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Browsing by master's degree program "Translationaalisen lääketieteen maisteriohjelma (Translational Medicine)"

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  • Adhikari, Sadiksha (2020)
    Structural variants comprise a large number of variations occurring in the human genome and are detected in many diseases including cancers. To a limited extent, whole exome sequencing (WES) is capable of detecting structural variations (SVs) using algorithms and tools utilizing local assembly, split-reads, discordant read-pairs and read depth methods. However, due to the significantly large size of SVs compared to the reads produced and the presence of repetitive regions in the genome, identification of SVs presents a major challenge. 10X Genomics has developed a technology that requires very low amounts of DNA and uses a linked-reads approach to produce long reads. Recently, linked-read technology has shown promising results in resolving complex SVs. In this thesis, we aimed to assess whether linked-read exome sequencing is able to infer more comprehensive information in SVs compared to WES in multiple myeloma (MM). The disease model was chosen based on the presence of high numbers of SVs in MM patient tumor cells. Here, we report that linked-read sequencing has led to the identification of a potential novel translocation t(1; 14) that significantly impacts the change in expression of genes and could potentially have impact on the prognosis and treatment of multiple myeloma patients. By Long Ranger analysis we detected t(1;14) in six out of eight samples. Further, to study whether the translocation differentially affects the expression levels of any genes, differential gene expression was performed between t(1;14) positive versus t(1;14) wild type samples. The analysis resulted in 107 differentially expressed genes where 4 upregulated and 103 downregulated genes were found in the translocation positive samples. Among the downregulated genes, we found S100A8 and S100A9 genes which are previously shown to be associated with chemoresistance to PAD (bortezomib, doxorubicin and dexamethasone) therapy. The related breakpoints of the event were identified by Manta tool (SV caller) using both linked-read and WES. Therefore, linked-read information does not appear necessary to detect this event. In this study, we found that linked-read sequencing has certain advantages over WES such as low input DNA, increased number and quality of calls and breakpoint information. However, linked-read sequencing technique is limited to the detection of certain SV types in addition to increased cost of sequencing. These two factors must be considered before choosing linked-read sequencing over WES. Somatic mutations and clinically relevant SV were detected equally efficiently by both techniques.
  • Wartiovaara, Linnea (2023)
    Renewal of the intestinal epithelium is driven by the actively dividing and strictly regulated Lgr5-expressing intestinal stem cells (ISCs). As uncontrolled proliferation may lead to colorectal cancer (CRC), ISCs and the regulatory circuit around them could elucidate new targets for colorectal cancer therapy. The regulatory crosstalk between the stem cells and surrounding stroma is under intensive investigation, but little is known about the neuronal control of the intestinal epithelium. Adrenergic signals from sympathetic nervous system regulate hematopoietic and melanocyte stem cells and promote tumorigenesis in e.g., pancreatic, and prostate cancer. Adra2a, one of the nine different adrenergic receptors, is expressed in the ISCs, and adrenergic neurons project neurites to the stem cell niche suggesting a paracrine signaling role. However, whether the adrenergic signaling plays a role in ISC regulation and/or in colorectal cancer remains unknown. The aim of this study was to develop tools to investigate the role of adrenergic signaling in ISC regulation. First, I set up a protocol to delete Adra2a and other genes in intestinal organoids recapitulating the stem cell hierarchy of the intestinal epithelium in vitro, using CRISPR-Cas9 technology. This led to successful deletion of the Ret proto-oncogene while an Adra2a-deficient organoid pool could not be established with these efforts. I differentiated neuroblastoma cells to a catecholaminergic phenotype and cultured them together with intestinal organoids to address the effect of catecholaminergic signaling on the intestinal epithelium in organoid cocultures. The coculture with catecholaminergic cells induced upregulation of the regeneration markers Ly6a and Clu in intestinal organoids, while Reg3b as well as the stem cell markers Lgr5, Olfm4, and Adra2a and the Paneth cell marker Lyz1 were reduced. Third, I assessed the direct effect of a 3-hour norepinephrine (NE) pulse on wild-type organoids with 3’RNA sequencing, however, this did not induce significant changes in the expression levels of the respective regeneration marker genes. Altogether, my work established a CRISPR-Cas9-based method to delete genes of interest in primary intestinal organoids. Further investigation is needed to verify if NE contributes to the regulation of intestinal regeneration and stem cell maintenance.
