Tag: biomarker

Role of pharmacological activity of autoantibodies in ME/CFS

Abstract:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a condition characterised by extreme fatigue, memory impairment, pain and other symptoms that vary from patient to patient. It affects about 0.9% of the population and is often triggered by an acute viral or bacterial infection, such as Epstein-Barr virus. The underlying physiological and molecular basis of ME/CFS is unknown, and no effective treatments exist.

One proposed mechanism is that the blood flow is altered by autoantibodies against receptors involved in blood flow regulation. Antibodies are generated by the immune system to recognise intruders and under normal conditions, our immune system is trained not to attack our own tissues. However, during a severe infection, the immune system adopts an “all hands on deck” approach, which results in some of the newly-produced antibodies escaping quality control and targeting our own tissues, autoantibodies. Receptors regulation blood flow are located in walls of blood vessels and cause a blood vessel to dilate or contract as the demand for oxygen and nutrients to tissues such as the brain or muscles changes. Research has found increased levels of these autoantibodies in ME/CFS patients and initial trials removing these autoantibodies from the blood using a technique called immunoadsorption have shown improvement in symptoms.

In this project, we will test the hypothesis that autoantibodies can activate or inhibit the receptors responsible for the blood flow regulation, in a similar way medical drugs are used to regulate blood pressure.
We aim to profile serum samples from 325 ME/CFS patients and 130 healthy individuals to determine the presence of autoantibodies against all thirty receptors involved in blood pressure regulation. Importantly, we will study the ability of autoantibodies detected in each sample to activate or inhibit these receptors in order to test the hypothesis that the activity of these autoantibodies is a decisive factor in the disease.
If our hypothesis is correct, we will be able to develop an accurate blood test that may be able to detect ME/CFS earlier or to independently confirm the diagnosis. Ultimately, we hope that these results may also indicate a possible route for therapeutic intervention to counteract the effects of autoantibodies and alleviate the ME/CFS symptoms using a combination of already existing drugs, specific for each individual case.

 

Technical Summary:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a condition of extreme tiredness and brain fog, often triggered by an acute infection. Its prevalence is ca 0.9% and here is no effective treatment. Competing theories for the root cause of ME/CFS include metabolic or redox homeostasis disruption, and presence of autoantibodies (AABs) against G protein coupled receptors (GPCRs) involved in regulation of blood flow.
Triggered by acute infection, autoimmunity is a result of reduced immuno-vigilance during severe infections, when an “all hands on deck” approach confers survival advantage. About 30% of ME/CFS patients show increased titre of autoantibodies against beta2-adrenoceptor and M3/4 muscarinic receptors controlling vasodilation/vasoconstriction, but this could become higher if all 30 receptors controlling blood flow would be taken into account.
In this project, we will test a hypothesis that the pharmacological activity of AABs against GPCRs is the key to their involvement in ME/CFS. Similar to medical drugs, AABs can be stimulatory (agonistic) or inhibitory (antagonistic) and induce a therapeutic or an undesired side effect.
We will profile 325 patient samples and 130 control plasma samples for AABs and their pharmacological activity using a state-of-the art GPCR drug screening pipeline we have established, against all 30 GPCRs involved in blood pressure regulation. We also have machine learning expertise that would allow us to interpret this extensive dataset, extract the most salient features. This will advance the understanding of the molecular basis of ME/CFS and could form the basis of a robust diagnostic blood test for ME/CFS. Ultimately, our findings may point in the direction of developing combination therapy using repurposed drugs to counteract the effects of autoantibodies and mitigate ME/CFS symptoms and stimulate the development of specific B-cell elimination strategy to cure ME/CFS.
Source: Lead Research Organisation: University of Nottingham, Department Name: School of Life Sciences. https://gtr.ukri.org/projects?ref=MR%2FY003667%2F1&pn=0&fetchSize=25&selectedSortableField=date&selectedSortOrder=ASC

Characterization of subgroups of myalgic encephalomyelitis/chronic fatigue syndrome based on disease onset, symptoms and biomarkers

Abstract:

Myalgic encephalomyelitis, also called chronic fatigue syndrome (ME/CFS), is an acquired multisystem disease. The core symptoms include fatigue, exercise intolerance and pain as well as cognitive, autonomic and immunological manifestations. The diagnosis of ME/CFS is based on clinical criteria. Specific biomarkers do not currently exist, but studies suggest a role for soluble cluster of differentiation 26 (sCD26) and autoantibodies (AAK) against G protein-coupled receptors (GPCR). In many cases, the disease begins as a result of infections. 

