Doctoral Theses / Doktorsavhandlingar Institutionen för biomedicin

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On blood and cerebrospinal fluid biomarkers of HIV-1 in the central nervous system
(2025-09-08) Renborg, Linn
Infection of the central nervous system (CNS) occurs early after transmission of human immunodeficiency virus (HIV), establishing a chronic infection which, if left untreated, can cause neurocognitive impairment in people living with HIV (PWH). In this thesis, biomarkers in blood and cerebrospinal fluid (CSF) were used to investigate aspects of pathogenesis, response to antiretroviral treatment (ART), and effects of treatment switch regarding HIV infection of the CNS. In paper I, the astroglial biomarker YKL-40 was measured in CSF in 120 PWH (7 with HIV-associated dementia, 85 untreated asymptomatic, and 28 on ART). YKL-40 was strongly correlated with neurofilament light (NfL), a biomarker of neuronal injury, implicating astrocyte activation as a part of the neuropathogenesis of HIV. In paper II, NfL, together with a panel of biomarkers of neuroinflammation, was measured in longitudinal archived CSF samples from 99 neuroasymptomatic PWH initiating ART. We found that all biomarkers declined significantly after three as well as twelve months of treatment, most pronouncedly in participants with elevated NfL levels at baseline. Paper III evaluated switching from one prodrug of the antiretroviral drug tenofovir; tenofovir disoproxil fumarate (TDF), to tenofovir alafenamide fumarate (TAF), a newer prodrug associated with less renal and bone density side effects. NfL was measured in plasma at baseline and after 24 and 84 weeks in 272 participants who switched to TAF and 144 continuing with TDF. There was a small but significant decrease of plasma NfL in the TAF group, resulting in significantly lower plasma NfL levels compared to the TDF group, after 84 weeks. In paper IV, treatment simplification to a two-drug regimen of dolutegravir and lamivudine was evaluated in 113 PWH by measuring plasma NfL before and one year after switch and by analyzing CSF in a subgroup of 20 participants with a sensitive multiplex immunoassay of 127 biomarkers. We found no significant changes in NfL or inflammatory biomarkers, indicating the CNS safety of switch to this regimen. In conclusion, astrocyte activation is associated with neuronal injury in HIV, ART effectively reduces signs of neuroinflammation and neuronal injury and regimen switch to both TAF and dolutegravir/lamivudine appear safe regarding CNS.
Decoding Glycosylation Signatures of Viral Envelope Proteins: From Structural Analysis to Vaccine Development Strategies
(2025-09-05) Könighofer, Ebba
Glycoproteins incorporated into the viral envelope are critical for virus-host interactions as they mediate viral attachment to cells as well as membrane fusion between the cellular and viral membranes, allowing viral entry into the cell. The exposure of glycoproteins on viral surfaces makes them key targets for the immune system. Despite the biological significance of viral glycoproteins, there is a lack of information regarding their glycan distribution, composition, and immunomodulatory properties. This thesis investigates the glycoprofile of selected viral glycoproteins and the impact of the glycosylation on antibody reactivity and adaptive immune activation, using native and recombinant glycoproteins from tick-borne encephalitis virus (TBEV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), varicella zoster virus (VZV) and herpes simplex virus type 2 (HSV-2). We characterized the glycosylation sites and glycoform distribution of both N-linked and O-linked glycans on a key viral envelope glycoprotein from each virus studied. This included the identification of novel O-linked glycans and confirmation of previously reported O- and N-linked glycans. In addition, we analysed convalescent serum samples from previously infected patients to assess antibody reactivity toward glycoproteins with varying glycan profiles, revealing individual differences in B-cell epitope recognition patterns. Lastly immunization with a recombinant form of glycoprotein G from HSV-2 (mgG- 2) showed that its glycans are facilitating protection against neuronal spread and severe disease, and that the recombinant viral glycoprotein show potential as a vaccine candidate due to the ability to elicit strong B- and T-cell responses. To verify this finding, we utilized a murine genital challenge model and showed that neuronal spread of HSV-2 is hampered in a mgG-2 deficient HSV-2 strain. The data presented in this thesis emphasizes that the development of effective glycoprotein-based vaccines requires a comprehensive understanding of glycosylation patterns, the factors shaping the glycan landscape, and their impact on antigenicity and immunogenicity.