  • Grönman, Jasmin (2023)
    High-grade serous ovarian cancer (HGSOC) is the most common histological type of epithelial ovarian cancer with the five-year survival rate of approximately only 40%. Platinum-based chemotherapy together with surgical cytoreduction is the standard of care in HGSOC. Platinum compounds bind to DNA causing intra- and interstrand cross-links ultimately leading to severe DNA damage in the form of DNA double-strand breaks. Functional DNA repair mechanisms, most importantly homologous recombination (HR), are crucial in repairing these double-strand breaks and maintaining genomic integrity. HR deficiency is found in approximately 50% of HGSOC tumours making these tumours sensitive to platinum-based chemotherapy and poly(ADP-ribose) polymerase (PARP) inhibitors. The androgen receptor (AR) has been reported to be expressed widely in the normal ovarian surface epithelium as well as in malignant ovarian cancer cells. With the current evidence pointing towards the involvement of AR signalling in the maintenance of HR as well as driving radiation resistance, combined or sequential use of anti-androgen therapies together with platinum-based compounds might function as a novel therapeutic approach and lead to the accumulation of DNA damage within HGSOC tumours. Blocking AR signalling in prostate cancer cells receiving androgen deprivation therapy has already been shown to functionally impair HR and result in the upregulation of PARP mediated pathways. To investigate the expression levels of AR within the human primary HGSOC tumour specimens, immunofluorescent immunohistochemistry together with reverse transcriptase polymerase chain reaction were performed. The androgen receptor was found to be expressed in approximately 75% of the patient samples with ≥10% nuclear AR staining. Intriguingly, the AR expression did not differ between primary and metastatic tumours or between chemo-naïve and chemotherapy treated samples. Moreover, the effects of AR inhibition and stimulation on the functional HR capacity of HGSOC cells was examined to elucidate the role of AR signalling in the homologous recombination DNA repair pathway. Receptor manipulation with an antagonist enzalutamide prior to gamma radiation led to a significant decrease in the HR capacity of HR- proficient and AR positive commercial HGSOC cell line OAW28. Furthermore, agonist treatment with dihydrotestosterone increased the HR capacity within these cells. A deeper understanding on the interactions between HR and AR signalling can be utilized in the development of future therapeutic strategies against platinum resistant HGSOC. Pharmaceutically induced HR deficiency has the potential to provide opportunities for a wide range of combination treatment strategies using standard of care chemotherapy in conjunction with molecularly targeted agents towards DNA damage response proteins as well as widen the therapeutic possibilities for the use of PARP inhibitors.
  • Valkonen, Konsta Valentin (2021)
    Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motoneuron disease. ALS is characterized by a progressive loss of upper and lower motoneurons, resulting in muscle atrophy, paralysis and ultimately in death. Approximately 30,000 people die of ALS annually. There is no cure for ALS, and only two drugs - riluzole and edavarone - have been approved for the treatment of the disease. The complex pathology of ALS contributes to the lack of effective treatments. Several cellular pathologies have been suggested to contribute to the pathogenesis, including ER stress, disruption of calcium homeostasis, oxidative stress and excitotoxicity. Here we describe the cytoprotective effects of C-terminal fragments of the novel proteins with neurotrophic factor properties MANF (mesencephalic astrocyte-derived neurotrophic factor) and CDNF (cerebral dopamine neurotrophic factor) on a toxin model of ALS in vitro. Unlike the classical neurotrophic factors, MANF and CDNF are predominantly localized to the endoplasmic reticulum (ER) and have been shown to alleviate ER stress by keeping the unfolded protein response (UPR) transducers inactive. ER stress is a major component in many neurodegenerative diseases, including ALS, and is a promising therapeutic target for MANF and CDNF. However, the potential of these proteins in ALS treatment remains to be insufficiently described. We used differentiated motoneuron-like NSC-34 cells treated with a range of toxins, modelling different cellular pathologies linked to ALS. After the toxin addition, we treated the cells with MANF and CDNF variants and riluzole and measured the cell viability. The toxin panel consists of tunicamycin, ionomycin and staurosporine. Tunicamycin causes cell death by activating proapoptotic branches of the UPR. Ionomycin is an ionophore and depletes the ER of calcium, thus inducing both UPR-dependent and UPR-independent apoptosis. Less is known about the mechanisms of staurosporine, but it has been shown to induce caspase-3-dependent apoptosis, increase intracellular calcium levels and cause oxidative stress. We hypothesized that both MANF and CDNF variants protect the cells against UPR-dependent apoptosis but not against UPR-independent cell death. We show that MANF and CDNF variants protect the cells against apoptosis induced by tunicamycin, ionomycin and staurosporine. Interestingly, the protein variants mediated the highest protection against ionomycin-induced stress, and they exhibited mild protective effects against staurosporine as well. These findings suggest that MANF and CDNF variants might have a role in maintaining intracellular calcium homeostasis. However, it is possible that staurosporine induces ER stress as well, which would explain the protection conferred by the protein variant. We report that the CDNF variant mediates higher protection at lower concentrations compared to the MANF variant in every toxin assay, whereas the MANF variant mediates higher protection at the highest tested concentration compared to the CDNF variant. We also show that the CDNF variant-mediated protection against staurosporine-induced stress peaked at lower concentrations, and the highest concentration provided distinctively lower, yet significant effect. These data lead us to hypothesize that the protein variants may have a slightly different mode of action, and that they might provide an additive effect when administered simultaneously. We tested a combination of MANF and CDNF variants in cells treated with tunicamycin, ionomycin and staurosporine. However, the combination treatment did not increase the viability more than MANF and CDNF variants independently did. The results answered our questions as well as raised new ones. In the future, the putative calcium-regulating effects of the protein variants should be investigated. The UPR-modifying effects of the drug candidates and toxins need to be assessed by quantifying changes in the UPR marker mRNA and protein expression levels. If it is revealed that the variants have a different mode of action, the possible additive protective effects must be assessed. Finally, a wider toxin panel is needed to fully explore the potential of MANF and CDNF variants in ALS treatment. This study demonstrates the potential of MANF and CDNF variants in protecting motoneurons against several pathological pathways contributing to ALS pathology. However, the mechanisms of action of the variants need further investigation to fully understood their therapeutic potential.
