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

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  • 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.
  • Pohjankoski, Tiina (2024)
    Amomum villosum Lour. (Zingiberaceae) has been a fundamental component of traditional Chinese medicine for centuries, primarily employed in managing gastrointestinal disorders. The commonly utilized component of the plant is the dried and mature fruit known as Amomi fructus, which has been shown to contain multiple active chemical components including volatile oils, polysaccharides and polyphenols. Despite extensive research into the medicinal properties of A. villosum, which encompasses anti-inflammatory, antioxidant, antimicrobial, antitumoral, hypolipidemic and hypoglycaemic effects, much of the focus has been on local inflammatory disease models such as inflammatory bowel disease. However, investigations into its potential efficacy in systemic diseases like rheumatoid arthritis are notably lacking. Rheumatoid arthritis is a chronic autoimmune disease characterized by inflammation of the synovial joints, leading to joint damage and disability. The effects of novel water and methanol extracts of A. villosum was studied on murine RAW 264.7 macrophages and human dermal fibroblasts (HDF). RAW 264.7 cells are widely used as a model for studying inflammation and immune responses, while HDF cells exhibit relevance to RA through their similarities to synovial fibroblasts and involvement in inflammatory processes, angiogenesis and tissue remodelling. High-performance liquid chromatography analysis revealed the presence of vanillic acid in our extracts, a potent antioxidant with potential therapeutic applications in various inflammatory conditions. Vanillic acid served as a baseline for subsequent in vitro experiments with our extracts. Results from the PrestoBlue cell viability assay demonstrated that water and methanol extracts from A. villosum enhanced HDF cell proliferation. Furthermore, both extracts exhibited antioxidant effects against hydrogen peroxide stimulation in both cell lines. Additionally, the water extract from A. villosum, inhibited the secretion of interleukin-6 in lipopolysaccharide-stimulated RAW 264.7 cells. Vanillic acid provided similar results. These findings suggest that the anti-inflammatory properties of A. villosum may offer a novel therapeutic approach for chronic inflammatory diseases.
  • Rinne, Nea (2022)
    Aims: Reading ability is a fundamental skill in the modern society, yet some individuals have difficulties in learn-ing to read and write. There is a lot of variability in reading skills, and one reason that can cause reading difficulty is a neurodevelopmental disorder called dyslexia. It is the most common learning disability, and the core deficit in dyslexia lies in word decoding, which is the process of connecting letter combinations into their corresponding auditory representations. Dyslexia is familial and is recognized to have strong genetic background. A dozen dyslexia susceptibility genes have been suggested, but DYX1C1, DCDC2 and KIAA0319 have been associated with dyslexia most commonly. The function of these genes is however not yet fully understood. In previous studies variation in these genes have been linked to struc-tural brain alterations in left hemispheric regions where language is mostly processed. The aim of this study was to examine the connection between dyslexia susceptibility genes DCDC2, DYX1C1 and KI-AA0319 and variation in brain activity during reading tasks in the left middle temporal gyrus (MTG), infe-rior Frontal Gyrus (IFG) and intraparietal sulcus (IPS), by combining functional magnetic resonance imag-ing data and genetic data in a neurotypical population. Previous studies have reported that weaker reading skills are associated with decreased brain activity in these regions, and reading incongruent sentences has been associated with increased brain activity in the left IFG and MTG. Methods: During fMRI, participants were presented with sentences with illogical and logical endings, and judged them as either congruent or incongruent, in distracted and undistracted conditions. Auditory speech stimuli were used as distractor. Regions of Interest analyses were conducted to examine brain activation in the aforementioned brain regions during distracted and non-distracted reading separately for different allelic groups in single nucleotide polymorphisms of the three genes. Results and Conclusions: DYX1C1 showed significant interaction with brain activation in the IPS. A significant interaction of DCDC2 with logic was found in the IFG and IPS showing that individuals carrying susceptibility alleles have reduced brain activation when reading incongruent sentences. Additionally, DCDC2 showed inter-action with distraction in the IFG, as individuals carrying susceptibility alleles had reduced brain activa-tion when a speech distractor was presented. In the MTG, there was a significant interaction of DCDC2 with logic and distractor showing that in different allelic groups, speech distractor modulated the activa-tion elicited by incongruent sentences in different ways. These results provide a link between variation in dyslexia susceptibility genes and brain activation during reading. Previous studies have mostly linked dyslexia susceptibility genes to structural brain alterations, and dyslexia and lower reading skills have been linked to variation in brain activity. The current study therefore expands the current understanding of genetic basis on reading and linguistic processing.