Genetic Contributions to Invasion and Biofilm Disruption in a Microbial Model Community
(2025-09-04) Burman, Emil
This thesis investigates how a synthetic microbial community respond to environmental stress and microbial invasion, using the model community THOR—comprising Pseudomonas koreensis, Bacillus cereus, and Flavobacterium johnsoniae. Through microbiological assays, transposon mutagenesis (INSeq), and proteomics, the work links environmental conditions and genetic determinants to community stability and disruption. The aim was to explore how cooperative traits emerge and how pathogens like Pseudomonas aeruginosa interfere with these dynamics. Paper I shows that cooperative biofilm formation in THOR is highly temperature-sensitive, with peak synergy at 18 °C and collapse at higher temperatures, suggesting fragility in microbial interactions. Paper II demonstrates that P. aeruginosa invasion disrupts biofilm formation without altering species composition, indicating a mode of functional interference rather than direct competition. Paper III identifies chromosomal genes conferring resistance to ciprofloxacin and tetracycline, including mexH, rhlI, and cysA, though these did not influence community invasion. Paper IV reveals invasion-specific genes such as wzz, nqrD, and pvdE, essential for successful colonization of THOR biofilms. Paper V explores the role of pvdE in depth, showing that its overexpression enhances invasion and triggers broad regulatory changes beyond iron acquisition. This work highlights how microbial interactions depend on both environmental context and genetic regulation. Pathogens like P. aeruginosa can destabilize communities through subtle interference mechanisms, with specific genes enabling invasion. These findings advance our understanding of microbial ecology, virulence, and resilience, with implications for managing microbiomes in environmental and clinical settings.
Proteomic analysis of chronically inflamed intestine and disease-related strictures in Crohn’s disease patients
(2025-09-04) Alfredsson, Johannes
Crohn’s disease (CD) is a chronic inflammatory bowel disease characterized by recurrent episodes of dysregulated immunity in segments of the gastrointestinal tract, leading to progressive tissue damage. Despite advances in medical therapies to reduce inflammation, many patients experience lifelong suffering and undergo repeated surgeries due to long-term complications, with no cure currently available. One severe CD complication is intestinal strictures, characterized by muscular hypertrophy and fibrosis in chronically inflamed intestine, causing successive narrowing and obstruction. Despite its severity, the mechanism underlying stricture formation remains poorly understood. This thesis advances understanding of stricture-associated mechanisms in CD using unbiased mass spectrometry and targeted spatial proteomics. In the first study, we identified proteome signatures in FACS-sorted immune- and epithelial cells from inflamed mucosal tissue from CD patients compared to controls. Proteins linked to mitochondrial translation and neutrophil degranulation were enriched in immune cells from CD mucosa, while epithelial cells showed prominent changes in glycosylation and secretory pathways. The second study examined CD patients with intestinal strictures and compared the proteomes of laser-microdissected submucosa (SM) and muscularis propria (MP) tissue layers in stricture versus control. Significant proteome shifts were observed in both tissue layers, with more pronounced alterations in the SM. In the SM, a transition from homeostasis to inflammation, fibrosis and muscular expansion characterized the proteome of strictures versus controls. Differential expression (DE) analysis revealed distinct protein clusters associated with each of these pathological states in stricture SM. In the MP layer, increases in immunity and ECM-associated proteins were accompanied by reductions in mitochondrial and key muscle regulatory proteins. Beyond layer-specific alterations, interlayer analysis highlighted concordant changes in subsets of proteins in the SM and MP, suggesting parallel processes occur in these layers in stricture formation. Top increased DE proteins shared between the layers included immune-related proteins and ER chaperones, while top shared decreased DE proteins were lipid transporters. Building on these insights, spatial proteomics was used for cellular profiling of strictured intestinal wall. Distinct spatial distributions and cellular associations of top DE proteins were apparent in strictured tissue, suggesting their potential roles in pathogenesis. In conclusion, this thesis provides detailed protein-level data and cellular profiling of defined anatomic regions of strictured intestine, an area that thus far is largely unexplored. The insights gained through this exploratory approach reveal several protein targets potentially involved in stricture pathogenesis, and opens avenues to explore new treatments for CD-related strictures. Finally, we show that differential protein expression is spatially restricted, emphasizing the importance of compartmentalized analysis of human tissue to optimize drug development.
Molecular Mechanisms and Therapeutic Targeting of Ribosome Assembly
(2025-08-28) Sjövall, Daniel
Protein synthesis is a tightly regulated cellular process regulated by major intracellular signaling pathways. In cancer, including acute myeloid leukemia (AML), these pathways are frequently dysregulated. Despite this, the role of protein synthesis control in AML remains incompletely understood. Ribosome assembly, where ribosomal subunits form a functional ribosome, is essential for protein synthesis. Disruption of this process causes Shwachman-Diamond Syndrome (SDS), a developmental disorder affecting multiple organs, underscoring the critical need for precise protein synthesis regulation. Here we show that immature fractions of leukemia cells have increased protein synthesis and ribosome biogenesis. Disrupting ribosome assembly in AML cells using a transgenic mouse model sharply reduced leukemia burden and prolonged survival. Single-cell RNA sequencing (scRNA-seq) revealed that leukemia cells adapt by upregulating ribosome biogenesis and downregulating myeloid transcription factors. Building on this, we set out to assess the effect of the clinically relevant protein synthesis inhibitor homoharringtonine. Transplantation assays showed improved survival without affecting normal hematopoietic stem cells counts. scRNA-seq revealed increased ribosome biogenesis, cell cycle activity, and upregulation of Myc and its targets in treatment-resistant leukemia cells. We examined ribosome assembly in the context of SDS using RNA and polysome-sequencing as well as subcellular fractionation, revealing a novel layer of spatial regulation. Dysregulation of key ribosome assembly players SBDS and eIF6 leads to a relative increase in translation of endoplasmic reticulum-targeted mRNAs. This shift is accompanied by a transcriptional adaptation characterized by increased ribosome biogenesis and downregulation of NMNAT2. This work validates protein synthesis and ribosome assembly as relevant therapeutic targets in AML. Additionally, by exploring the role of ribosome assembly in an SDS-like context, we add to the understanding of the cell’s response to insults to the translational machinery. Together, these findings highlight strategies to manipulate ribosomal machinery, either by correcting defects or exploiting them.