  • Nguyen, Ngoc Anh (2019)
    Immunophenotyping by flow cytometry (FC) is an established practice to identify immune cells and their cellular changes at the single-cell level. Since preserving the structural integrity of cellular epitopes is vital for immunophenotyping, samples should be processed shortly after being collected. However, the requirements of complex facilities and trained personnel for flow cytometry make it challenging to handle samples immediately. Fixation and cryopreservation extend sample shelf life and allow analysing longitudinal samples simultaneously while minimizing technical variation. Nevertheless, usage of whole blood cryopreservation in flow cytometry is limited due to challenges in preserving epitope structures during fixation and detecting dim antigens. This thesis investigates the performances of four commercial whole blood cryopreserving kits; 1) Cytodelics, 2) Stable-Lyse V2 and Stable-Store V2 (SLSS-V2), 3) Proteomic stabiliser (PROT-1), and 4) Transfix. Peripheral blood samples were processed with these stabilising buffers immediately after the collection and cryopreserved until further analysis by flow cytometry. Here, we measured the stability of major immune lineages, T cell subpopulations, and activated neutrophil profiles in samples treated with these commercial whole blood stabilisers. Our flow cytometry data showed that PROT-1, Transfix and Cytodelics maintained the distribution of major leukocyte subsets – granulocytes, T cells, natural killer cells and B cells, comparable to unpreserved samples despite the attenuation of fluorescence intensities. Moreover, these three stabilisers also preserved phenotypes of activated neutrophils upon stimulation with N-Formylmethionyl-leucyl-phenylalanine and Lipopolysaccharides. The upregulation of adhesion molecules (CD11b), Fc receptors (CD16) and granule proteins (CD66b) as well as the shedding of surface L-selectin (CD62L) on activated neutrophils was conserved most efficiently in PROT-1, followed by Cytodelics. On the other hand, none of the stabilisers provided a reliable detection of CCR7 for accurate quantification of T cell subpopulations. COVID-19 is caused by a highly transmissible and pathogenic coronavirus, so-called severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). To test the potential of whole blood cryopreservation kits for flow cytometry in COVID-19 research, we studied the detectability of major leukocyte lineages and granulocyte subsets in longitudinal patient samples processed with Cytodelics. High dimensional analysis with Uniform Manifold Approximation and Projection (UMAP) and Self-Organising Maps (FlowSOM) clustering revealed remarkable stability of CD3, CD15, and CD14 expression in samples stored with Cytodelics. It allowed the detection of lymphopenia and emergency granulopoiesis often found during the acute phase of severe SARS-COV-2 infection. Nonetheless, we could not determine signatures of granulocyte subsets, notably suppressive neutrophils, during the acute and convalescent phases of COVID-19. Variable detection of lowly expressed markers and diminished fluorescence intensities in Cytodelics - preserved samples might have hindered the analysis. In conclusion, this study demonstrates that PROT-1, Transfix, and Cytodelics enabled reliable detection of highly expressed leukocyte markers, whereas SLSS-V2 preservation resulted in the most inaccurate identification of studied markers. Notably, our results show that Cytodelics can be applied in COVID-19 studies to immunophenotype major immune lineages by flow cytometry. Nevertheless, more optimisation is needed for less abundant or fixation-sensitive epitopes to enhance the efficacy of whole blood cryopreservation for flow cytometry.
  • Khalife, Ghida (2024)
    Lung cancer is a significant global health concern, as it remains the leading cause of cancer-related deaths worldwide. While all Nordic countries have shown improvement in the 5-year survival rates for lung cancer patients during the last decade, Finland has lagged in terms of survival outcomes. Previous studies have explored the variations in lung cancer guidelines across Nordic countries. Still, there is limited research that addresses the differences in the countries’ practices that could affect the survival outcomes. This thesis aims to address this knowledge gap by conducting a comprehensive benchmarking comparison of the current care and process practices for lung cancer patients across all Nordic countries. This thesis employed a non-interventional, prospective observational study design with three distinct phases. First, an expert workshop involving lung cancer experts from Finland (N=6) was organized to formulate relevant questions for the structured survey. Second, the survey was constructed via REDCap and distributed to healthcare professionals in Nordic hospitals and primary care units. Lastly, after analyzing the survey results, a Nordic expert workshop (N=8) was conducted to validate the results and identify the most important factors influencing the outcomes. Four main areas of differences in care practices between the Nordic countries were identified: a) discrepancies in access and resources available in primary care units, b) absence or presence of fast-track and structured pathways, c) medication adoption based on reimbursement policies, and d) outcome tracking and monitoring. While the differences in specialized care were minimal, this study emphasizes the need to develop primary care in Finland, in terms of resourcing and processes, to improve lung cancer outcomes. In conclusion, all countries follow similar care guidelines. Thus, differences in early recognition and timely access to care for lung cancer patients may explain the differences in the 5-year survival rates.
