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Browsing by study line "Cross-disciplinary translational medicine"

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  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Their, Anna (2021)
    The contact site between the endoplasmic reticulum and mitochondria, also known as the mitochondria endoplasmic reticulum contact sites (MERCS), have a crucial role in maintaining the homeostasis within the cell. Across the MERCS multiple functions, such as regulation of calcium (Ca2+) homeostasis, lipid metabolism, ER stress, mitochondrial quality control (MQC) and regulation of unfolded protein response (UPR) take place. These processes have been shown to be implicated in numerous different neurodegenerative diseases, such as Parkinson’s disease. Parkinson’s disease is the second most common neurodegenerative disease that at the moment has no cure. The main obstacle in developing a neuroprotective treatment for the disease is the limited understanding of the key molecular events leading to neurodegeneration. One of the things in Parkinson’s disease that has eluded scientists for years is the selective death of the dopaminergic (DA) neurons in substantia nigra pars compacta. One hypothesis that could explain the selective death is the Ca2+ hypothesis, looking at the Ca2+ vulnerability of SNpc DA neurons as a plausible cause leading to the selective cell death. This project focused looking at the protein level and morphological changes of the ER and MERCS in stressed neurons, hypothesizing these as possible sites that contribute to the neuron vulnerability, as they are known to be the key modulators of the intracellular Ca2+ homeostasis. This study looked closer at two MERC proteins, GRP75 and BAP31, and one ER protein, SERCA2, to see how they are affected in stressed dopamine-like neurons. Firstly, the in vitro model was established by differentiating SH-SY5Y neuroblastoma cells to dopamine-like neurons expressing tyrosine hydroxylase. Three different molecular compounds were tested as possible stressors affecting the Ca2+ homeostasis within the neurons, and we concluded that thapsigargin, a commonly used stressor to model PD like pathology, leads to the highest measurable ER Ca2+ depletion. Lastly, we quantitatively and qualitatively analyzed the effect of 24-hour treatment with each stressor on the differentiated SH-SY5Y neurons. Thapsigargin treatment lead to an increased level of GRP75 and SERCA2. A slight increase in BAP31 was also detected after thapsigargin treatment, but no apparent changes of the ER morphology were detected. The results, together with previous research, show GRP75 to be a possible contributor to the pathology of the disease, but further research is needed to see if it could be a possible target for treatment.
  • Jagdeo, Joanna (2020)
    Merkel cell carcinoma (MCC) is a rare, neuroendocrine carcinoma of the skin that is known to have poor prognosis. It is associated with the Merkel cell polyomavirus (MCPyV) and majority of cases harbor this infection. Other risk factors include older age, the male sex, Caucasian skin and increased ultraviolet exposure. Increased lymphocyte invasion into the MCC tumor microenvironment has been reported to infer better survival, but better mechanisms understanding why this occurs this is needed. CCL3 is a chemokine that is implicated in a variety of inflammatory conditions like viral infections and exhibits pro-inflammatory activity mainly through its chemoattractant abilities. In cancer specifically, it functions within the tumor microenvironment by encouraging the trafficking of leukocytes to the tumor site. Transcriptomic data of CCL3 was studied in a cohort of 102 Finnish MCC patients to observe its association with survival, and a variety of clinical-pathological features. The presence of CCL3 in cells was later investigated via immunohistochemistry in 30 formalin-fixed paraffin-embedded Finnish MCC primary tumor tissue samples with varying mRNA expression of CCL3. Macrophages and lymphocytes were found to stain positively for CCL3 and were found exclusively in tumor surroundings. CCL3 was also found to exhibit a MCC-specific survival benefit in patients that harbored higher expression (p=0.031), and was found to be associated with MCPyV positivity (p=0.032). These preliminary findings help establish CCL3’s role in the immune response against MCC and support the need for further studies looking at CCL3 both as a prognostic marker and potential adjuvant therapeutic.