Approaches to Enhancing Mucus Production to Counteract Infection
(2025-08-27) Santos, Licínia
Mucosal surfaces are covered with mucus that protects the underlying epithelium from pathogens and particles. As the primary routes through which pathogens enter the host, mucosal surfaces rely on this mucus layer as a critical first line of defense. Mucins - highly O-glycosylated glycoproteins - are synthesized and secreted by mucous and goblet cells, and they play key roles in pathogen binding, clearance, and modulation of microbial growth and virulence. Helicobacter pylori is the most common gastric pathogen, it is associated with chronic inflammation of the gastric mucosa, and in some cases progression to gastric adenocarcinoma. While H. pylori can attach to gastric epithelial cells, it predominantly resides within the mucus layer where it interacts with mucins. Similarly, Aeromonas salmonicida is an opportunistic pathogen responsible for furunculosis in rainbow trout and other fish. Mucins can bind to A. salmonicida and regulate its growth. In this thesis, I investigated the use of compounds known to affect mucin production to enhance and restore mucin biosynthesis and assessed how these changes influence pathogen localization and host inflammation. Mice infected with H. pylori were treated with Interleukin-4, (R)-α-methylhistamine, Rebamipide, Roxatidine, or a combination of the latter two compounds. These treatments exhibited gastroprotective effects by reducing inflammation scores. Using metabolic labeling by GalNAz incorporation, we observed increased mucin biosynthesis following treatments. This increase in mucin biosynthesis correlated with a reduction in H. pylori colonization in the gastric pits. In rainbow trout, lipopolysaccharide treatment resulted in an increased mucin production in the stomach and intestine. Furthermore, mucins isolated from rainbow trout regulated A. salmonicida virulence and reduced the bacterium’s ability to auto-aggregate. These findings suggest that targeting mucosal surfaces to enhance mucin biosynthesis represents a promising strategy to counteract infections caused by mucosal pathogens such as H. pylori and A. salmonicida. However, further research is necessary to elucidate the regulatory mechanisms of mucin production and secretion, as well as to better understand the functional characteristics of the secreted mucus.
Development of targeted therapies against FET oncogene sarcomas
(2025-08-27) Vannas, Christoffer
FET oncogene sarcomas are fusion-driven malignancies characterized by gene fusions involving one of the genes FUS, EWSR1 or TAF15, fused to a transcription factor partner. FET oncogene sarcomas comprise more than ten different sarcoma entities, with myxoid liposarcoma (MLS) and Ewing sarcoma (EWS) being the most prevalent. Current treatments for advanced disease in MLS and EWS rely on chemotherapy, but outcomes remain poor. New therapies and improved methods to assess treatment efficacy are urgently needed. The aim of this project was to explore potential targeted therapies in preclinical models of MLS and EWS. Initially, we analyzed receptor tyrosine kinase (RTK) signaling in MLS and tested the efficacy of targeted RTK inhibitors. Overall, they showed limited therapeutic benefit, likely due to compensatory crosstalk between different RTKs facilitated by the chaperone protein HSP90. We subsequently evaluated the HSP90 inhibitor 17-DMAG in both MLS cell lines and a patient-derived xenograft (PDX) model of MLS, observing potent anti-tumor effect. Various HSP90 inhibitors then demonstrated comparable efficacy in vitro, while the in vivo effects varied significantly between inhibitors. Next, we investigated tumor vessel formation in MLS and found that the tumor vasculature was enriched with pericytes. Additionally, we observed increased PDGFB-PDGFRB signaling, suggesting the presence of an autocrine loop that may influence both cell survival and vascular architecture. Targeting PDGFRB with the multi-RTK inhibitor imatinib caused tumor regression in vivo, though it did not noticeably alter the vascular morphology. We then pursued another treatment strategy targeting epigenetic dysregulation caused by the FET fusion oncoproteins. Combined inhibition of BRD4 and histone deacetylases, both epigenetic proteins, resulted in synergistic effects in vitro and significant tumor regression in vivo. Finally, we explored the use of circulating tumor DNA and inflammatory protein profiles as informative blood-based biomarkers for treatment monitoring. We demonstrated that simultaneous ctDNA and inflammatory protein quantification can be used to monitor treatment response and provide complementary tumor-related data in a patient with metastatic undifferentiated pleomorphic sarcoma. In conclusion, our findings add to the understanding of dysregulated signaling pathways in FET oncogene sarcomas and potential targeted therapies to be further evaluated in clinical trials. Furthermore, incorporating quantification of novel blood-based biomarkers may enhance the precision and adaptability of future clinical trials, ultimately improving patient outcomes through real-time response assessment.