  • 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.
  • Suominen, Peppi Lotta Josefiina (2023)
    Multiple myeloma (MM) is a malignancy of antibody-secreting plasma cells in the bone marrow (BM). MM is the second most common hematological malignancy, accounting for about 14% of blood cancers. Despite the improvements in the treatment of MM, the disease remains incurable, and essentially all patients end up relapsing. Deletion of chromosome 16q, the location of tumor suppressor CYLD, occurs in 35% of MM patients. CYLD is a deubiquitinase, most recognized for its function as a negative regulator of the nuclear factor kappa B (NF-κB) pathway. The loss of CYLD is associated with disease progression and worse survival in MM, but its significance in drug response is unknown. As the loss of CYLD is common in MM, determining its effect on drug response is essential. Analysis of data gained from MM patient samples were used to study the effect of CYLD copy number status on drug response. The treatments were selected based on previous research performed by our group. The effect of homozygous deletion was most significant in inducing resistance to BMS-754807, an inhibitor of insulin-like growth factor 1 receptor. To investigate the role of loss of CYLD on drug response in vitro, cell lines with CYLD knockout (KO) were created using the clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR associated protein 9 (Cas9) technology. An established CYLD-KO cell line was treated with carfilzomib, bortezomib, dexamethasone and BMS-754807 to assess the effect of CYLD-KO. The CYLD-KO slightly increased the sensitivity to BMS-754807, but essentially no differences were detected in response to the drugs. The effect of CYLD-KO was additionally explored to NF-κB - and Wnt-pathway activation by Western blot analysis, but due to technical difficulties, the results were inconclusive. The loss of CYLD is a common genetic aberration in MM, giving a survival benefit for the malignant cells. Based on the results from patient data analysis, the loss of CYLD could promote drug resistance to BMS-754807, and the effect should be further studied with more cell lines with CYLD-KO. As the population ages, and as the median age of newly diagnosed patients is 70, the need for efficient MM therapies increases. Studying the mechanisms behind drug resistance and sensitivity is essential in the aim of improving the efficacy of MM therapy and, in the end, the overall survival of the patients.
  • 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.
  • Martikainen, Enni Maria (2024)
    Although approximately 80% of children with cancer survive five years after diagnosis, many childhood cancer survivors suffer from side effects and long-term health complications caused by the treatments. In addition, the diseases can often recur due to ineffective therapy. In pediatric sarcoma treatment, the recurrence of the disease is one of the major challenges. In rhabdomyosarcoma (RMS) 30% of cases end up relapsing and the cure rate is low with current therapies. Functional precision medicine approach is of interest especially with children since childhood cancers are likely diseases of upregulation or downregulation of protein expression with an overall low mutational burden and a lack of identifiable targets. In functional precision medicine the information from drug sensitivity and resistance testing (DSRT) of patient-derived cells (PDCs) is combined with other molecular tumor data, such as genomic and transcriptomic data, to better guide the selection of personalized treatments. In this study, we first established the PDC culture conditions for two longitudinal tumor samples of a young patient with RMS. DSRT was performed to age-matched healthy control fibroblasts and aortic smooth muscle cells, four RMS cell lines with different molecular backgrounds, and as a proof-of-concept study, to two longitudinal tumor samples of the RMS patient, to find compounds able to inhibit RMS cell viability. In addition, the clinically relevant genetic findings of the patient were highlighted. Specific drug responses relating to the molecular characteristics of the RMS cell lines were observed, such as MEK inhibitors in RAS mutated fusion-negative RMS cells. The findings of this work show that the DSRT assay can be utilized to discover RMS specific drug vulnerabilities, and it reveals the importance of proper control cells when using DSRT. This study provides evidence that combination of DSRT and NGS-based diagnostics may provide additional value for cancer treatment.
  • 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.