The aim of this work was to determine the pathophysiological significance of potential biomarkers, assuming different development mechanisms in patients with infection-associated disease onset compared to those with other triggers. In a first study, sCD26, also called dipeptidyl peptidase-4 (DPP-4) due to its enzymatic activity, was analyzed and compared in the serum of 205 ME/CFS patients and 98 controls. This was followed by a comprehensive correlation analysis between sCD26 and clinical and laboratory parameters for ME/CFS patients, separated by type of disease onset. In addition, CD26 expression on lymphocyte subpopulations was determined for 12 patients and 12 controls. 

In another study, a correlation analysis was carried out between AAK against vasoregulatory GPCR and symptoms in 116 ME/CFS patients, separated by type of disease onset. It was shown that in ME/CFS patients with infection-associated disease onset, sCD26 correlated with numerous immunological and metabolic parameters, the changes of which have also been described in connection with DPP-4 inhibitors. In addition, there were inverse correlations with AAK against alpha1-adrenergic and M3-acetylcholine receptors. 

In this subgroup, the second study found correlations between numerous GPCR-AAK and the severity of fatigue, muscle pain and cognitive symptoms as well as greater functional impairment relevant to everyday life. None of these correlations were found in patients without infection-associated disease onset. 

Here, sCD26 correlated inversely with orthostatically induced heart rate increases and AAK against alpha- and beta-adrenergic receptors with the severity of orthostatic symptoms. Different correlation patterns between AAK against GPCR and symptoms allow us to assume that in patients with ME/CFS, an altered function of the AAK or its receptors or signaling pathways has occurred as a result of an infection. The association of sCD26 and GPCR-AAK also indicates the dysregulation of other parts of the immune system with potentially pathological consequences. The differences presented compared to patients with non-infectious genesis suggest two definable subgroups.

Source: Szklarski, Marvin. Characterization of subgroups of myalgic encephalomyelitis/chronic fatigue syndrome based on disease onset, symptoms and biomarkers. Charité – University Medicine Berlin, dissertation. https://refubium.fu-berlin.de/handle/fub188/40276

Dysregulation of the Kynurenine Pathway, Cytokine Expression Pattern, and Proteomics Profile Link to Symptomology in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Abstract:

Dysregulation of the kynurenine pathway (KP) is believed to play a significant role in neurodegenerative and cognitive disorders. While some evidence links the KP to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), further studies are needed to clarify the overall picture of how inflammation-driven KP disturbances may contribute to symptomology in ME/CFS.

Here, we report that plasma levels of most bioactive KP metabolites differed significantly between ME/CFS patients and healthy controls in a manner consistent with their known contribution to symptomology in other neurological disorders. Importantly, we found that enhanced production of the first KP metabolite, kynurenine (KYN), correlated with symptom severity, highlighting the relationship between inflammation, KP dysregulation, and ME/CFS symptomology.

Other significant changes in the KP included lower levels of the downstream KP metabolites 3-HK, 3-HAA, QUIN, and PIC that could negatively impact cellular energetics. We also rationalized KP dysregulation to changes in the expression of inflammatory cytokines and, for the first time, assessed levels of the iron (Fe)-regulating hormone hepcidin that is also inflammation-responsive. Levels of hepcidin in ME/CFS decreased nearly by half, which might reflect systemic low Fe levels or possibly ongoing hypoxia.

We next performed a proteomics screen to survey for other significant differences in protein expression in ME/CFS. Interestingly, out of the seven most significantly modulated proteins in ME/CFS patient plasma, 5 proteins have roles in maintaining gut health, which considering the new appreciation of how gut microbiome and health modulates systemic KP could highlight a new explanation of symptomology in ME/CFS patients and potential new prognostic biomarker/s and/or treatment avenues.