The importance of membrane order in T cell signalling and viral infection
(2025-05-20) Huang, Ainsley
The plasma membrane is a dynamic and heterogeneous structure, whose lateral organization into lipid nanodomains critically regulates cellular processes such as signaling, adhesion, and pathogen entry. In T cells, membrane order, which reflects the degree of lipid packing, plays a pivotal role in organizing signaling platforms and modulating immune responses. In this thesis, membrane order was investigated using the environment-sensitive fluorescence probes Laurdan and C-laurdan, with generalized polarization (GP) used to quantify lipid packing. Paper I presents an optimized GP imaging approach for C-laurdan-labeled T cells by introducing a systematic deconvolution protocol. Deconvolution improved the spatial resolution of fluorescence images, enabling more accurate delineation of plasma membrane regions of interest (ROIs) and enhancing the sensitivity for detecting differences in membrane order between plasma and intracellular membranes. A detailed protocol for optimizing deconvolution parameters was established, allowing for reproducible and higher-precision GP measurements. Paper II investigates how different substrate types and environmental conditions influence T cell plasma membrane order at the equatorial plane and examines how these factors relate to early activation events. Laurdan-based imaging revealed that membrane order at the equatorial plane did not differ significantly across conditions, including CD3- and CD45-functionalized supported lipid bilayers, PLL-coated surfaces, hydrogels, and microfluidic suspension. Although CD3-functionalized SLBs showed a non-significant trend toward increased membrane order, changes were subtle overall. Cells in suspension exhibited a wider range of GP values, while those in hydrogels showed no significant increase in order compared to TESPA. Calcium imaging demonstrated that CD3-functionalized SLBs triggered significantly faster responses than CD45-functionalized SLBs, despite both supporting similar activation levels. Additionally, PLL-coated surfaces induced a higher fraction of activated cells compared to TESPA, though with similar timing. These findings indicate that while substrate composition and receptor engagement strongly influence early signalling dynamics, they do not induce major changes in global membrane order at the equator. Paper III examines the effects of simvastatin on plasma membrane organization and SARS-CoV-2 infection. Treatment with simvastatin reduced plasma membrane order, as assessed by C-laurdan GP imaging, and led to a significant decrease in both intracellular viral load and extracellular virus release in lung epithelial cells. These effects were not solely explained by alterations in ACE2 expression, suggesting that modulation of membrane order contributed directly to limiting viral infection and propagation. Overall, this thesis demonstrates that plasma membrane order is a key determinant in T cell signaling and viral infection. By advancing imaging methodologies and elucidating the interplay between membrane organization and cellular function, this work provides new insights into the biophysical regulation of immune responses and identifies membrane order as a potential target for therapeutic intervention.
Exploring the genetics of hereditary cancer, with a focus on colorectal cancer
(2025-05-13) Eiengård, Frida
Hereditary cancer syndromes are characterized by an inherited genetic predisposition that increases an individual's risk of developing specific types of cancer. Hereditary cancer accounts for about 5-10 % of all cancer cases, and most hereditary cancer syndromes follow an autosomal dominant inheritance pattern. The genetic mecha-nisms underlying predisposition to hereditary cancer syndromes include errors in tu-mor suppressor genes, DNA repair genes, oncogenes and other genes involved in cellu-lar processes. This thesis aimed to enhance diagnostic yield and advance understanding of the ge-netics underlying hereditary cancer syndromes, with a specific focus on hereditary colorectal cancer, through the application of novel technologies and AI-integrated analysis. Applying next-generation sequencing and broad gene panels led to the identification of pathogenic variants in novel genes, POLE and GREM1, which now are established as high risk genes for hereditary colorectal cancer. We also found that bi-allelic non-sense variants in MLH3 give rise to colorectal cancer. The investigation of genotype-phenotype relationships across different types of hereditary cancer syndromes re-vealed overlapping manifestations between syndromes, suggesting broader gene pan-els in clinical diagnostics. Families with a pattern of hereditary colorectal cancer sur-prisingly harbored pathogenic variants in genes BRCA1 and RAD51C, primarily asso-ciated with hereditary breast- and ovarian cancer. Additionally, we quantified the transcript level from promoter 1B of the APC gene. Significantly lower expression levels were observed in patients with sporadic colorectal cancer compared to healthy individuals. In conclusion, combining advanced sequencing with AI-driven analysis improves the handling of large genomic datasets, enhancing detection, classification, and interpre-tation of variants in hereditary cancer syndromes. In addition, the transcript level generated from promoter 1B of the APC gene is proposed as a biomarker for early detection of colorectal cancer.