  • Sylgren, Inka (2022)
    Patients over 65 years are more prone to face difficulties in their care due to not only complex and chronic multimorbid conditions, but also fragmentation of health care services. Current healthcare systems are designed for single-disease conditions that do not align with the care needs of the multimorbid elderly. Multimorbid long-term home care clients use a wide range of home care services but also other health and social services outside of home care, which can lead to fragmentation. The study aimed to map out services used by multimorbid home care clients, present disruptions related to their care, and suggest feasible and scalable solutions for the identified disruptions. Home care professionals (N=10) and clients (N=5) were interviewed focusing on the services and disruptions, and a focus group workshop was conducted for health and elderly care specialists (N=9) for creating the solutions. A total of 38 individual disruptions were discovered, of which 58% (22 cases) mentioned more than one interviewee. The results indicate that multimorbid home care clients faced the most care disruptions when care was prescribed outside of home care and caused fragmentation in the care coordination and sharing of patient information between multiple care providers and actors. Other disruptions were caused by a lack of co-creation of health, inconsistencies within home care protocols, and other factors outside of home care such as rapid workforce turnover. The disruptions discovered were mainly related to healthcare service networks rather than social-related care, due to the healthcare services’ lack of care-related integration at multiple levels and dimensions which was not necessarily needed with social services. Possible solutions suggested by health and elderly care specialists included adapting current healthcare systems to the needs of the home care, improving care coordination through various means, utilizing digital solutions, creating tools to track the status of the client’s care, and increasing co-creation of health with the client. With the current challenges in recruiting and maintaining health care personnel in the home care and constant training of personnel; this study suggests that different types of technological solutions are needed to improve care coordination and integration.
  • Koivula, Julia (2023)
    Wilms tumor is the most common kidney cancer in children. The origin of Wilms tumor is thought to arise from disturbed embryonic development. Wilms tumor is characterized by so called nephrogenic rests consisting of undifferentiated metanephric mesenchyme, that are precursor lesions for tumorigenesis. In addition, the tumors themselves contain tissue types normally found only in the developing kidney and they resemble embryonic kidneys transcriptionally. Since the tumors are heterogenous and contain blastemal, stromal, and epithelial components, all three progenitor populations of the developing kidney, nephron, collecting duct and stromal progenitors, are possible origins of tumors. MAPK/ERK pathway plays a significant role in kidney development for example by affecting nephron progenitor self-renewal and regulating collecting duct progenitor maintenance. Previous studies suggest that MAPK/ERK activity plays a role in Wilms tumor by mediating the intracellular effects of IGF2 overexpression. The aim of this master’s thesis is to compare transcriptional profiles of Wilms tumor to the transcriptional profiles in mouse MAPK/ERK deficient nephron progenitors and ureteric bud epithelium. This is done by utilizing internet-based tool ToppFun and R/Bioconductor tool gage (Generally Applicable Gene-set Enrichment for Pathway Analysis) for Gene Ontology (GO) and KEGG pathway analysis. My analysis revealed several shared GO and KEGG pathways that compose of differentially expressed genes with opposite expression patterns in Wilms tumor and MAPK/ERK deficient renal progenitors. The identified pathways include those previously validated by the host laboratory and some interesting new pathways that will be studied further in the future. The most interesting novel pathways affected in both Wilms tumor and MAPK/ERK deficient kidneys were related to the extracellular matrix (ECM) and chromatin. ECM related GO terms were specifically altered in nephron progenitors and Wilms tumor suggesting that Wilms tumor transformation involves dysregulation of ECM possibly downstream of MAPK/ERK pathway. MAPK/ERK pathway also mediates chromatin level regulation which is also demonstrated by my results. Chromatin related GO terms were upregulated in Wilms tumor and downregulated both in ureteric bud epithelium and nephron progenitors. The second aim of my thesis is to verify the observed gene expression changes by utilizing mouse embryonic kidney cultures and qPCR. The validation was only initial trial and requires further optimization. It did not show significant downregulation in selected validation genes that were chosen for validation. Future goal of my research is to carry out similar analysis in chemotherapy naïve dataset and at different Wilms tumor stages. This will allow avoiding possible chemotherapy induced changes in the outcome and better sorting of tumor progression and its correlation with specific renal progenitor types. In summary, my current results suggest both ECM and chromatin regulation as promising fields for future research.
  • Sundaresan, Srividhya (2024)
    Epithelial Ovarian cancer remains to be one of the leading causes of cancer-related deaths in women, and 70-80% of EOC-related deaths are attributed to High-Grade Serous Ovarian Cancer (HGSC). The 5-year survival rate of HGSC remains concerning at 50%. Approximately 50% of HGSC patients exhibit impaired Homologous Recombination (HR) DNA repair, a high-fidelity double-stranded DNA damage repair mechanism. Patients with Homologous Recombination Deficient (HRD) tumors exhibit better responses to DNA-damaging platinum agents and Poly (ADP-ribose) polymerases (PARP) inhibitors. The success of these treatments is attributed to the inability of HRD tumors to repair DNA damage, ultimately leading to cell death. It is expected that Homologous Recombination Proficient (HRP) tumors have functional repair mechanisms to eliminate this damage. Therefore, they derive no durable benefit from DNA-damaging agents and PARP inhibitors, resulting in a poor prognosis. DNA damage can be identified by the presence of pHistoH2AX (phosphorylated H2AX protein). This protein is formed at an early stage in response to the presence of Double-Strand Breaks (DSBs), and its interaction with MDC1 is critical for the recruitment of subsequent DNA repair proteins. Interestingly, Immuno-Fluorescent Imaging has revealed the presence of pHistoH2AX in chemo naïve HRP tumors. This raises questions about the origin of this endogenous DNA damage in HRP tumors and whether it signifies underlying vulnerabilities that could be exploited for therapeutic purposes. To address these questions, my thesis aimed to identify the co-expression markers of pHistoH2AX. To achieve this objective, I analyzed existing data from Cytometry by Time of Flight and Cyclic Immunofluorescence experiments to identify interesting molecular candidates. These experiments gave rise to several interesting candidates including pSTAT3, PDL1, pAkt, pEGFR, Cyclin E1 and Vimentin as potential co-expressed markers in HRP tumors. I further validated the co-expression of pSTAT3 and pHistoH2AX in HGSC patient tissue sections by immunofluorescence staining. This study holds significant relevance for identifying alternative molecular targets that could potentially improve the survival outcomes of patients with HRP HGSC. Future studies, encompassing larger cohorts of HRP patients, may unveil suitable subpopulations that could benefit from targeting these identified proteins, paving the way for the development of more effective therapeutic strategies in the management of HRP HGSC.
