Browsing by master's degree program "Magisterprogrammet i translationell medicin"

The APOE4 gene variant of apolipoprotein E (apoE) has been identified as a predisposing factor for late-onset Alzheimer’s disease (AD). ApoE is known to interact with one of the classically characterized hallmarks of AD, the amyloid-beta (Aβ) protein. Aβ activates the classical complement pathway by binding to C1q that may sustain inflammation, hamper Aβ clearance and therefore promote accumulation of Aβ deposits in the brain and affect the integrity of the blood-brain barrier. Depending on context, binding of apoE to C1q can either activate or inhibit complement activation. ApoE can also interact with the complement regulator factor H (FH) which colocalizes with C1q on Aβ plaques. The interactions between these proteins and their role in the AD pathogenesis has not been fully explored. This thesis aimed at unraveling the complex formation between Aβ1-42, apoE, FH, and C1q, and their role in complement activation to provide novel insights on the mechanisms contributing to AD pathophysiology. By conducting enzyme-linked immunosorbent assays and Western Blotting of native PAGE, I was able to show that FH bound to apoE and forms a complex with Aβ1-42/apoE in an isoform- specific manner (apoE2 > apoE3 > apoE4) whereas C1q bound all Aβ1-42/apoE complexes with same affinity. FH did not bind Aβ1-42 alone, thus the results indicated that binding of FH in Aβ1-42/apoE complex took place via apoE2 and apoE3. In the absence of FH and C1q, immobilized Aβ1-42/apoE4 complexes led to higher serum complement activation levels when compared to Aβ1-42/apoE2 and Aβ1-42/apoE3 complexes or Aβ1-42 alone. C1q activated serum complement especially in the presence of Aβ1-42/apoE3 and Aβ1-42/apoE4. Further functional analysis with flow cytometry revealed that in fluid phase the presence of FH could restrict Aβ1-42-induced complement activation, the subsequent formation of C5a and activation of C5a receptor (C5aR) expressed on Human embryonic kidney (HEK) 293 cells. The presence of all apoE isoforms on Aβ1-42/C1q complexes led to reduced C5aR activation. Surprisingly, however, the reduction was the largest with apoE4, and the presence of C1q on Aβ1-42 alone could also decrease C5aR activation. The results of this thesis elucidated the effects of Aβ1-42, apoE, C1q and FH in complement activation and proposed a mechanism by which FH regulates complement activation and cell-mediated inflammation through isoform-specific binding to apoE associated with Aβ1-42. Further work is required to analyze the functional effects of these complexes, whether Aβ1-42, apoE, FH, and C1q can all bind simultaneously to form a large complex and whether this complex affects C5aR activation. This study provides important insights on how APOE4 may predispose to the neurotoxicity and neuroinflammatory changes in AD pathophysiology early in the disease process. Yet, the findings are only preliminary and therefore further studies are required to validate these results and to demonstrate whether promoting FH binding to apoE/Aβ complex could provide a novel target for AD therapy.

IER3IP1 is a protein located in the endoplasmic reticulum (ER) transmembrane, and it is highly expressed in pancreatic beta cells and developing brain cortex. The loss-of-function mutations in IER3IP1 cause monogenic neonatal diabetes together with brain linked diseases such as epilepsy and microcephaly. The aim of this thesis is to study the role of IER3IP1 in the development and function of human beta cells using hESC-derived pancreatic islets. Using CRISPR/Cas9, IER3IP1 knockout (KO) and IER3IP1 loss-of-function mutation knock-in (KI) hESC clones were generated. For KO, the first exon of IER3IP1 was deleted whereas for KI, the 21. valine of IER3IP1 was changed to glycine. The clones together with their unedited controls (H1), were differentiated into pancreatic stem cell (SC)-islets following the optimized 7-stage differentiation protocol. The differentiation was followed during the protocol and the SC-islets were tested at the end of the protocol. In vitro, IER3IP1 KO-islets contained less beta cells and more alpha cells when compared to the H1-islets, as shown by immunostainings for insulin and glucagon. The beta cells of KO-islet accumulated more proinsulin compared to H1-islets and had significantly higher level of ER-stress shown by elevated ER-stress marker BiP. Moreover, the KO-islets showed drastically lower amount of insulin secretion and diminished insulin content. The IER3IP1 KI-islets did not significantly differ from H1-islets. Thus, this master’s thesis shows that IER3IP1 is essential for maintaining normal ER homeostasis and beta cell function in vitro. In future, these results should be confirmed using in vivo model.

Ovarian cancer remains a significant health challenge worldwide, with high-grade serous carcinoma (HGSC) representing its most aggressive subtype. Despite advancements in treatment, disease recurrence remains high, with most patients relapsing within a few years, necessitating the investigation of novel and combination therapies. This study presents a robust framework for evaluating the efficacy of immuno-oncology agents in HGSC through the establishment of a high-throughput patient-derived immunocompetent culture (iPDC) drug screening platform. Using an in-house analysis pipeline to harmonize the complex data outputted by our high-throughput model, we found significant reductions in live tumor cell abundance in response to ataxia telangiectasia and Rad3-related inhibitor (ATRi) in targeting tumor cells irrespective of homologous recombination (HR) status. However, certain samples exhibit immune cell toxicity, emphasizing the importance of assessing immune cell-specific responses for comprehensive evaluation of therapy outcomes. The tissue-based investigation further validates these findings, revealing elevated DNA damage and replication stress biomarker, pRPA32-RPA2(Ser8), expression in responders to ATRi combinations, corroborating previous research and suggesting promising therapeutic targets. Moreover, analysis of immune cell abundance and functional states underscores the predominance of myeloid cells in the tumor microenvironment (TME), with limited variation observed in T-cell activation between responders and non-responders to ATRi. Spatial analysis reveals distinct intertumoral immune infiltration patterns, potentially influencing treatment responses. Overall, these results shed light on the intricate interplay between tumor biology and therapeutic response in HGSC, offering insights into tailored treatment strategies and emphasizing the need for personalized approaches in oncology.

