastic deformation on the potential drop and consequently the measured crack length. It is concluded that the nominal calibration equation obtained in the undeformed geometry can be used at large plastic deformations. However, two conditions must be met: the reference potential must be taken in the deformed geometry and the reference potential needs to be adjusted at every major change of plastic deformation. The potential drop technique is further used in Paper D and Paper E for crack length measurements at monotonic LSY. Constant amplitude loads are considered in Paper D and two different variable amplitude block loads are investigated in Paper E. The crack tip opening displacement is concluded in Paper D to be an objective parameter able to characterize the load state in two different geometries and at the present load levels. Furthermore, if the crack tip opening displacement is controlled in an experiment and the local load ratio set to zero, then only monotonic LSY will appear due to extensive isotropic hardening, i.e. elastic shake-down. This is also the reason why the linear elastic stress-intensity factor successfully could merge all growth rates, extending from SSY to monotonic LSY along a single line in a Paris law type of diagram, even though the generally accepted criteria for SSY is never fulfilled. For the variable amplitude loads investigated in Paper E, the effect of plastic deformation on measured potential drop is more pronounced. However, also here both the crack tip opening displacement parameter and the linear elastic stress-intensity factor successfully characterized the load state.
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| 3. |
- Lundgren, Linda, 1973-
(författare)
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Incidental Gallbladder Cancer : Incidence, predictors, management and outcome in a Swedish population
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Cholecystectomy is a common surgical procedure and incidental gallbladder cancer is a rare and unexpected finding at a cholecystectomy performed upon benign indications. Whether to perform routine or selective histopathology of the gallbladder specimen is still a subject for discussion. The prognosis of gallbladder cancer is largely affected by tumour stage and treatment.Aims: The overall aim was to study whether routine histological examination of the gallbladder specimen is of clinical and health economic value; determine if there are any predictive factors of incidental gallbladder cancer at benign cholecystectomy and compare the management and outcome of incidental gallbladder cancer patients in Sweden.Methods: All studies were based on registry data from GallRiks (The Swedish Registry for Gallstone Surgery and Endoscopic Retrograde Cholangiopancreatography) between 2007 and 2016, with some modifications between studies. Complemental cross-linkage was made to national registries, and medical records were reviewed. Papers I, II and III were population-based observational studies with prospectively and retrospectively collected data. Paper IV was a health economic evaluation based on the results from papers I and III.Results and conclusions: Hospitals submitting >75 per cent of gallbladder specimens diagnosed a higher proportion of incidental gallbladder cancer than did hospitals submitting ≤25 per cent of samples (paper I). Incidental gallbladder cancer was more prevalent in older patients, women and patients with acute or previous cholecystitis, as well as ongoing jaundice. The risk model based on predictive preoperative factors was further improved by adding a macroscopic assessment of the gallbladder (paper II). Predictive factors for gallbladder cancer appeared to have an impact on which specimens were submitted in hospitals with a selective approach of histopathology (paper I). For pT2 and pT3 patients, re-resection improved diseasespecific survival, although these groups differed in terms of age and comorbidity (paper III). Residual disease was an independent factor for impaired survival. A change to routine histopathology of gallbladder specimens in Sweden would lead to increased costs with little improved health outcomes. Instead, a more standardized approach to selective histology would be needed (paper IV).
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| 4. |
- Qian, Xiaoyan, 1989-
(författare)
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Towards comprehensive cellular atlases : High-throughput cell mapping by in situ sequencing
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- With recent technological advancements in single-cell biology, many aspects of individual cells are characterized with unprecedented resolution and details. Cell types in human and model organisms are redefined, and multiple organ-wide atlases are proposed to integrate different types of data to provide a comprehensive view of biological systems at cellular resolution. Incorporating location information of cells in such atlases is crucial to understanding the structure and functions. Several spatially resolved transcriptomics technologies may serve this purpose, and in situ sequencing (ISS) is among the most powerful ones.ISS detects the expression of tens to hundreds of genes in situ, i.e. inside preserved cells and tissues. ISS is a targeted approach, using probes designed to identify specific transcripts. Its key advantages, as compared to other spatially resolved gene expression analysis methods, are high throughput, cellular resolution and tissue compatibility, making it a tool ideally suited for spatial cell mapping. The work included in this thesis aims to develop tools and methods for this application.In paper I, a network analysis tool was developed to analyze ISS and other spatially resolved data. The tool enables smooth visualization of large datasets and generates networks based on colocalization. It also includes functions to test statistical significance and resolve tissue heterogeneity.In paper II, we studied spatio-temporal patterns of immune response in tuberculosis granuloma by targeting immune markers with ISS. Using the tool developed in paper I together with other methods, we established an immune response time course at the granuloma sites and found histologically different granulomas based on transcriptional information. The paper demonstrated that ISS can robustly detect transcripts in formalin-fixed paraffin-embedded tissues across biological samples and reveal biologically relevant structures.In paper III, we developed probabilistic cell typing by in situ sequencing (pciSeq), a method to spatially map cell types defined by single-cell RNA-sequencing. pciSeq is an integrated pipeline that includes gene selection, image analysis, barcode calling and cell type calling. We mapped closely related interneuron cell types of the mouse hippocampal CA1 region in 14 coronal sections and validated the results against ground truth.In paper IV, we investigated the quantification bias of ISS resulting from the probe target selection. We developed a method to sequence in situ synthesized cDNA and found that the read coverage of in situ cDNA library reflected ISS counts more closely than conventional RNA sequencing, making it possible, to some extent, to predict a probe’s performance and guide the probe design.Taken together, the developments described in this thesis comprise several tools that make ISS suitable for building cellular atlases via large-scale spatial mapping.