  • Fan, Qiuyu (2020)
    Alzheimer’s disease (AD) is a neurodegenerative brain disorder in which the disease process may take decades until the symptoms become evident. To date, no ideal biomarker has emerged that would enable early detection of AD. Environmental and lifestyle factors are thought to affect the risk of developing AD, possibly through epigenetic mechanisms such as DNA methylation (DNAm). DNAm has been shown to differ in the blood and brain of subjects with AD compared with subjects without AD, suggesting that DNAm may be involved in the pathogenic process of AD. This study aims to detect the difference in blood DNAm at baseline between cases who later developed AD and controls who remained AD diagnosis-free during follow-up in a sample selected from a Finnish population-based cohort. Leucocyte genome-wide DNAm was profiled on approximately 850,000 CpG sites by using Infinium MethylationEPIC assay. Each CpG was regressed on the outcome of AD diagnosis during follow-up, controlling for subjects’ age at sampling, sex, smoking status, blood cell counts, working stress level, slide, and array. Specific differentially methylated positions (DMPs) were further explored using pathway analysis. Finally, the methylation level of the candidate gene (APOE) selected from the literature was compared with the sample of this study. After correction for multiple testing, the later diagnosis of AD was not significantly (adjusted p-value < 0.05) associated with methylation level at the baseline at any DNAm site. There was, however, a robust hypomethylation of DMPs among the cases, as 90.9% of the DMPs (p-value < 0.05) were hypomethylated in the case group. The 200 genes annotated by DMPs with the smallest p-values were involved in two neuronal pathways: “Axon guidance associated with semaphorins Homo sapiens” (p-value = 0.0058, adjusted p-value = 0.065) in Panther 2016 and “Semaphorin interactions Homo sapiens” (p-value = 0.00005, adjusted p-value = 0.078) in Reactome 2016. No significant difference existed in DNAm of the candidate gene (APOE) between cases and controls, while cg26190885 at the promoter region of APOE showed nominal significance (p-value = 0.04). In conclusion, no strong evidence was found to support the hypothesis that systemic changes in DNAm are involved in the pathogenesis of AD or that DNAm marks could be detected in blood before the symptoms become evident. A genome-wide pattern of hypomethylation measured by the Infinium MethylationEPIC assay was observed in the case group, serving as a venue for further investigations.
  • Montonen, Reetta (2023)
    Since the beginning of the Coronavirus Disease 2019 (COVID-19) pandemic, there has been a need for developing an efficient vaccine against the SARS-CoV-2 virus and its emerging variants. In this thesis, humoral immune responses induced by either two doses of mRNA Pfizer-BioNTech (Pfizer) vaccine or one dose of adenoviral vector-based Oxford-AstraZeneca followed by a second dose of Pfizer were studied. Levels of anti-spike protein (anti-S1) IgG, IgA and IgM antibodies were measured with enzyme-linked immunosorbent assay. In addition, neutralizing antibody titers against the original Wuhan-Hu-1 strain, the Beta variant and the Delta variant were studied with a pseudovirus neutralization assay. The study used 180 serum samples from a cohort of vaccinated Finnish healthcare workers. Sera were collected from vaccinees before their first vaccination, after which vaccinees provided sequential samples at timepoints of three weeks, six weeks, three months, and six months. The results showed that both vaccination regimes produce high levels of anti-S1 IgG antibodies in vaccinees, and the antibodies persist in blood at least for six months. Anti-S1 IgA levels were lower compared to IgG levels, but were long-lasting, as 95% of vaccinees were IgA seropositive six months after the first vaccine dose in both study groups. Anti-S1 IgM levels resembled the levels of IgA, but the IgM seropositivity after six months was only 50% in the Pfizer-only group and 70% in the “mix-and-match” vaccination group. Neutralization assays demonstrated that the heterologous vaccination induces higher neutralizing antibody titers compared to the homologous vaccination. After six months, the neutralization in the homologous vaccination group was 6-fold reduced against Wuhan-Hu-1, 4-fold reduced against the Beta variant and 5-fold reduced against the Delta variant compared to the heterologous regime. The results are in line with previous findings that have proven the “mix-and-match” vaccination to be more immunogenic than the homologous prime-boost vaccination. By combining different booster COVID-19 vaccines to the primer vaccine dose, it would aid in cases of vaccine shortages and provide options to individuals that respond poorly to a specific type of vaccine.