Enteric infections in Rwandan children under 5 years
(2025-05-13) Munyemana, Jean Bosco
Infectious diarrhea is a main cause of illness and death among children in poor countries, and enteric infections may also cause stunting as a result of impaired uptake of nutrients. This thesis investigated a broad range of enteric pathogens among children under five in Rwanda, focusing on their role in diarrhea, the rates of pathogen acquisition and clearance, and the potential impact of IFNL4 and FUT2 genetic variants on infection susceptibility. The findings in Paper I show high infection rates among 794 Rwandan children with Shigella and ETEC as the primary causes of diarrhea, while rotavirus was no longer the predominant etiology. However, as shown in Paper II, rotavirus remains an important cause of diarrhea despite vaccination since 2012. The longitudinal cohort study of 120 children described in Paper II also shows that new enteric infections occur very frequently, indicating heavy exposure, particularly in rural areas, but also that most infections are cleared within one month. Genetic variation in the IFNL4 gene has recently been associated with the rate of diarrhea in a study from Mali, but Paper III did not identify any significant association between the rs12979860 genotypes and any of a broad range of enteric pathogens. Paper IV showed that ‘non-secretors’ with a FUT2 stop codon variant had a lower frequency of rotavirus but identified no association with other pathogens. The results show that Rwandan children were heavily exposed to enteric pathogens, that this exposure was greater among children in rural areas, that Shigella, ETEC, and rotavirus were the main causes of diarrhea, and that children with inactivated FUT2 (non-secretors) were less susceptible for rotavirus. The findings suggest that reducing exposure by improving living conditions should be a main priority to improve childhood health and growth in Rwanda.
NOX-inflicted oxidative stress in neurodegeneration and inflammation
(2025-05-13) Törnell, Andreas
Further understanding of mechanisms that promote neuronal decay in neurodegenerative diseases may pave the way for new therapies. Aberrant activation of the reactive oxygen species (ROS)-generating enzyme NADPH oxidase 2 (NOX2) in myeloid cells is suggested to contribute to neurodegeneration in experimental models. However, its exact role in human disease is not known. We aimed to define the impact of NOX2 activity on neurodegeneration and identify potential therapeutic strategies for NOX2-inflicted pathologies. To this end, we examined single nucleotide polymorphisms (SNPs) that affect the magnitude of NOX2-derived ROS formation in the context of the neurodegenerative and neuroinflammatory diseases multiple sclerosis (MS), Guillain-Barré syndrome (GBS), and Parkinson’s disease (PD). Furthermore, we investigated the NOX2-inhibitory potential of inhibitors of Bruton’s tyrosine kinase (BTKi), which regulates myeloid cell activation. We identified two SNP alleles (rs4673 A and rs1049254 G in CYBA, encoding the NOX2 subunit p22phox) that were associated with reduced NOX2-derived ROS production. In MS, these low-ROS alleles heralded reduced disease severity and a markedly delayed onset of secondary progressive MS (paper I). Patients with GBS carrying low-ROS alleles were less likely to require assisted ventilation during the acute phase and experienced a rapid recovery of motor function (paper II). Furthermore, an analysis of clinical milestone cumulation in idiopathic PD revealed that patients with low-ROS alleles showed a reduced rate of disease progression (paper III). In paper IV, we demonstrated that BTKi effectively blocked activation of NOX2 in myeloid cells in response to surface receptor stimulation. This translated into potentiated natural killer cell-mediated clearance of malignant cells in the presence of immunosuppressive myeloid cells in vitro and in vivo. In conclusion, our results suggest that NOX2-derived ROS may contribute to neuronal death in MS, GBS, and PD. This implies that NOX2 might serve as a generic driver of neurodegeneration and invites research on its role in additional neurodegenerative diseases. The NOX2-inhibitory potential of BTKi makes them conceivable candidates to target myeloid immunosuppression in both hematological and solid cancers, as well as to alleviate other NOX2-dependent pathologies.
Comparisons between new and established methods for analysis of response to treatment in acute leukemia
(2025-05-08) Johansson Alm, Sofie
Acute leukemia is a severe hematologic malignancy that affects both adults and children. Although prognosis has improved over recent decades, relapse remains one of the major challenges. A key reason for relapse is that small amounts of leukemic cells, in numbers well below the sensitivity of routine morphology, have survived treatment and remain in the bone marrow: measurable residual disease (MRD). Presence of MRD after initial treatment is one of the strongest predictors of relapse, and sensitive MRD analysis is cru-cial for identifying patients at risk of relapse. MRD is routinely assessed at treatment of acute leukemia, using various methods depending on the specific leukemia subtype. How-ever, none of the current techniques is fully optimal; many require further evaluation, and for some patients, no applicable method is available. This thesis aims to improve MRD diagnostics in acute leukemia by evaluating existing methods and developing novel tech-niques, with the goal of contributing to improved clinical management and, ultimately, a better prognosis for patients with acute leukemia. The main method used for MRD analysis in this thesis was deep sequencing, a highly sensitive next generation sequencing-based technique that quantifies mutations present in leukemic cells at diagnosis. Other molecular methods, including reverse transcription quantitative polymerase chain reaction (RT-qPCR), were also applied, as well as flow cytometry for comparison and complementary MRD assessment. The material consisted of blood and bone marrow samples analyzed at diagnosis and during and after treatment, from adult and pediatric patients with acute mye-loid leukemia (AML), and from pediatric patients with precursor B-cell acute lympho-blastic leukemia (pre-B ALL). Paper I shows that RT-qPCR of ETV6::RUNX1 fusion transcript can be used as an alternative and valuable complementary MRD method to flow cytometry in children with pre-B ALL. Paper II shows that there is a strong correlation between RNA- and DNA-based methods for MRD analysis of NPM1 mutations in adults with AML, and that DNA-based methods can complement, or replace, RT-qPCR. Paper III shows that deep sequencing of mutated NPM1 during and after treatment predicts relapse and poorer survival in adult AML. Paper IV shows that deep sequencing of FLT3-ITD is a highly sensitive method for MRD detection in pediatric AML, enabling monitoring of treatment response and early relapse detection. In conclusion, this thesis contributes new knowledge about MRD analysis in acute leukemia, highlighting the clinical value of deep sequencing. The implementation of these findings in clinical practice could support more precise risk stratification, guide treatment decisions, and may contribute to improved prognosis for patients with acute leukemia.