Every childbirth is a unique experience for a mother and the whole family. While there is growing evidence that childbirth has long-term implications for a mother’s life, and that a personal childbirth experience is in a major role in determining those implications, personal birth experiences have not yet come to the centre of labour care in Finland. In this qualitative study I investigated the constitutive elements of personal childbirth experiences. The material consisted of 29 birth stories written by 20 mothers, collected as a part of a larger research project Battles over Birth – Finnish Birth Culture in Transition (2020-2023), funded by the Kone Foundation. I divided the stories into positive and negative experiences based on how mothers described the events and their implications. I analysed the role of pain management in all experiences, after which other elements were identified from each story and compared within category and finally between categories. In addition, I analysed the long-term implications of positive and negative experiences for mothers. Systematic content analysis suggested three elements as crucial in determining whether the experience was valued as positive or negative, and these were: 1) pain and pain management, 2) interaction with professionals, and 3) sense of control and self-determination. The birth experience had implications on the mother-infant bond, mental health, trust toward health care professionals, number of subsequent children, general well-being, and confidence in motherhood. The birth experience thus constitutes of several aspects and can have significant long-term implications. By considering mothers’ wishes and treating them respectfully, not only is their constitutional right for self-determination respected, but a safe and comfortable environment can be created for labour. The findings of this study can contribute to developing maternity and childbirth care toward patient-centred care, where personal birth experiences have more value and positive birth experiences can be ensured.

Lipid droplets (LDs) are ubiquitous intracellular storage organelles, consisting of a core of energy rich neutral lipids surrounded by a phospholipid monolayer. Research in the past decade has expanded the view on LDs from simple, passive cytosolic inclusions to dynamic organelles which play an important role in many cellular processes. Furthermore, there is mounting evidence for links between LD biology and human pathologies, such as metabolic disorders, non-alcoholic fatty liver disease and cardiovascular diseases. Thus, understanding the basic biology of LD formation is crucial. LD biogenesis is thought to occur in the microdomains of endoplasmic reticulum (ER), due to the accumulation of neutral lipids between the two leaflets of the ER bilayer before budding into the cytosol. Many proteins are involved in this early formation, but no single indispensable protein has been discovered. After assembly, these early LDs grow through lipid deposition from the ER, and with lipid synthesis on the droplet monolayer. During LD growth, LDs are thought to retain connection to the ER. A protein important for LD biogenesis is seipin. This oligomeric ER protein has been found to localize at contact sites between the ER and LDs. Mutations in seipin give rise to three distinct diseases in humans; BSCL2, seipinopathy and Celia’s encephalopathy. The role of seipin in the formation of LDs and the pathogenesis of these diseases is still unknown. Work from numerous model systems has shown seipin to be important for LD biogenesis and adipocyte differentiation. LD formation is a complex process which is still poorly understood, and seipin likely collaborates with other proteins during LD assembly. In this thesis, APEX2-mediated proteome mapping combined with LC-MS/MS, is set up to identify proteins involved in LD biogenesis. In this technology, an engineered ascorbate peroxidase, APEX2, is genetically inserted to the intracellular region of interest where it rapidly biotinylates nearby endogenous proteins upon exposure to biotin-phenol and hydrogen peroxide. Biotinylated proteins can then be enriched by using streptavidin beads and identified with a mass spectrometry. The aim using this technology is to unravel new interaction partners of seipin and proteins important for LD formation, which is a crucial step for understanding LD formation and diseases related to it.

Antibiotic-resistant bacteria present a severe threat to global health. The future treatment of common bacterial infections relies on the identification of novel antibiotics and targets in the present. One area of antimicrobial research is the study of bacteriophage (Petrovic Fabijan et al.) mechanisms and the identification of phage-derived antimicrobials. Sequenced phage genomes are largely (>70%) annotated as “hypothetical proteins of unknown function” (HPUFs) and investigation into HPUFs with a toxic effect on host bacteria (toxHPUFs) aims to reveal new antibacterial targets and antimicrobials. Next-generation sequencing and plating-based toxicity screening of Staphylococcus phage Stab21 HPUFs identified nine HPUFs that incurred toxicity to Escherichia coli. In this study, the tightly controlled tetracycline-inducible plasmid pRAB11N was used as a shuttle vector and verified the toxicity of five out of nine HPUFs to E. coli and revealed that no HPUFs caused toxicity to the Stab21 natural target and clinically relevant Staphylococcus aureus. These results suggest that screening for toxHPUFs should be carried out in closely related bacterial species or the phages’ natural host. The five toxHPUFs of E. coli were further characterised by protein function and structural predictions. Only one toxHPUF, g024, returned a reliable model with homology to Bacillus phage SPO1 homing endonuclease I-HmuI, yet the role of this DNase in bacterial host toxicity is still unknown. To determine the bacterial targets of the toxHPUFs, spontaneous toxin-insensitive mutants of the five toxHPUFs were investigated. For three toxHPUFs, the toxin insensitivity was ascribed to the elimination of the toxin-encoding gene. However, toxin-insensitive g172 and g187 sequences revealed mutations in the tetR gene of pRAB11N that led to the inability of tetracycline binding and thus no induction of gene expression and did not aid in identifying the bacterial targets of these toxHPUFs. This study highlights the experimental complexities of phage-derived antimicrobial research. It also maintains the value of this research strategy, with the verification of HPUFs with a toxic effect on E. coli and accompanied future studies of bacterial target determination having the potential to uncover novel antimicrobial mechanisms that can be exploited for therapeutic application.