  • Bouhlal, Jonas Otto Vilhelm (2022)
    Despite of great advancements in the field of cancer therapy in the past decades, the 5-year survival of acute myeloid leukaemia (AML) patients remains low with high mortality especially in elderly patients, in whom the disease is most often observed. Poor prognosis often results from complex heterogenous molecular abnormalities defining the progress of the disease, while making it more difficult to treat due to intensive treatments only being feasible for younger patients. Our increased understanding of cancer immunology and the potential of immunotherapy has, however, led to promising therapeutic innovations, which give hope for discovering long-lasting and effective treatment options. Natural killer (NK) cell-based immunotherapies are amongst the emerging novel therapeutic approaches that aim to target malignant cells with less toxicity and improved applicability. Using high-throughput drug sensitivity and resistance testing combined with single cell RNA (scRNA) sequencing, this study focused on finding drug compounds that could synergise with NK cells to improve their effectiveness in killing leukemic cells. In this study, many drugs showed promising results in being able to potentiate NK cell cytotoxicity, with daporinad and pevonedistat showing the most notable differences when compared to controls. The potentiating effect of Janus kinase (JAK) inhibitors also suggested a method of increasing NK cell activity against leukemic cells through downregulation of major histocompatibility complex (MHC) class I molecules. In conclusion, findings shed light on the synergetic potential of drugs and NK cells, giving hope for clinically relevant findings following further validation and testing.
  • 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.
  • Karttunen, Konsta Eemeli (2023)
    Transposable elements (TEs) are repetitive DNA elements that have an autonomous capability of replicating and inserting themselves into new loci within genomes. TEs have often been thought to be a part of the non-functional “junk DNA”, but advances in next-generation sequencing technology has revealed that TEs have rich biochemical functions: Specific TEs bind a large fraction of transcription factors and harbor marks of active chromatin in human genomes. However, there have been few genome-wide functional enhancer activity studies on the role of TEs in gene regulation, especially in the context of human cancers. In normal cellular homeostasis, TE activity is tightly controlled by epigenetic mechanisms. In contrast, the cell state in cancer genomes is often permissive for TE activation: genome instability and mutations, notably p53 inactivation, and nonmutational epigenetic reprogramming such as DNA hypomethylation are common characteristics of cancers. TE transcription, somatic retrotransposition and activation of cryptic promoter elements occur frequently in tumors. TE activation is highly heterogeneous between cancer types: for example gastrointestinal tract cancers such as colorectal cancer show high somatic insertion activity, whereas retrotransposition is rare in hematolymphoid malignancies. Due to the widespread TE activation in cancers, we posited that this may also be seen in the activity of cryptic TE enhancers, with specific active families of TEs characteristic to different cell types due to lineage-specific TF binding. We asked if TEs contribute to the enhancer landscape of cancers and to which extent, what are the differences between cancers in the activity and transcription factor binding and whether TE enhancers may have a role in tumorigenesis and the regulation of cancer-specific genes. To functionally study TEs, we utilized a high-resolution, unbiased, genome-wide massively parallel reporter assay (MPRA). We utilized colorectal and hepatocellular cancer cell lines to study the differences and similarities between TE activation and combined the MPRA data with orthogonal epigenetic data to study the in vivo signatures of the TEs. We found that both cell lines show common and highly enriched TE subfamilies that were mostly specific for p53, as well as TEs that were highly unique in both cell lines. By using in vitro methylated MPRA libraries, we found that CpG methylation has relatively minor a role in regulating the enhancer activity of some TE subfamilies in the reporter assay. By comparing the epigenetic context of the TEs, we found that especially colorectal cancer has specific highly active TE subfamilies with signatures of canonical active enhancers. We also used an in silico model to predict TE enhancer to gene contacts and found that these subfamilies regulated genes that were frequently overexpressed. Thus, we present the widespread functional activity of TE enhancers in cancers, providing evidence for further functional validation of TEs and their effects on transcriptional programs and especially dysregulation of gene expression in cancer.