Source: Kavyani B, Ahn SB, Missailidis D, Annesley SJ, Fisher PR, Schloeffel R, Guillemin GJ, Lovejoy DB, Heng B. Dysregulation of the Kynurenine Pathway, Cytokine Expression Pattern, and Proteomics Profile Link to Symptomology in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Mol Neurobiol. 2023 Nov 28. doi: 10.1007/s12035-023-03784-z. Epub ahead of print. PMID: 38015302. https://pubmed.ncbi.nlm.nih.gov/38015302/

Investigation into the Plasma Proteome Signature in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Abstract:

Background: ME/CFS is a complex disease with unclear etiology. Current diagnostic criteria lack objective laboratory measures.

Aims: This study aimed to investigate the plasma proteomic profile of ME/CFS patients and determine any differentially expressed proteins compared to controls.

Methods: Plasma samples obtained from 19 ME/CFS patients and 9 controls underwent analysis (Somalogic, Inc, CO). The ME/CFS patients met the National Academy of Medicine criteria for the disease. Samples were collected from a mixed venous compartment. Statistical analysis and a Mixed Graphical Model were used to identify candidate biomarker.

Results: Among ~7000 proteins detected, ~400 were differentially expressed between patients and controls (False Discovery Rate<0.05 and Absolute Fold Change ≥1.5). Selectin E (SELE), ATP Synthase Subunit F6 (ATP5PF), and Transcobalamin 2 (TCN2) were identified as top candidates. A classifier of these proteins in pulmonary artery blood of patients were distinguishable from controls (AUC =0.99).

Conclusion: The study highlighted potential biomarkers for ME/CFS, the top candidates of which are involved in inflammation, cellular energy metabolism, and Vitamin B12 transport. The plasma proteomic signature identifies ME/CFS from normals and suggests that the disease’s pathophysiology is driven by abnormalities of aerobic metabolism, vascular dysregulation, and Vitamin B12 metabolism.

Source: Johanna Squires, Sarra Al-Zayer, Peng Li, Wenzhong Xiao, David Systrom. Investigation into the Plasma Proteome Signature in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). European Respiratory Journal Sep 2023, 62 (suppl 67) PA2960; DOI: 10.1183/13993003.congress-2023.PA2960 https://erj.ersjournals.com/content/62/suppl_67/PA2960.abstract

Bioimpedance spectroscopy characterization of Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) peripheral blood mononuclear cells

Abstract:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a disabling and chronic disease, importantly related to the current COVID-19 pandemic. Currently, there are no specific laboratory tests to directly diagnose ME/CFS. In this work, the use of impedance spectroscopy is studied as a potential technique for the diagnosis of ME/CFS. A specific device for the electrical characterization of peripheral blood mononuclear cells was designed and implemented.

Impedance spectroscopy measurements in the range from 1 Hz to 500 MHz were carried out after the osmotic stress of the samples with sodium chloride solution at 1M concentration. The evolution in time after the osmotic stress at two specific frequencies (1.36 kHz and 154 kHz) was analyzed.

The device showed its sensitivity to the presence of cells and the evolution of the osmotic processes. Higher values of impedance (around 15% for both the real and imaginary part) were measured at 1.36 kHz in ME/CFS patients compared to control samples. No significant difference was found between patient samples and control samples at 154 kHz. Results help to further understand the diagnosis of ME/CFS patients and the relation of their blood samples with bioimpedance measurements.

Source: Sara Martinez Rodriguez, Alberto Olmo Fernandez, Daniel Martin Fernandez, Isabel Martin-Garrido. Bioimpedance spectroscopy characterization of Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) peripheral blood mononuclear cells. Biomedical Letters, Volume 9, Issue 2: 121-128. http://thesciencepublishers.com/biomed_lett/v9i2abstract6.html (Full text available as PDF file)

A Systematic Analysis of the Effectiveness of Mitochondrial-Based Therapies for the Management of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Abstract:

Background: This study aimed to compile and analyze an assortment of research findings concerning potential therapeutic strategies for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). The understanding of the multifaceted nature of ME/CFS and the need for varied and personalized therapeutic approaches were central to this investigation.