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| 5. |
- Sundqvist, Nicolas, 1993-
(författare)
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Mathematical Modelling of Cerebral Metabolism : From Ion Channels to Metabolic Fluxes
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The brain is the most metabolically active organ in the human body and therefore rely on a continuous supply of oxygen and glucose. Neuronal stimulation in specific regions of the leads to the firing of action potentials, a process facilitated by voltage-gated ion channels in the neurons’ cell membranes. This activation of the ion channels significantly elevates the brain’s metabolic energy demand, compelling neurons to ramp up their metabolic activity in response. Concurrently, this neuronal activation also initiates a signalling cascade that induces vasodilation and increases blood flow, thereby ensuring that regions with elevated neural activity are adequately supplied with oxygen and nutrients. This dynamic interplay between neuronal activity and cerebral blood flow (CBF) regulation constitutes the neurovascular coupling (NVC). The NVC is a cornerstone in interpreting functional Magnetic Resonance Imaging (fMRI) Blood Oxygen Level-Dependent (BOLD) responses. The BOLD response is an indirect, non-invasive, and highly sensitive indicator of neuronal activity, reflecting changes in blood oxygenation and flow associated with the neuronal and metabolic activity in the brain. By examining these responses, we can gain insights into the complex interactions between neuronal activity, energy metabolism, and CBF.Additionally, techniques such as 13C Metabolic Flux Analysis (13C MFA) makes it possible to gain further insight into the cerebral metabolism. This method enables a detailed examination of metabolic pathways and fluxes by tracking the incorporation of 13C-labelled substrates into various metabolites. By using 13C MFA, researchers can quantify the flow of substrates through metabolic networks, offering a deeper understanding of how cell such as neurons adapt their metabolism during different functional states and conditions.Central to exploring these multifaceted aspects of cerebral metabolism is the use of mathematical modelling and systems biology. These disciplines provide a framework for integrating diverse biological data, allowing for the simulation and prediction of complex neurovascular interactions under various physiological and pathological conditions. Mathematical models can encapsulate the dynamics of ion channel kinetics, metabolic pathways, and neurovascular coupling, offering a comprehensive view of the interplay between neuronal activity, metabolism, and cerebral blood flow. This approach is instrumental in bridging the gap between molecular-scale events and observable physiological phenomena, enhancing our understanding of cerebral metabolism and its critical role in the brain’s function.Paper I sets the foundation by developing a mechanistic model that integrates the mechanisms of the NVC with the metabolism. This model connects cerebral regulation of blood flow and metabolism, using small mechanistic model to represent the central metabolism. By integrating experimental data based on nuclear magnetic resonance spectroscopy (NMRS), the model successfully captures the dynamics of metabolites in response to neuronal stimuli, providing a crucial link between metabolic changes and NVC.Paper II extends the investigation to the realm of ion channel kinetics. By developing a generic model structure for voltage-gated ion channels, this paper explores how ion channel activity, a fundamental aspect of neuronal function, influences cerebral metabolism. The model, validated against experimental data and existing kinetic models, accurately predicts various channel behaviours and action potential characteristics. It includes mechanisms like voltage sensor movements and rate constants dependent on membrane voltage, offering a universal approach for studying all types of voltage-gated ion channels in neural networks and other applications.Paper III further explores the neurovascular relationship by examining the influence of inhibitory neurons on CBF and metabolism. This study introduces an expanded mathematical model that integrates the effects of γ-aminobutyric acid(GABA)ergic inhibitory neurons on vascular responses, aligning with new experimental evidence and enhancing understanding of neurovascular coupling (NVC). The model, validated with data from various studies, not only captures vascular changes triggered by inhibitory neuron activation but also reveals how these neurovascular responses vary with stimulation frequency, underscoring the important role of inhibitory neuron in the NVC.Paper IV tackles the critical aspect of accurately measuring metabolic fluxes in cells, focusing on 13C MFA. This study introduces a novel approach for model selection in MFA, ensuring that the chosen models accurately represent the underlying metabolic processes. This method enhances our ability to identify and understand key metabolic pathways and reactions, providing deeper insights into various metabolic conditions. Connecting back to the cerebral metabolism, application of 13C MFA to neuronal systems offers a powerful tool for studying metabolically linked neuropathology such as the development of Alzheimer’s disease.In Conclusion, this thesis establishes key components for understanding the mechanisms of the cerebral metabolism. The integration of mathematical modelling across different scales, from ion channels to cerebral blood flow, is used to provide a comprehensive perspective on how cerebral metabolism is regulated and how it interacts with other physiological processes. This work not only advances our basic scientific knowledge but also holds significant potential for improving our understanding of neurological disorders where metabolism and neurovascular function are impaired.