  • Jalkanen, Nelli (2020)
    Mitochondrial aminoacyl tRNA-synthetases (mt-aaRS) catalyse the charging of tRNAs with their cognate amino acids in mitochondria. Mutations in mt-aaRS cause tissue-specific mitochondrial diseases, especially affecting tissues with high energy expenditure like the nervous system, heart, and kidneys. However, disease mechanisms for the heterogeneous group of diseases have not yet been fully elucidated. Harnessing CRISPR-Cas9 genome editing in induced pluripotent stem cells (iPSC) provides an opportunity to model mt-aaRS mutations in vitro and investigate the effects of individual mutations on cellular phenotype. SARS2 encodes mitochondrial seryl tRNA-synthetase, and its c.1347 G>A mutation causes severe childhood-onset progressive spastic paresis. Here, CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology directed repair (HDR) the genome of iPSC and knock-in the patient mutation. Guide RNAs were designed and tested for efficiency before electroporation into wild type iPSC. Clonal cell lines were made by low-density seeding and manual colony picking. The expression of pluripotency markers was measured by RT-qPCR. RT-qPCR and Western blot measured SARS2 mRNA expression and protein level respectively. The success and precision of genome editing were analysed by Sanger sequencing, comparing the performance of the different guide RNAs, and screening regions of potential off-target genome editing. Two genome-edited iPSC lines with the SARS2 c.1347 G>A mutation were successfully generated to model the patient mutation. The iPSC lines expressed pluripotency markers and contained no off-target genome editing and modelled the patient’s decrease in SARS2 protein level and mRNA expression. More evidence of differentiation ability is needed before differentiation into the affected cell type (motor neurons) and further disease modelling. The efficiency of CRISPR-Cas9 for genome editing, especially harnessing HDR in iPSC, is an area of future research.
  • Patpatia, Sheetal (2020)
    Antibiotic resistance of pathogenic bacteria has increased in recent years. When antibiotics do not work, alternative therapies are developed to prevent major bacterial epidemics. Phage therapy is one of the alternative possibilities to cure infections caused by antibiotic resistant bacteria. Due to the narrow host range of phages, hundreds or even thousands of phages are required to cover the various bacterial pathogens. For a reliable selection process, high-throughput rapid host range screening of phages is needed to cover the future demands. In addition, collaboration between laboratories is highly important, as the collections of phages of individual laboratories are not broad enough. Thus, the transportation of phages between laboratories is one of the key elements to provide successful phage therapy for patients. The aim of the study was to use gel-based products as protective matrix in phage host-range screening and transportation. The optimal conditions were selected to set a baseline for high-throughput rapid host range screening process, and to set up a ready-to-screen plate assay for phage transportation. In addition, the purpose of the study was to evaluate whether hydrogels could be used as a long-term storage matrix for phages and future product development. Fourteen Escherichia coli phages were used to optimize the liquid culture assay for the E.coli strains. The hydrogel based assays were conducted with two Escherichia and two Staphylococcus phages. For long-term storage, phages were mixed with different consistencies of hydrogels and stored in three different conditions for up to six months at +4oC. The transportation experiments were conducted with phages stored with optimized hydrogel consistencies. The phage viability was measured using liquid culture method. Results show that liquid culture method on microtiter plate is a convenient way to screen bacteriophages in high-throughput assay and that phages can be stored reliably in hydrogel format. When stored in microcentrifuge tubes, phage stability was shown to last for at least six months. When stored as drops on microtiter plate, the phages retained their viability for up to two months. These plates can be used as a robust means for phage transportation.
  • Ranta-aho, Johanna (2022)
    Distal myopathies are a group of inherited diseases that cause progressive muscle weakness primarily in the hands and feet. Variants in various different genes have been identified as disease-causing, but most recently, variants in ACTN2, a gene previously associated with cardiomyopathy, have been shown to cause a distal myopathy phenotype. ACTN2 encodes alpha-actinin-2, an important structural protein that links actin and titin to the sarcomere Z-disk. In ACTN2-related diseases, actininopathies, several variants have been identified as disease-causing, however, new variants are continuously discovered, and the significance of many variants remain unknown. Thus, lack of clear genotype-phenotype correlations in actininopathies persists. Further, the molecular mechanisms underlying actininopathies are largely unknown, especially in recessive actininopathies. Here, a reanalysis of the previously reported ACTN2-related molecular and clinical findings is conducted, with a subsequent reclassification of variants according to American College of Medical Genetics and Genomics (ACMG) guidelines. The results indicate that ACTN2 serves an important function in the muscle tissue and is involved in the pathomechanisms of myopathy, which is supported by a growing body of clinical, genetic, and functional evidence. However, distinct genotype-phenotype correlations are currently clearly understood only in some actininopathies, and limited segregation and functional data are still available to support the pathogenicity of most previously reported missense variants. Additionally, functional characterization of ACTN2 variants identified in recessive actininopathies suggest that the underlying molecular mechanisms of recessive actininopathies are different form the dominant from of the disease, as they do not produce detectable alpha-actinin-2 aggregates in the cell models. Thus, alternate methods should be used to investigate the molecular mechanisms of recessive actininopathies. Also, the results suggest that multiple different molecular mechanisms are involved in dominant actininopathies.