Methods: A comprehensive review and analysis of various studies conducted on ME/CFS was undertaken. These studies covered a wide array of interventions, including pharmacological treatments, nutritional supplements, dietary changes, physical therapies, and lifestyle modifications. The analysis pertained to the effectiveness of these interventions, potential physiological and biochemical markers, and the response of ME/CFS patients to different treatment strategies.

Results: The 22 selected papers investigated demonstrated varied responses to the multitude of interventions. While some interventions showed significant improvement in fatigue and biochemical parameters, others found no significant differences between the treated and control groups. Potential physiological and biochemical markers for ME/CFS, such as impaired T cell metabolism, reduced flow-mediated dilation, and decreased work rate at the ventilatory threshold, were highlighted.

Conclusion: The findings underscored the complexity of ME/CFS and the need for personalized treatment strategies. Despite mixed results and several limitations, these studies collectively contributed to understanding ME/CFS’s complex pathophysiology and treatment, laying the groundwork for future research towards more effective therapeutic strategies for this debilitating disease.

Source: Keferstein, L.G. A Systematic Analysis of the Effectiveness of Mitochondrial-Based Therapies for the Management of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Preprints 2023, 2023100637. https://doi.org/10.20944/preprints202310.0637.v1 https://www.preprints.org/manuscript/202310.0637/v1 (Full text available as PDF)

THU581 Possible Markers For Myalgic Encephalomyelitis / Chronic Fatigue Syndrome Developed In Long Covid: Utility Of Serum Ferritin And Insulin-like Growth Factor-I

Abstract:

Almost three years have passed since coronavirus disease 2019 (COVID-19) pandemic broke out, and along with the number of acute COVID-19 patients, the number of patients suffering from chronic prolonged symptoms after COVID-19, long COVID, or post COVID-19 condition, has also increased.

We established an outpatient clinic specialized for COVID-19 after care (CAC) in Okayama University Hospital in Japan in February 2021. Our recent study has revealed that the most common symptom is “fatigue”, a part of which potentially may develop into myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). However, the pathogenesis and specific prognosticator have yet to be elucidated. The aim of this study was to elucidate the clinical characteristics of patients who developed ME/CFS after COVID-19.

This retrospective observational study investigated the patients who visited our CAC outpatient clinic between February 2021 and March 2022. Of the 234 patients, 139 (59.4%) had fatigue symptoms, of whom 50 (21.4%) met the criteria for ME/CFS (ME/CFS group), while other 89 did not (non-ME/CFS group); 95 patients had no fatigue complaints (no-fatigue group). Although the patients’ backgrounds were not significantly different among the three groups, the ME/CFS group presented the highest scores on the self-rating symptom scales, including the Fatigue Assessment Scale (FAS), EuroQol, and Self-Rating Depression Scale (SDS).

Of note, serum ferritin levels, which were correlated to FAS and SDS scores, were significantly higher in the ME/CFS group (193.0 μg/mL; interquartile range (IQR), 58.8-353.8) than those of non-ME/CFS (98.2 μg/mL; 40.4-251.5) and no-fatigue (86.7 μg/mL; 37.5-209.0) groups, and this trend was prominent in the female patients. Endocrine workup further showed that the ME/CFS group had higher thyrotropin levels but lower growth hormone levels in the serum, and that insulin-like growth factor (IGF)-I levels were inversely correlated with ferritin levels (R = -0.328, p < 0.05).

Collectively, we revealed that serum ferritin levels could be a possible predictor for developing ME/CFS related to long COVID, especially in female patients. Earlier studies have suggested that hyperferritinemia is a clinical feature in the patients of long COVID, in which hepcidin-like effects could also be involved. Our present study also uncovered a relationship between hyperferrinemia and endocrine disorders among patients developing ME/CFS after COVID-19, although further investigations are necessary to understand the characteristics of ferritin metabolism.