  • Nowlan, Kirsten Helene Anna-Marie (2020)
    Mucosal associated invariant T (MAIT) cells are a fairly recently described population of innate-like T cells. In humans, MAIT cells represent an exceptionally abundant population in the blood, where they account for 1–10% of all T cells. However, compared to conventional T cells, which can display an almost unlimited T cell receptor (TCR) repertoire, the specificities of MAIT cell TCRs are limited. This evolutionarily conserved subset displays a semi-invariant TCR which recognises riboflavin metabolites, produced by a wide range of bacteria and fungi, and presented on the major histocompatibility complex (MHC)-class I related (MR1) molecule. The function and significance of MAIT cells in health and disease have only started to be unravelled, and it is becoming increasingly clear that MAIT cells are also modulated in non-microbial diseases. Interestingly, MAIT cells have been shown to exhibit a relatively high expression of the transcription factor, Helios, compared to most other T cell subsets. The function of this translational activator and repressor, encoded by the gene IKZF2, in the MAIT cell population remains obscure. This study focused on the functional role Helios may play in regulating the activation of MAIT cells. Thus, by using siRNA to silence Helios expression, and flow cytometry to analyse any potential alterations in MAIT cell activation markers, we aimed to be able to characterise the kinetics and functional role of Helios in peripheral MAIT cells of healthy individuals. Here, we clearly established a striking upregulation of Helios in MAIT cells following 24 hours of stimulation. Moreover, we were able to achieve a >50% knockdown of Helios at the protein level, in this subset of T cells. Nevertheless, no significant difference in any of the activation markers we investigated was present between the MAIT cells with reduced Helios expression and their controls. This could, however, of occurred as a result of the toxicity that the transfection had on the functionally of the T cells. From these results, it is difficult to conclude any clear role for Helios in the activation of MAIT cells, and consequently, further research needs to be performed before any clear conclusions can be drawn.
  • Lillback, Victoria (2024)
    Many rare skeletal muscle disease patients have remained unsolved after initial analysis, prompting reanalysis with the hope that recent findings and increased knowledge would resolve previously ambiguous variants and genes. We have an enrichment of especially unsolved singletons; patients with no segregation data to assist in the variant interpretation. Sequencing singletons have led to larger cohorts of unsolved exomes that are now laborious to manually reanalyse. Standardization and automation could streamline analysis and reanalysis. Bioinformatic tools such as the gene prioritization tool Exomiser leverage structured clinical annotations (Human phenotype ontologies, HPOs) and variant in silico predictions to rank genes and variants according to pathogenicity, reducing potential variants to a handful. By comparing phenotypes to animal models and protein-protein interactions it is also able to prioritize phenotypically relevant genes not yet associated with disease. By benchmarking with solved cases, we could also conclude that Exomiser performs well without patient specific HPOs and prioritizes the correct variant in top 10 results in 97.4 % of 117 cases with the same three HPOs describing skeletal muscle disease in general when the corresponding percentage for patient specific HPOs was 96.7 %. This automatizes analysis for large cohorts wherein assigning specific HPOs individually for every sample would be a time-consuming step. Additionally, by benchmarking with exomes mimicking a finding in a skeletal muscle candidate gene not yet associated with disease we gained 57.1 % genes prioritized in the top 10 results, confirming Exomiser ability to prioritize novel findings. By combining unsolved singletons to a 278-sample cohort, we gain, in addition to prioritization of genes, support to rule out internally occurring variants not of interest. A cohort approach also gives support to novel findings if the same gene is seen as pathogenic in several samples sharing a phenotype. To date this re-analysis approach has streamlined and reduced manual labour significantly. Nevertheless, diagnostic yield remained low (1 %) in our already extensively studied cohort, clinicians have confirmed the causative variant(s) in four patients and additionally we have 17 findings requiring further investigation to determine pathogenicity or to be shared in larger research community efforts for further cohort support. We recommend this method as an initial analysis and a periodic reanalysis of unsolved exomes, which will benefit from literature updates. This will help in solving cases with minimum effort. A similar approach can be used with whole genome sequencing data to enable interpretation of variants beyond the coding region.