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| 6. |
- Svedlund, Jessica
(författare)
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Wnt/β-catenin Signaling and Epigenetic Deregulation in Breast Cancer and Parathyroid Tumours
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Wnt/β-catenin signaling pathway is often deregulated in cancer. Here we investigate Wnt/β-catenin signaling, aberrant accumulation of β-catenin, and epigenetic deregulation in breast cancer and parathyroid tumours.An aberrantly spliced Wnt coreceptor LRP5 (LRP5Δ) is important for accumulation of nonphosphorylated active β-catenin and tumour growth in parathyroid tumours. Paper I demonstrated frequent expression of LRP5Δ in breast tumours and substantiated that breast tumour cell growth was dependent on continuous activation of the Wnt/β-catenin pathway by LRP5Δ. A LRP5 antibody reduced the levels of active β-catenin, inhibited tumour cell growth and caused apoptosis in breast cancer cells. Antibody therapy may have a significant role in the treatment of breast cancer.Paper II revealed lost expression of the tumour suppressor gene APC in parathyroid carcinomas, likely due to CpG methylation. Also accumulation of nonphosporylated active β-catenin was observed, indicating activation of Wnt/β-catenin signaling. Treatment of primary parathyroid carcinoma cells with the demethylating agent 5-aza-2’-deoxycytidine reduced the levels of active β-catenin, inhibited cell growth and caused apoptosis, suggesting that adjuvant epigenetic therapy could be considered in patients with metastatic or recurrent parathyroid carcinoma.In paper III we showed that the expression of the tumour suppressor gene HIC1 was generally reduced in parathyroid tumours of primary and secondary origin, and parathyroid carcinomas. Overexpressing HIC1 reduced cell viability and suppressed colony formation, supporting a tumour suppressor role in the parathyroid gland. Results suggested that the observed underexpression of HIC1 could be explained by epigenetic deregulation involving histone methylation rather than CpG methylation.Paper IV demonstrated increased expression of the histone methyltransferase EZH2 in parathyroid tumours of primary and secondary origin, and most apparent in parathyroid carcinomas. Decreasing EZH2 resulted in reduced cell viability and colony formation capacity suggesting that EZH2 may function as an oncogene in parathyroid tumours. Furthermore, depletion of EZH2 also reduced the amount of active β-catenin. EZH2 may represent a novel therapeutic target in parathyroid tumours.The fact that HIC1 was underexpressed and EZH2 overexpressed in parathyroid tumours regardless of the hyperparathyroid disease state may represent a possibility for a common pathway in parathyroid tumour development.
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| 7. |
- Boekel, Carolina, 1977-
(författare)
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Integration and topology of membrane proteins
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins comprise around 20-30% of most proteomes. They play important roles in most biochemical pathways. All receptors and ion channels are membrane proteins, which make them attractive targets for drug design. Membrane proteins insert and fold co-translationally into the endoplasmic reticular membrane of eukaryotic cells. The protein-conducting channel that inserts the protein into the membrane is called Sec61 translocon, which is a hetero-oligomeric channel that allows transmembrane segments to insert laterally into the lipid bilayer. The focus of this thesis is how the translocon recognizes the transmembrane helices and integrates them into the membrane.We have investigated the sequence requirements for the translocon-mediated integration of a transmembrane α-helix into the ER by challenging the Sec61 translocon with designed polypeptide segments in an in vitro expression system that allows a quantitative assessment of membrane insertion efficiency. Our studies suggest that helices might interact with each other already during the membrane-insertion step, possibly forming helical hairpins that partition into the membrane as a single unit. Further, the insertion efficiency for Nin-Cout vs. Nout-Cin transmembrane helices and the integration efficiency of Alzheimer’s Aβ-peptide fragments has been investigated.Finally, detailed topology mapping was performed on two biologically interesting proteins with unknown topology, the human seipin protein and Drosophila melanogaster odorant receptor OR83b.