  • Hämäläinen, Lina-Maria (2023)
    Antiepileptic drugs (AED) are vital for treating epilepsy, but their use in pregnancy carries significant risks. Prenatal exposure to some AEDs like valproic acid increases the risk of teratogenicity and deficits in cognitive development in children. The impact of AEDs at different ages has been studied but their effect on the trajectory of cognitive development remains unknown. This study assessed the cognitive performance of children with prenatal AED exposure and unexposed controls using Bayley scales of infant and toddler development III (BSID-III) at 2 years and Weschler intelligence scales for children (WISC-IV) at 6 years of age. The association between 2- and 6-year outcomes was analyzed with separate ANCOVA models for each WISC-IV subscale. BSID-III subscales were used as the covariates and the models were adjusted for confounding factors. BSID-III scores in cognitive (B=6.70, p=0.01) and language (B=6.92, p=0.008) subscales were significantly associated with the WISC-IV working memory scores in controls but not in the exposed group. The groups were also found to differ in their intercorrelations between BSID-III subscale scores. Scores at 2 years were not associated with later results in exposed children as they were in controls. This may suggest that the cognitive development trajectory of AED-exposed children differs from that of unexposed controls, possibly following an alternate projection. Developmental trajectories should be considered when investigating the cognitive effects of prenatal AED exposure. The finding of lacking correlations of BSID-III subscales also raises cause for future investigation of the structure of cognition in AED exposed children.
  • Nestaite, Ernesta (2023)
    Intestinal epithelium is capable of rapid regeneration, which is associated with transient changes in cellular identity. Some of these changes involve an enrichment of fetal-like gene expression and simultaneous suppression of adult stem cell signature. Interestingly, the upregulation of fetal-like marker Stem cell antigen 1 (Sca1) is modulated by extracellular matrix (ECM) which is known to guide epithelial cells during regeneration. Our recently published decellularized small intestinal ECM (iECM) system retains the composition and topology of natural ECM. This makes it an attractive system for ex vivo studies addressing regeneration. This thesis aimed to gain insight into the fetal-like identity and its dynamics using an ex vivo iECM system. Intriguingly, Sca1 expressing cells on iECM displayed migratory features, such as a leading edge and changes in nuclear morphology. Curiously, these features are typical for epithelial cells during development. Furthermore, based on marker gene expression during iECM re-epithelization, fetal-like state was upregulated while adult stem cell state was downregulated, revealing a gradually emerging inverse correlation. Additionally, data suggests that circadian rhythms may have a role in modulating the fetal-like state. iECM from an active-state mice indicated a reduced capability to induce fetal-like identity and overall re-epithelization compared to the rest-state iECM. The results of this thesis suggest further potential of iECM system in studying emergence of fetal-like state during re-epithelization and circadian rhythm impact on it.
  • Ahveninen, Lotta (2022)
    Objectives. Ageing is accompanied by neurobiological changes, such as changes in grey matter (GM) volume and cortical thickness, that mediate a gradual cognitive decline, which can, in turn, be potentially offset by stimulating leisure activities. Choir singing is an especially feasible musical activity with positive effects on physiological, psychological, cognitive, and social functioning in old age. Research investigating the effects of choir singing on the ageing brain is limited. As part of the Brain, Ageing, and Vocal Expression (BRAVE) project, this study aimed to investigate the effect of ageing and choir singing on GM structure. Methods. Using a cross-sectional design and voxel-based morphometry (VBM) and surface-based morphometry (SBM), this study compared GM structure between young (20-39 years; n=35), middle-aged (40-59 years; n=34), and old (60-90 years; n=31) participants and investigated the interaction of age and choir singing on GM structure with amateur choir singer (n=54) and controls (n=46). Results and conclusions. Age had a significant and widespread effect on GM structure, with old participants showing lower GM volume and cortical thickness than young (in bilateral sensorimotor, auditory/language, visual, and limbic areas, midbrain, and cerebellum) and middle-aged (in right visual cortex, thalamus, hippocampus and left auditory cortex) participants. Middle-aged participants also showed lower GM volume and cortical thickness than young participants (in bilateral sensorimotor, language, and visual areas, basal ganglia, cerebellum, and right hippocampus and amygdala). These results corroborate the current understanding of neurobiological ageing. No significant interaction of age and choir singing was found on GM structure, which could be explained by methodological factors. Further research is needed to determine whether choir singing can support brain structure or function across healthy ageing.