  • Mölsä, Riikka (2023)
    Lung cancer is the number one cause of cancer-related deaths in the world every year. Of all non-small cell lung cancers, lung adenocarcinoma is the most prevalent subtype. There is a great need for better treatment options for lung cancer, of which cancer immunotherapy is an attractive option due to the high mutational burden of lung tumors. Patient-derived lung cancer organoids (lung PDTOs) could provide a new testing platform for these studies as the 3D models better represent the original tumor and its microenvironment compared to often used 2D cell lines. One interesting field is immunopeptidomics, which focuses on discovering tumor peptides presented in the HLA-I molecule on the tumor surface that could elicit an immune response. Using lung adenocarcinoma PDTOs, this study aimed to analyze the immunopeptidome of five PDTOs to discover tumor-specific and immunogenic peptides using PeptiCHIP purification and LC-MS analysis. These findings could be used in PeptiCRAd, a novel cancer vaccine comprised of an oncolytic adenovirus coated with tumor peptides. To elucidate the applicability of PDTOs for virotherapy, three oncolytic adenoviruses, D102, Ad5/3Δ24 and Ad5Δ24-RFP, and their ability to infect, kill, and replicate in lung PDTOs was studied. PDTOs were characterized as epithelial, as they presented epithelial cytokeratin and epithelial layer structures, as indicated by cytoskeletal F-actin staining. The three oncolytic adenoviruses were studied by infecting PDTOs and a difference in killing capacity of the three viruses was shown, potentially due to differences in receptor interactions and expressed transgenes. In addition, D102 and Ad5Δ24-RFP were shown to replicate in PDTOs, which is necessary to induce a strong enough immune response against the virus for immunotherapy efficiency. HLA-I expression was high in all tested models, which indicated that antigens could be presented in the tumor cells. Immunopeptidome analysis did not result in a high yield of peptides, likely due to challenges in sample preparation and patient material being scarce. As the HLA-type of each patient was unknown during this study, more data analyses still need to be done to determine the best immunogenic peptides, which could then be further studied in vitro. However, peptides overexpressed in lung cancer and with cancer benefiting properties were found from PDTOs, which already gives promising results. In conclusion, though additional immunopeptidome studies with an increased yield of peptides are needed to select tumor-relevant immunogenic targets for therapeutical use, as well as additional testing on the optimal oncolytic virus for lung cancer targeted PeptiCRAd immunotherapy, this study proved that oncolytic viruses can infect and kill lung PDTOs, and that HLA-I expressed tumor peptides can be identified from them. This is also one step towards finding better and patient-specific research models for testing therapies and discovering and developing personalized cancer treatments.
  • Stadelmann, Christian (2019)
    Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by transgenically expressing the four transcription factors OCT4, SOX2, KLF4, and C-MYC. This technology has revolutionised the stem cell field, yet cellular reprogramming is still inefficient and slow. To become fully applicable in regenerative medicine, the robust generation of safe and high-quality iPSCs from patient samples is essential. Various methods and potent reprogramming factors have been described to date. Yet, none have been able to circumvent these limitations markedly. The recently published activator-mediated approach (CRISPRa) is considered to be more physiological compared to the forced transgenic expression as the cell’s own genes are activated. Here, guide RNAs (gRNAs) mediate sequence-specific recruitment of non-cutting Cas9 (dCas9) activator proteins to the promoter region. Unlike other methods, it holds great multiplexing capacity and can also target enhancer and non-coding sequences. CRISPRa reprogramming still needs to be optimised since its efficiency is low. Thus, we aimed at enhancing this aspect and the temporal kinetics by targeting the micro RNA (miRNA) clusters 302/367 and miR-371-373, which both have been described as powerful cell fate regulators. We demonstrate successful reprogramming by targeting the miR-302/367 promoter alongside OCT4, SOX2, KLF4, C-MYC, LIN28A, REX1, NANOG, and EEA-motifs with CRISPRa. Activating the miRNA cluster results in a 2.5 fold efficiency increase in human foreskin fibroblast (HFF) reprogramming compared to the published basal CRISPRa system, quantified by staining for alkaline phosphatase. In HFFs, the CRISPRa efficiency is now comparable to the commonly used transgenic approach. Aiming to clarify the molecular mechanisms of these results, we characterised the expression of direct and downstream targets of miR-302/367 at different time points throughout the reprogramming process. Furthermore, validated with immunocytochemical stainings, the generated bona fide iPSCs express pluripotency markers and spontaneously differentiate into the three germ-layers, both signs of high-quality iPSCs. Beyond that, we report that miR-302/367 activation appears to result in earlier iPSC colony formation resulting in faster proliferating stem cell colonies shown with live-cell imaging. Employing a conditionally stabilised activator construct, we further show that with miR-302/367 targeting, the dCas9 activator expression seems to be required for only a short time period, sufficient to induce pluripotency. At the end of the project, the miR-302/367 cluster targeting was optimised and the best-working gRNAs were selected for further studies, which when combined further increase the CRISPRa-induced expression of the miR-302/367 cluster markedly. All in all, this study demonstrates that non-coding genetic elements like the miR-302/367 cluster can be targeted with CRISPRa, and its targeting significantly improves the reprogramming efficiency. Implications of the study for regenerative medicine and future steps are discussed.