  • Lapinsuo, Ella (2023)
    The extraembryonic placenta is composed of trophoblast cells consisting of the proliferative cytotrophoblasts (CTB) and its differentiated subtypes syncytiotrophoblast (SCT) and extravillous trophoblast (EVT). A normal trophoblast development is important as disruptions can lead to pregnancy complications such as pre-eclampsia. Therefore, it is crucial to investigate the underlying causes behind these abnormalities to discover treatments for patients suffering from pregnancy related disorders. Previously placental research was conducted largely on animal models and despite shared conservative pathways with humans, there are differences that exist. Only recently have researchers managed to successfully isolate and culture primary trophoblast stem cells (TSC)s by creating a TSC medium. Due to limited access to placental cells, pluripotent stem cells (PSC)s can be differentiated to TSCs by using the TSC medium. Naïve and primed states are described to be PSCs in different developmental stages, the former representing the pre-implantation state and the latter the post-implantation state. There lacks a consensus on whether both PSC states can be used to generate TSCs that correspond to primary trophoblasts. It has been argued that naïve cells possess more potential to differentiate into TSCs compared to the primed ones. The primed cells have been induced with the bone morphogenic protein (BMP) 4 to generate TSCs. This method is controversial as some suggest the induction resulting in other than TSCs, such as amniotic cells. Therefore, the aim of this thesis was to investigate whether both PSC states could be used to generate TSCs and its subtypes, if at all. Further, the effect of BMP4 was examined in the prime- derived differentiation protocol. The generated cells were then characterized and analyzed using imaging, immunocytochemistry (ICC) and quantitative reverse transcription PCR (RT-qPCR). The thesis found that although TSCs and its subtypes could be successfully generated from both PSC states, differences were observed. In addition to morphological differences, the most significant finding was the expression of the HLA-G gene, an EVT-specific marker, in the prime-derived TSCs (TSC(BMP4)). HLA-G was also significantly more expressed in the prime-derived EVTs (EVT(p)) compared to the naïve-derived EVTs (EVT(n)). Further, MMP2 which is also an EVT specific marker, was significantly more expressed in the EVT(n) compared to the EVT(p). As a result, the research question regarding the validity of the TSCs using both methods and the effect of BMP4 remains open. Further studies are required including single-cell RNA sequencing to obtain a better and broader view of the trophoblast profile and functional assays for subtype differentiation. Additionally, the role of BMP4 should be investigated in more depth.
  • Arffman, Maare (2021)
    Uterine leiomyomas are common smooth muscle tumours, with a prevalence as high as 80%. Even though they are benign, they present severe symptoms such as heavy menstrual bleeding, pelvic pain and reproductive dysfunction. Uterine leiomyomas can be classified to conventional tumours and leiomyoma variants based on their histopathology. The tumours usually harbour one of the three driver alterations: MED12 mutations, HMGA2 overexpression or biallelic FH inactivation. Known risk factors for leiomyoma development are African ancestry, family history and age. Uterine leiomyomas are most typically treated by surgery, through either uterus preserving myomectomy or by definitive hysterectomy. This Master’s thesis is continuation of a study from Äyräväinen et al. 2020, a retrospective study of 234 patients undergoing myomectomy at Helsinki University Hospital during 2009-2014. The aim of this study was to analyse how many of these patients had developed recurrent leiomyomas and how often the tumours in subsequent operations were potentially clonally related. In addition, clinical characteristics associated with the operations were analysed. In total 18% of these patients had recurrent operations, leading to the screening of 77 individual uterine leiomyomas from 32 patients. The mutational statuses were studied systematically with molecular screening using Sanger sequencing and immunohistochemistry. Altogether 33 tumours from 21 patients were found to have identical mutational status with a tumour from the original study. Of these tumours, 14 had a MED12 mutation. All the MED12 mutations were found in exon two affecting either codons 44 or 36. Six tumours had HMGA2 overexpression, and eight tumours were FH deficient. Five tumours were triple negative for all studied alterations. Whereas 81% of the patients had had two removal operations, the rest of them had had three to five operations. The years between operations ranged from performing them on the same year to performing them ten years apart. Even though most of the recurrent tumours were sporadic, almost half (43%) of them had identical mutations, suggesting that though uterine leiomyomas usually arise independently, some might be clonally related. The mutational distribution was different in the recurrent tumours than in uterine leiomyomas in general, indicating that in addition to germline predisposition, the driver related characteristics might also contribute to the potential of recurrence and to the likelihood of developing clonal lesions. Tumours harbouring MED12 abnormalities were the least probable to be clonally related. The tumours showing identical HMGA2 overexpression were likely clonally related. The number of identical FH deficient ULs was high, but not unexpected, since all the patients harbouring the mutation in the recurrent tumours had HLRCC, and therefore having a predisposition. Most surprisingly, all patients with recurrent triple negative tumours had identical mutation statuses in the recurrent tumours, which points to previously unknown clonal development of these lesions. Most of the patients with more than two surgeries had recurrent mutations, suggesting that multiple surgeries might indicate the development of clonally related tumours. However, further research is required to confirm the clonal relationships and to investigate the pathological nature of the tumours with different driver alterations.