Presentation: Thursday, June 15, 2023

Source: Yukichika Yamamoto, Yuki Otsuka, Kazuki Tokumasu, Naruhiko Sunada, Yasuhiro Nakano, Hiroyuki Honda, Yasue Sakurada, Toru Hasegawa, Hideharu Hagiya, Fumio Otsuka, THU581 Possible Markers For Myalgic Encephalomyelitis / Chronic Fatigue Syndrome Developed In Long Covid: Utility Of Serum Ferritin And Insulin-like Growth Factor-I, Journal of the Endocrine Society, Volume 7, Issue Supplement_1, October-November 2023, bvad114.1370, https://doi.org/10.1210/jendso/bvad114.1370 (Full text available as PDF file)

Unsupervised cluster analysis reveals distinct subtypes of ME/CFS patients based on peak oxygen consumption and SF-36 scores

Abstract:

Purpose: Myalgic encephalomyelitis, commonly referred to as chronic fatigue syndrome (ME/CFS), is a severe, disabling chronic disease and an objective assessment of prognosis is crucial to evaluate the efficacy of future drugs. Attempts are ongoing to find a biomarker to objectively assess the health status of (ME/CFS), patients. This study therefore aims to demonstrate that oxygen consumption is a biomarker of ME/CFS provides a method to classify patients diagnosed with ME/CFS based on their responses to the Short Form-36 (SF-36) questionnaire, which can predict oxygen consumption using cardiopulmonary exercise testing (CPET).

Methods: Two datasets were used in the study. The first contained SF-36 responses from 2,347 validated records of ME/CFS diagnosed participants, and an unsupervised machine learning model was developed to cluster the data. The second dataset was used as a validation set and included the cardiopulmonary exercise test (CPET) results of 239 participants diagnosed with ME/CFS. Participants from this dataset were grouped by peak oxygen consumption according to Weber’s classification. The SF-36 questionnaire was correctly completed by only 92 patients, who were clustered using the machine learning model. Two categorical variables were then entered into a contingency table: the cluster with values {0,1} and Weber classification {A, B, C, D} were assigned. Finally, the Chi-square test of independence was used to assess the statistical significance of the relationship between the two parameters.

Findings: The results indicate that the Weber classification is directly linked to the score on the SF-36 questionnaire. Furthermore, the 36-response matrix in the machine learning model was shown to give more reliable results than the subscale matrix (p – value < 0.05) for classifying patients with ME/CFS.

Implications: Low oxygen consumption on CPET can be considered a biomarker in patients with ME/CFS. Our analysis showed a close relationship between the cluster based on their SF-36 questionnaire score and the Weber classification, which was based on peak oxygen consumption during CPET. The dataset for the training model comprised raw responses from the SF-36 questionnaire, which is proven to better preserve the original information, thus improving the quality of the model.

Source: Lacasa M, Launois P, Prados F, Alegre J, Casas-Roma J. Unsupervised cluster analysis reveals distinct subtypes of ME/CFS patients based on peak oxygen consumption and SF-36 scores. Clin Ther. 2023 Oct 4:S0149-2918(23)00352-1. doi: 10.1016/j.clinthera.2023.09.007. Epub ahead of print. PMID: 37802746. https://www.clinicaltherapeutics.com/article/S0149-2918(23)00352-1/fulltext (Full text)

Long read sequencing characterises a novel structural variant, revealing underactive AKR1C1 with overactive AKR1C2 as a possible cause of unexplained severe fatigue

Abstract

Background: Causative genetic variants cannot yet be found for many disorders with a clear heritable component, including chronic fatigue disorders like myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). These conditions may involve genes in difficult-to-align genomic regions that are refractory to short read approaches. Structural variants in these regions can be particularly hard to detect or define with short reads, yet may account for a significant number of cases. Long read sequencing can overcome these difficulties but so far little data is available regarding the specific analytical challenges inherent in such regions, which need to be taken into account to ensure that variants are correctly identified.

Research into chronic fatigue disorders faces the additional challenge that the heterogeneous patient population likely encompasses multiple aetiologies with overlapping symptoms, rather than a single disease entity, such that each individual abnormality may lack statistical significance within a larger sample. Better delineation of patient subgroups is needed to target research and treatment.