  • Sahlman, Sara-Sofia (2022)
    Mitochondrial disorders form a heterogenous disorder group with symptoms widely varying. They exist because of mutations in mtDNA or nuclear DNA, which encode mitochondrial proteins or regulate their functions. Because mitochondria are present in almost all cells in the human body, symptoms of mitochondrial disorders vary and can manifest in all tissues. The most common manifestation of mitochondrial disorders in adult patients is mitochondrial myopathy, which leads to muscle weakness and fatigue. Pathological findings of mitochondrial myopathy include changed activities in mitochondrial respiratory chain, centrally located nuclei and increased expressions of genes related to integrated mitochondrial stress responses. One of the neurological symptoms of mitochondrial disorders is parkinsonism. Parkinson’s disease is the world’s second most common neurodegenerative disease. Its clinical symptoms include bradykinesia and resting tremor, and its pathological hallmarks include alphasynuclein protein accumulations and loss of dopaminergic neurons in substantia nigra. Despite being described first time over a hundred years ago, the mechanisms behind Parkinson’s disease are still unclear. However, several genes have been associated with Parkinson’s disease. PINK1, encoding PINK1 protein, and PRKN, encoding Parkin protein, are genes, which mutations have been associated to early onset Parkinson’s disease. According to suggested theories, their normal physiological functions include mediating a special form of autophagy, mitophagy, and suppressing mitochondrial antigen presentation. It has been suggested that loss of PINK1 or PRKN could disrupt mitophagy, leading to accumulation of dysfunctional mitochondria in the cell, disrupting its homeostasis and leading to its death. This PINK1/Parkin-mediated mitophagy and its disruption could explain the death of dopaminergic neurons. In addition, loss of PINK1 and Parkin could also activate mitochondrial antigen presentation, which activates autoimmune response and destroys dopaminergic neurons presenting mitochondrial antigens. This suggests that Parkinson’s disease could have autoimmune-like mechanisms behind it. Together, these theories could explain the onset of Parkinson’s disease when PINK1 or PRKN are absent. In this thesis, the main objective was to study, how loss of PINK1 and PRKN would affect on muscle of aged mouse. More precisely, the effects of these genes to mitochondrial homeostasis were studied in terms of mitophagy and mitochondrial respiratory chain activity. In addition, it was studied, if these genes could induce mitochondrial myopathies or affect to existing condition by studying typical signs associated with mitochondrial myopathy, such as changes in activity of mitochondrial respiratory chain subunits, location of nuclei in muscle, changes in lipid and glycogen accumulation and expressions of genes associated to mitochondrial integrated stress responses. Finally, the theory of mitochondrial antigen presentation was studied. The results of this thesis provide more evidence to theory of PINK1/Parkin mediated mitophagy, but also suggest, that these proteins could be compensated. Based on the results, PINK1 seems to be more crucial for healthy muscle, but in already existing mitochondrial myopathy, loss of either of PINK1 or Parkin seems to improve the condition. According to this thesis, both PINK1 and Parkin, and mitophagy seem to be crucial in induction of mitochondrial myopathy. Despite more evidence is now provided to support the roles of PINK1 and Parkin in mitochondrial maintenance, their roles in mitochondrial antigen presentation did not receive support from this thesis.
  • Sinkko, Matilda (2021)
    In this master’s thesis, in vitro neuromuscular junction (NMJ) model was set up using microfluidic devices. Additionally, the effect of R878H/R878H mutation in MCM3AP gene that causes an early-onset peripheral neuropathy on NMJ formation and maintenance was studied. To study human NMJs that significantly differ from other mammal NMJs is challenging and new models to study the function of these complex and highly specialized structures are needed. Induced pluripotent stem cells (iPSC) and motor neurons were characterized with gene expression studies using qRT-PCR and with immunocytochemistry studies using commonly known markers for pluripotency and motor neurons. NMJs were studied in 2D co-cultures and with microfluidic devices. Gene expression studies were conducted from 2D co-cultures and co-cultures in microfluidic devices provided detailed information of the localization and morphology of NMJs. Expression of essential genes for NMJ formation together with immunocytochemistry results with alpha-bungarotoxin (BTX) staining showed that NMJs were formed in both control and R878H/R878H mutant cell line co-cultures. There was a trend of lower gene expression levels of NMJ essential genes in the R878H/R878H mutant line compared to the control line and also immunocytochemistry results indicated impairment in NMJ formation in the mutant line, but further studies are needed to validate the effect of R878H/R878H mutation on the NMJ formation. In future, functional studies could be conducted to investigate whether these NMJs are functional and the information from the motor neuron terminal is conveyed to the muscle membrane.
  • Kaaja, Ilse (2020)
    Bone marrow failure (BMF) is a condition where the bone marrow fails to produce enough functional blood cells leading to peripheral blood cytopenias. Inherited BMF is often a consequence of germline mutations in DNA repair pathway, telomere maintenance, or ribosome biogenesis -related genes and results in up to 20-40% risk of developing a hematological malignancy. Recently, biallelic germline mutations in the gene ERCC6L2 have been identified to cause inherited BMF leading to the accumulation of somatic TP53 mutations and acute myeloid leukemia (AML M6) with dire prognosis. ERCC6L2 is a DNA repair protein that has also been indicated in mitochondrial function. The aim of this thesis was to study the ERCC6L2 protein expression and cellular metabolism in ERCC6L2-derived BMF. The metabolic profile in ERCC6L2-derived BMF was studied in patient-derived fibroblasts using a Seahorse XFe96 Analyzer. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured at multiple time steps when cells were in standard cell culture (10mM) glucose concentration or low (1mM) glucose concentration. The protein expression was studied in fibroblasts and peripheral blood mononuclear cells (PBMCs) with immunofluorescence assay and Western blotting. The results of this thesis demonstrate a differential metabolic profile in the patient-derived cells. In normal glucose, they thrive exhibiting a higher basal OCR, ATP-related respiration, mitochondrial reserve capacity, and maximal respiratory capacity compared to the control. Contrarily, in low glucose the patient-derived cells struggle and show a lower basal OCR, ATP-related respiration, reserve capacity, and maximal respiratory capacity than the control implying decreased substrate availability in the mitochondrial respiratory chain or mitochondrial dysfunction. Immunofluorescence assay suggests that ERCC6L2 is expressed in the patient-derived cells supporting truncating mutations observed in RNA sequencing. In order to improve the treatment and clinical outcomes in inherited BMF, understanding the role of altered mitochondrial metabolism in ERCC6L2-derived BMF and its progression to AML M6 calls for further studies.