  • Pällijeff, Pieti (2021)
    Recently, several novel post-translational modifications (PTMs) have been identified as important regulators in biology. Succinylation, the reversible addition of a succinyl group from a free succinyl-CoA into a protein lysine, is one such novel PTM. The last decade of research has unveiled succinylation as a powerful regulator of metabolism, prevalent in every organism it has been studied in and with functional effects on target proteins in several key metabolic pathways. A major contribution of this thesis is to catalogue the recent advances in succinylation research into the most comprehensive literary review currently available on succinylation. While the biological role of this PTM is being established, the relevance of succinylation in human disease has remained unclear. Meanwhile, mitochondrial DNA depletion syndrome caused by defective SUCLA2 (SUCLA2 disease) is a progressive hereditary mitochondrial disease with no available treatment. SUCLA2 disease is caused by defective mutations in the ß-subunit SUCLA2 of the TCA cycle enzyme succinyl-CoA synthetase. While the characteristic manifestations, including impairment of respiratory complexes, and the etiological mutations in this disease are well established, the pathogenic model for SUCLA2 disease has remained incomplete. As succinyl-CoA synthetase shares a substrate, succinyl-CoA, with succinylation, this thesis set out to probe SUCLA2 mutants for a potential succinylation phenotype. An extensive hypersuccinylation phenotype was characterized in fibroblasts and tissue samples from SUCLA2 mutant patients by immunochemical methods. The hypersuccinylation target identities in SUCLA2 mutants were revealed with proteomics by mass-spectrometry. Hypersuccinylation in SUCLA2 mutants was shown to be enriched in proteins participating in mitochondrial energy metabolism, including respiratory complex proteins. In addition, several novel metabolic phenotypes were characterized in SUCLA2 mutants with metabolomics by mass-spectrometry, most prominently a significant depletion of aspartate metabolism. While identification of extensive hypersuccinylation in SUCLA2 mutants establishes a novel concept of succinylation relevance in human metabolic disease, the prospect of altered regulation of the respiratory complexes due to hypersuccinylation lays the foundation for a novel pathogenic model for SUCLA2 disease. Meanwhile, the observed novel metabolic phenotypes significantly contribute to the current understanding on SUCLA2 mutant metabolism and inspire a hypothetical model on how the defective succinyl-CoA synthetase could be circumvented in the TCA cycle of SUCLA2 mutants.
  • Saario, Maria (2024)
    Adaptive immunity is a specialized immune system, responsible for pathogen-specific immunity and the creation of memory cells. This system is activated in secondary lymphoid organs, such as lymph nodes, by antigen presentation conducted by professional antigen-presenting cells (APCs) to T cells. The activated T cells migrate to inflamed tissue for the elimination of pathogens. Recently, it has been shown that lymphatic endothelial cells (LECs) of the lymphatic system also partake in immune regulation alongside professional APCs. LECs accomplish this by internalizing soluble antigens from the lymph. The internalized antigens can be either presented on the LEC plasma membrane with major histocompatibility complex I for induction of T cell anergy, or transferred to migrating professional APCs for complete T cell activation. Outside of direct immune regulation, the antigens can also be transcellularly transported through the LECs, which contributes to the transport of soluble antigens to lymph nodes. Through all these functions, LECs have been shown to play a role in the maintenance of peripheral T cell tolerance, impairment of tumor-specific immune responses, and conversely, the creation of stronger secondary immune response due to stimulation of T memory cell generation. However, the mechanisms of antigen internalization in LECs remain highly uncharacterized. To further study the role of LECs in adaptive immunity regulation, this thesis investigates the intracellular routes of antigen internalization and trafficking, in LECs. In this thesis, I set up antigen feeding assay on two primary cell culture models, and identify vesicular compartments involved in antigen internalization. To study antigen internalization, I set up extraction and cell culture methods for mouse lymph node primary cells and performed antigen feeding on the cultured cells. In addition, I optimized antigen feeding assay and identified means to increase antigen internalization in primary human dermal LECs (HDLECs). As LECs internalize antigens in both cell culture models, both models offer an opportunity to study antigen internalization and fates of antigens, in the context of LEC-performed adaptive immunity regulation. To investigate the pathways involved in antigen internalization and trafficking in LECs, I utilized RAB GTPases, major regulators of vesicular traffic, each specific for a distinct vesicular compartment. By observing and quantifying the colocalization between antigens and fluorescently stained RAB GTPases, I identified the antigen-associated vesicular compartments. The identification of the antigen trafficking pathways was successful, as functional validation of the results by inhibition of the antigen colocalizing RAB GTPases led to decreased antigen internalization. In conclusion, in this thesis, I optimized antigen feeding assay in LECs and identified pathways involved in antigen internalization and trafficking. The results of this thesis provide ground for further identification of molecules involved in LEC-performed adaptive immunity regulation. Manipulation of these control molecules would provide opportunities to modify the role of LECs in adaptive immunity regulation, to a desired direction.