On COVID-19-Associated Central Nervous System Complications
(2025-04-29) Kanberg, Nelly
The acute and long-term neurological impacts of COVID-19 have raised important concerns regarding the involvement of the central nervous system (CNS) in SARS-CoV-2 infection. This thesis examines the biochemical and neurochemical effects of COVID-19 on the CNS, focusing on acute injury and persistent manifestations. The primary aims were to assess markers of CNS injury in plasma and cerebrospinal fluid (CSF), investigate neuroinflammation and immune activation, and explore the associations between acute biomarker profiles and persistent neurological sequelae. Four studies employing cross-sectional, case series, and longitudinal design were conducted to evaluate CNS involvement across varying COVID-19 severities. Biomarker analyses focused on neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and CSF markers of immune activation. Our findings demonstrated elevated plasma NfL and GFAP levels during the acute phase in moderate to severe COVID-19, indicating astrocytic activation followed by neuronal injury. Longitudinal follow-up showed normalization of biomarker levels within six months, even in individuals reporting symptoms such as fatigue and cognitive impairment. CSF analyses in patients with post-COVID-19 condition (PCC) revealed no evidence of viral presence or sustained CNS inflammation. This thesis highlights the transient neurochemical impact of COVID-19, characterized by astrocytic and neuronal injury during the acute phase. The normalization of biomarkers despite PCC symptoms suggests that these sequelae may result from initial inflammatory or hypoxic insults rather than ongoing CNS damage. These findings underscore the complexity of neuro-COVID, emphasizing the role of systemic and neuroimmune pathways over direct viral effects in both acute and chronic CNS complications.
On viral kinetics and immune responses following SARS-CoV-2 infection and vaccination
(2025-04-09) Marklund, Emelie
This thesis consists of two parts, exploring different aspects of Coronavirus Disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The first part examines viral kinetics and antibody responses in individuals with severe and mild COVID-19. The second part assesses different components of the immune response following mild SARS-CoV-2 infections and mRNA COVID-19 vaccinations. In Paper I, we examined the duration of viral RNA shedding with serial PCR tests from the upper respiratory tracts and investigated the association of viral load and disease severity in patients with mild (n=39) and severe (n=15) COVID-19. We observed no significant differences in median viral load between groups on day 7 and day 14, and the duration of viral shedding was similar between groups. Paper II assessed time to seroconversion, and levels of SARS-CoV-2 IgG against Nucleocapsid and Spike proteins in patients with mild (n=32) and severe (n=17) COVID-19. We found a significantly shorter time until seroconversion and significantly higher IgG levels in the severe group during early follow-up (< 35 days). In Paper III, we investigated the seroprevalence among a group of healthcare workers (HCWs) in Sweden during the first ten months of the pandemic. We found an increasing and significantly higher seroprevalence in the HCWs compared to matched blood donors. Moreover, we assessed the performance of different IgG assays targeting different antigens and analyzed CD4+ T cell reactivity in both infected and uninfected HCWs. In Paper IV, we profiled immune responses, including IgG and T cell cytokine (IFN-γ, IL-2, and TNF-α) responses, following up to four COVID-19 mRNA vaccine doses in HCWs (n=108). We found significantly higher IgG levels in previously infected than in uninfected HCWs up until the third vaccine dose, while differences in cytokine responses were less affected by previous infections or breakthrough infections. Together, the results provide insights into viral kinetics, antibody responses and T-cell responses following SARS-CoV-2 infections and mRNA vaccinations, contributing to the understanding of immunity in different phases of the COVID-19 pandemic.