Methods: We use nanopore sequencing in a case of unexplained severe fatigue to identify and fully characterise a large inversion in a highly homologous region spanning the AKR1C gene locus, which was indicated but could not be resolved by short-read sequencing. We then use GC-MS/MS serum steroid analysis to investigate the functional consequences.

Results: Several commonly used bioinformatics tools are confounded by the homology but a combined approach including visual inspection allows the variant to be accurately resolved. The DNA inversion appears to increase the expression of AKR1C2 while limiting AKR1C1 activity, resulting in a relative increase of inhibitory neurosteroids and impaired progesterone metabolism.

Conclusions: This study provides an example of how long read sequencing can improve diagnostic yield in research and clinical care, and highlights some of the analytical challenges presented by regions containing tandem arrays of genes. It also proposes a novel gene associated with a specific disease aetiology that may be an underlying cause of complex chronic fatigue and possibly other conditions too. It reveals biomarkers that could be assessed in a larger cohort, potentially identifying a subset of patients who might respond to treatments suggested by the aetiology.

Source: Julia Oakley, Martin Hill, Adam Giess, Mélanie Tanguy, Greg Elgar. Long read sequencing characterises a novel structural variant, revealing underactive AKR1C1 with overactive AKR1C2 as a possible cause of unexplained severe fatigue. ResearchSquare [Preprint] https://www.researchsquare.com/article/rs-3218228/v2 (Full text)

Investigating the potential role of circulatory extracellular vesicles in myalgic encephalomyelitis/ chronic fatigue syndrome

Abstract:

ME/CFS is a debilitating disease thought to affect millions of individuals. Still, the etiology of ME/CFS is unknown, and there are no standard treatments or established biomarkers. The current symptom-based diagnosis is extensive, and the use of different diagnostic criteria contributes to heterogeneity among patients and may problematize the comparison of findings. Thus, the discovery of a biomarker for ME/CFS is urgent and would benefit both patients and the ME/CFS research field.

Extracellular vesicles (EVs) are membrane limited vesicles secreted by all cells to the extracellular environment and can be collected through biofluids. EVs serve many functions, including transferring functional proteins, lipids, and nucleic acids between cells, thus mediating cell-to-cell communication. EV secretion and cargo may reflect disease state and EVs thus pose great potential as source of minimally invasive biomarkers.

The primary aim of this project was to study EVs in plasma from ME/CFS patients and assess the potential of EVs as source of biomarkers for the disease.

Using size exclusion chromatography, EVs were enriched from plasma from ME/CFS patients (n = 20) and healthy controls (n=20). Success of EV isolation was determined in representative patient- and control EV pools (n=5) using western blotting and transmission electron microscopy. Western blot experiments for detection of EV markers CD9, CD63 and TSG101, and albumin, were optimized and confirmed enrichment of EVs and presence of non-EV eluates in the isolated samples.

EV enrichment was further validated through observation of intact EVs on transmission electron micrographs, however few CD63-positive EVs were observed. Through analysis of nanoparticle tracking analysis data, the isolated EV population primarily consisted of small EVs (< 200 nm). Within this EV population, meanand mode EV size was similar between cohorts, but the EV concentration was significantly elevated in samples from patients compared to controls (p = 0.006). However, statistical tests may have been influenced by high variation within the ME/CFS cohort.

Early-stage analysis of tandem mass spectrometry data identified 663 EV associated proteins. The majority of detected proteins overlapped with registered EV proteins, but only few differences could be observed between patient- and control derived samples. However, differential expression was not analyzed.

A biomarker for ME/CFS could not be suggested at this stage of the study, however increased EV concentration suggests abnormality in EV secretion in patients which strengthens their potential as source of biomarkers and further motivates EV research in ME/CFS and related diseases.

Source: Elena Støvring Yran. Investigating the potential role of circulatory extracellular vesicles in myalgic encephalomyelitis/ chronic fatigue syndrome. Master Thesis [University of Oslo] https://www.duo.uio.no/bitstream/handle/10852/103812/1/Masterthesis_ElenaYran_May15th2023.pdf  (Full text)