  • Siskovs, Klims (2021)
    STK11/LKB1 is a tumor suppressor gene and mutated in 18% of lung adenocarcinomas. Tumor suppressor liver kinase B1 (LKB1) is known to activate adenosine monophosphate-activated protein kinase (AMPK) and 12 AMPK-related kinases (ARKs) by phosphorylating a conserved threonine residue in their T-loop region. A number of studies focused on investigating the influence of LKB1-AMPK signaling on cancer cell proliferation. However, there is no systematic study for identifying the critical LKB1 kinase substrates in suppressing lung cancer cell growth. In this project, the LKB1-deficient lung adenocarcinoma cell line A549 cells were sequentially overexpressed with constitutively active mutants of AMPKα1, AMPKα2, MARK1, MARK2, MARK3, MARK4, NUAK1, NUAK2, SIK1, SIK2, SIK3. The overexpression status was confirmed at both genetic and protein levels by qPCR and Western blotting, correspondingly. In vitro growth assays demonstrated up to 33% reduced growth rate of A549 cells overexpressing AMPKα1, AMPKα2 and NUAK1. Furthermore, siRNA knockdown of the selected substrates in LKB1-overexpressing A549 cells significantly rescued the cell growth defect. These findings suggest, that AMPKα1, AMPKα2 and NUAK1 kinases are critical for LKB1-mediated cell growth defect in lung adenocarcinoma.
  • Tripathi, Shivanshi (2020)
    Multiple Myeloma (MM) is the second most common hematologic malignancy. Despite the advancements in treatment approaches in the last decade, the prevalence of refractory disease leading to relapsed cases has been a major challenge. A wide range of intricate genetic heterogeneity demonstrated by myeloma patients is a credible explanation for the diverse treatment responses observed in patients sharing the same treatment regimens. Pertaining to this, the study aims to identify predictive gene expression biomarkers that forecast response to BCL2 inhibitor venetoclax and treatment outcome to proteasome inhibitor bortezomib. In this study, samples from MM patients were characterized into sensitive and resistant, (1) based on ex vivo response to venetoclax treatment (Resistant n=21; Sensitive n=21), and (2) based on their bortezomib treatment outcome in clinical profiles (Resistant n=12; Sensitive n=15). Associations between the different gene expressions and drug responses were studied using statistical and bioinformatic tools. As a result, we identified that significant (p-value <0.05) overexpression of 36 genes and downregulation of 38 genes appeared to confer resistance to venetoclax drug response in MM patients. Additionally, the functional association of these genes with pathways was determined using a pathway enrichment tool. Furthermore, the study provided evidence that cytogenetic alterations t(11;14) and t(4;14) are significantly (p-value <0.05) associated with differing venetoclax response in MM patients. These findings demonstrated that gene expression biomarkers and chromosomal translocations play a significant role in regulating venetoclax drug response in MM, which can be further utilized to personalize treatments for patients. The knowledge obtained from this work best applies in personalized medicine; whereby fitting treatments to an individual patient’s genomic landscape will enhance patient outcome.