Inflammation-related protein biomarkers in ischemic stroke
(2025-04-02) Angerfors, Annelie
Ischemic stroke is a leading cause of death and disability worldwide. Inflammation plays a role in both ischemic stroke pathophysiology and response to cerebral injury. However, the mechanisms remain inadequately understood and few inflammation-related proteins have been analyzed in clinical cohorts. This thesis aims to investigate a broad range of inflammation-related proteins in ischemic stroke and its subtypes, their associations with functional outcome and vascular recurrence, and their potential causal relationships with functional outcome. The studies rely on two hospital-based cohorts: the Sahlgrenska Academy Study on Ischemic Stroke (SAHLSIS) and its continuation (SAHLSIS2). Plasma levels of inflammation-related proteins were measured by a proximity extension assay. Paper I studied 65 plasma proteins in ischemic stroke patients compared to controls in a longitudinal study. It identified over 30 proteins that were elevated in cases across all timepoints, even in the long-term, which could reflect underlying pathophysiological processes. Subtype-specific patterns were also observed for some proteins. Paper II investigated the relationships between acute-phase levels of the 65 proteins and functional outcome 3 months post-stroke. It revealed 20 proteins that were associated with outcome, several with experimental evidence indicating a role in injury or recovery after stroke. Paper III identified S100A12 as a novel biomarker for vascular recurrence, showing that elevated levels increased the risk. Paper IV employed a two-sample Mendelian randomization design to investigate potential causal relationships between the 20 proteins identified in Paper II and functional outcome. This study provided evidence for causal associations between genetically determined levels of four proteins, including S100A12, and functional outcome. In conclusion, this thesis demonstrates that inflammation-related plasma proteins have diverse patterns after ischemic stroke and identifies several potential biomarkers for prediction of functional outcome and vascular recurrence. These results advance our understanding of the role of inflammation in ischemic stroke and may guide the development of novel therapeutic strategies for improved stroke care.
Memory B cell dynamics after respiratory viral infections
(2025-02-27) Axelsson, Hannes
Viral respiratory infections continue to be a global health burden with high mor- bidity and mortality. With two viral pandemics with only 10 years apart, by Influenza A virus, 2009, and SARS-CoV2 emerging 2019, and rapidly evolving viral pathogens, the next pandemic is not a question of if, but of when. As B cells provide critical protection against viral infections, this thesis aimed at stud- ying how B cells respond to viral respiratory infections and how they develop over time. Through a combination of flow cytometry and single cell sequencing and analysis of secreted and expressed antibodies, we created a detailed map of B cell development. We also studied whether secondary Streptococcus pneu- moniae infection influence the primary viral responses. In paper I, we found that memory B cells in the lung can originate from other lymphoid tissue and that high affinity B cells may generate both memory B cells and Plasma cells. In paper II, we discovered how a secondary infection disrupts the accumulation of memory B cells in the lungs and how that leads to a detri- mental outcome upon re-challenge. In paper III, we found a strong Plasmablast response after SARS-CoV2 infection that, based on their antigen specific and rapid onset points to differentiated memory B cell origin from endemic corona- viruses. This was further corroborated in paper IV, where, through clonal anal- ysis we could show rapid onset of highly mutated plasmablasts. Furthermore, we could show continued maturation of the B cell response over time. Taken together, this thesis contributes to better understanding of B cell development, and factors that influence B cell responses.
Weaving the Threads of Memory: How Pre-existing Immunity Shapes B Cell Responses to Influenza A Virus
(2025-02-05) Reusch, Laura
Influenza A virus (IAV) poses a persistent global health threat due to its ability to evolve rapidly, requiring annual updates to seasonal vaccines. Despite significant advancements, a universal vaccine capable of providing long-term and broad protection has not been developed yet. It was investigated here, how pre-existing immune components—antibodies (Abs), memory B cells (MBCs), and CD4 T cells—shape B cell responses to drifted IAV haemagglutinin (HA). We also explored innovative immunogen design strategies, targeting a conserved epitope to overcome immunodominance (ID) and enhance vaccine efficacy. While pre-existing CD4 T cells accelerated Ab and GC responses, pre-existing Abs were shown to mask epitopes and exhibit feedback mechanisms, thereby reshaping ID patterns of B cell responses. The valency of antigens used for vaccination influenced the extent of Ab-mediated modulation, with multivalent antigens showing greater effects compared to monovalent counterparts. Whereas MBC rapidly differentiated into antibody-secreting cells (ASC) rather than re-entering secondary germinal center reactions (GCs), this localized Ab secretion contributed to secondary responses rather than the presence of serum-Abs. Finally, MBC and naïve B cells were regulated differently after vaccination with a multivalent particle. To address challenges posed by antigenic variability and to induce broad immunity against IAV, a computationally designed HA stem mimetic was developed. This immunogen selectively engaged MBCs of IAV-experienced individuals and induced cross-reactive Ab responses against both group 1 and group 2 IAV strains after vaccination in mice. By combining insights into ID, the effect of pre-existing immunity on secondary B cell responses and rational antigen design, this work highlights key mechanisms driving protective and broad B cell responses to IAV, thereby providing valuable insights to inform the development of a universal IAV vaccine. These findings also offer broader implications for combating other highly variable pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human immunodeficiency virus (HIV).