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| 8. |
- Bogefeldt, Johan, 1969-
(författare)
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Low Back Pain : With Special Reference to Prevalence, Diagnosis, Treatment and Prognosis
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Objectives. Ascertain if there has been a secular trend in 3-months prevalence of casually reported back pain. Evaluate if such back pain predicts concurrent health as well as future sick leave, disability pension, hospitalization and survival. Study differences in diagnostic assessment and labelling between physicians. Evaluate if a comprehensive manual therapy programme reduces sickness absence. Materials and methods. Combined population samples from 1973 to 2003 with a total of 12,891 observations with self-reported back pain and covariates. 7,074 of these individuals were followed for an average of 8.5 years and outcomes were self-reported health as well as official register data on sick leave, disability pension, hospitalisation and mortality. The Gotland Low Back Pain Study, a randomised controlled trial with participation of two general practitioners and two orthopaedic surgeons treating 160 patients with acute/subacute low back pain, with 10 weeks diagnostic evaluation and treatment and a two-year follow up. Results. Back pain prevalence increased 16% per ten years (OR 1.16, 95%CI 1.11-1.22). Back pain was negatively associated with self-rated health (p<0.0001), increased the risk of disability pension (p<0.002), and hospital admissions (p<0.0005), but not number of days in hospital, sick leave or mortality. General practitioners used terms from manual medicine and reported more pseudoradicular pain, while orthopaedic surgeons used non-specific pain labels, reported more true radicular pain and used more x-ray examinations. Among those on sick leave at baseline, manual therapy patients showed faster return to work (HR 1.62, 95%CI 1.006–2.60) and a lower point-prevalence of sick leave than reference patients at end of treatment period (ratio 0.35, 95% CI 0.13–0.97) but not after two years. Conclusions. There was a strong secular trend towards increase in self-reported back pain from 1973 to 2003. Such pain had a negative effect on some of the health outcomes and does not appear to be harmless. Physicians from different specialities labelled the condition differently. The manual therapy programme proved to be more effective than the established treatment regarding return to work.
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| 9. |
- Bohlin, Jan
(författare)
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Enzymes and electron transport in microbial chlorate respiration
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Microbial chlorate respiration plays an important role in the turnover of oxochlorates in nature and industrial waste management. This thesis deals with the characterization of the molecular components of chlorate respiration in Ideonella dechloratans. Chlorate respiration utilizes two soluble periplasmic enzymes, chlorate reductase and chlorite dismutase, to convert chlorate to chloride and oxygen. The genes encoding the enzymes participating in the chlorate degradation have been sequenced, and are found in close proximity, forming a gene cluster for chlorate metabolism. This work also includes the successful recombinant expression of three genes from Ideonella dechloratans. Two of the gene products, chlorite dismutase and the C subunit of chlorate reductase, participate in the chlorate respiration. The third gene, which is found close to the gene cluster for chlorate metabolism, encodes a soluble c-type cytochrome. The localization of the gene suggests the corresponding protein as a candidate for a role as electron donor to chlorate reductase. Also, the role of soluble periplasmic c cytochromes of Ideonella dechloratans in chlorate respiration was studied. At least one of the soluble c cytochromes was found capable of serving as electron donor for chlorate reduction. This c cytochrome, and several others, can also donate electrons to a terminal oxidase for subsequent reduction of oxygen, as required for the branched electron flow during chlorate respiration.
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| 10. |
- Folkersen, Lasse, et al.
(författare)
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Genomic and drug target evaluation of 90 cardiovascular proteins in 30,931 individuals.
- 2020
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Ingår i: Nature metabolism. - : Springer Science and Business Media LLC. - 2522-5812. ; 2:10, s. 1135-1148
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Tidskriftsartikel (refereegranskat)abstract
- Circulating proteins are vital in human health and disease and are frequently used as biomarkers for clinical decision-making or as targets for pharmacological intervention. Here, we map and replicate protein quantitative trait loci (pQTL) for 90 cardiovascular proteins in over 30,000 individuals, resulting in 451 pQTLs for 85 proteins. For each protein, we further perform pathway mapping to obtain trans-pQTL gene and regulatory designations. We substantiate these regulatory findings with orthogonal evidence for trans-pQTLs using mouse knockdown experiments (ABCA1 and TRIB1) and clinical trial results (chemokine receptors CCR2 and CCR5), with consistent regulation. Finally, we evaluate known drug targets, and suggest new target candidates or repositioning opportunities using Mendelian randomization. This identifies 11 proteins with causal evidence of involvement in human disease that have not previously been targeted, including EGF, IL-16, PAPPA, SPON1, F3, ADM, CASP-8, CHI3L1, CXCL16, GDF15 and MMP-12. Taken together, these findings demonstrate the utility of large-scale mapping of the genetics of the proteome and provide a resource for future precision studies of circulating proteins in human health.
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