Ultrasensitive nucleic acids analysis using digital sequencing
(2025-01-24) Luna Santamaria, Manuel
Liquid biopsies offer a minimally invasive and affordable procedure to obtain valuable clinical insights through biomarker analysis that can aid to manage the care of different diseases and health conditions. Analysis of cell-free DNA that is shedded into blood plasma from different tissues and cell types is of particular interest, providing molecular information that can be used in clinical decisions. In the case of cancer, cell-free DNA includes a fraction of DNA liberated by tumoral cells, containing tumor-specific mutations. Analysis of circulating tumor DNA has applications like monitoring disease progression and determining therapy effectiveness to avoid under- or over treatment. However, in most clinical applications the levels of circulating tumor DNA are very low, hence it cannot be assessed with traditional analytical methods. In this thesis, we aimed to develop and optimize the SiMSen-Seq workflow, which is a digital sequencing method that enable ultrasensitive detection of DNA variants using molecular barcodes, also enabling variants detection in RNA. First, we studied the effects of preanalytical variables, including the type of blood collection tube, time between sampling and plasma isolation using blood collected from healthy individuals. Our results indicate that most preanalytical parameters are essential to consider in order to obtain high cell-free DNA yield and avoid cellular contamination. Then, we designed and tested the effects of structured elements in the molecular barcode that is used bioinformatically to correct errors in the sequencing data. Our results proved that assays using structured molecular barcodes outperformed those using standard non-structured barcode sequences, resulting in improved sensitivity and reduced amounts of off-target reads. Finally, we adapted the SiMSen-Seq workflow to include a reverse transcription step that enables the detection of RNA target sequences. Our data demonstrate the importance of using a reverse transcriptase and reaction condition that generate high yield of complementary DNA and produce few enzymatic sequence errors. This approach can detect RNA variants, including modifications, in clinical samples with the same sensitivity as for DNA. In conclusion, our protocols and data contribute to the field of liquid biopsy analysis and the ability to detect DNA and RNA molecules as biomarkers. We expect that biomarkers analysis in blood will be increasingly important in clinical decision and disease management with the goal of improving treatment outcome and quality of life.
Exploring the source of human cardiac regeneration
(2025-01-21) Sjölin, Linnéa
The human heart is known to have limited regenerative capacity, and the source of new cardiomyocytes remains enigmatic. The existence of stem cells in the adult human heart has been a topic of debate. Recently, a potential stem cell niche in the atrioventricular junction (AVj) of rats was identified. This region was further explored in human hearts, showing expression of cardiac stem cell related markers in the left AVj at the base of the mitral valve. In study I, the right AVj was investigated with immunohistochemistry (IHC) and RNA sequencing. Stem cell markers were expressed in the fibrous region and at the edge of the myocardium, decreasing with distance from the AVj. In study II, an alternative regenerative mechanism, cardiomyocyte dedifferentiation, was investigated through IHC on cardiac tissue from organ donors who have had cardiac arrest. A subpopulation of cardiomyocytes in the left ventricle (LV) reduced expression of cardiac troponin T, and upregulated stem cell markers, suggesting dedifferentiation. Study III utilized proteomics, supported with RNA sequencing and IHC, to explore the left AVj. Proteins involved with cardiac development and stem cell niches were expressed. In conclusion, the results of these studies suggest the AVj as a potential niche and source of cardiac progenitor cells. Additionally, dedifferentiation of cardiomyocytes might play a role in cardiac regeneration following injury, such as cardiac arrest.
Biomarker profiling in sepsis diagnostics
(2024-11-28) Irani Shemirani, Mahnaz
Effective and timely antibiotic therapy for sepsis requires a thorough understanding of the types and molecular characteristics of bacterial strains. Therefore, we investigated diagnostic strategies to facilitate faster classification of bacteria and identification of their molecular features. We benchmarked the 1928 Diagnostic platform (1928 Diagnostics, Gothenburg, Sweden) for characterizing Staphylococcus aureus (S. aureus) strains against an in-house bioinformatics (INH) pipeline and reference clinical laboratory methods, including MALDI-TOF MS and phenotypic antibiotic susceptibility testing. We observed a high agreement between the 1928 platform and the INH pipeline in predicting laboratory results. Notably, the 1928 platform exhibited a lower rate of false negative while showing slightly higher rates of false positive (Paper I). Additionally, our findings revealed that clindamycin, erythromycin, and fusidic acid exhibited efficacy against all methicillin resistance S. aureus strains, and vancomycin demonstrated susceptibility in all tested strains (Paper II). The challenge remains in predicting the bacterial type. Several studies highlighted the differences between blood markers of gram-positive and gram-negative bacterial sepsis. Using machine learning algorithms and Proximity Extension Assay (PEA), we discovered a set of informative proteins comprising 55 proteins, including 5 potential biomarkers, which distinguish patients with gram-positive or gram-negative bacteria from other cases, achieving AUCs of 0.66 and 0.69, respectively (Paper III). However, while the analysis of 55 proteins offered insights into classifying bacterial types, our method did not distinguish between specific bacterial strains. Employing a more comprehended approach utilizing whole blood microarray technology on septic patients infected with either S. aureus or Escherichia coli revealed 25 genes with high AUC values (0.98 and 0.96, respectively) that effectively distinguished these infections from other cases. These findings were consistent across two separate independent datasets, with AUC values ranging from 0.72 to 0.87 (Paper IV). In conclusion, efforts to improve diagnostic strategies and understand bacterial characteristics in sepsis continue. Platforms like 1928 Diagnostics and technologies such as the PEA show promise, with machine learning offering opportunities to tackle bacterial typing challenges. These advancements are crucial for evolving clinical practices in sepsis diagnosis and management.

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