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Browsing by master's degree program "Genetiikan ja molekulaaristen biotieteiden maisteriohjelma"

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  • Aho, Niina (2022)
    Breast cancer is the most prevalent cancer in women worldwide and in 2020 it was the fifth deadliest. In Finland 2019 more than 5000 breast cancer cases were diagnosed, 94% in women and 6% in men. Until now, the high-risk breast cancer susceptibility genes have been identified including BRCA1, BRCA2 and TP53 as well as many of the moderate risk genes. Still, together all the identified genes explain only approximately half of the familial breast cancer cases. Furthermore, all the known breast cancer susceptibility genes are linked to the DNA repair mechanism. Serpina3 stands out as a non-DNA repair gene but as a gene that encodes a protease inhibitor which belongs to the serpin superfamily. Serpina3 has been associated with various diseases before and especially changes in its expression levels are linked to the tumor prognosis in many cancers including breast cancer. However, a previous study proposed that Serpina3 c.918-1G>C is a susceptibility variant for breast cancer in the Northern Finland population. This thesis a case-control study to investigate whether Serpina3 c.918-1G>C variant is associated with breast cancer in the Southern Finland population. In addition, the tumor histology and cellular markers of Serpina3 c.918-1G>C carriers were examined. This study utilized DNA collected from breast cancer patients as well as DNA from blood donors and healthy biobank controls. Breast cancer patients included both familial and unselected cases. The prevalence of Serpina3 c.918- 1G<C variant was studied by genotyping the cases and controls. Genotyping was done by TaqMan real-time PCR and carriers were further confirmed by Sanger sequencing. Moreover, statistical tests were used in the data analyses. The studied Serpina3 c.918-1G>C variant was not found to be significantly (p>0.05) enriched in the breast cancer cases. The variant was found in 0.23 % of familial and 0.36 % of unselected cases, altogether in 0.28 % of all studied breast cancer cases, the frequency in controls was 0.27 %. The tumor histology was found to be ductal in 73 % of the Serpina3 c.918- 1G>C variant carriers and only 9 % had lobular tumor. In other words, the tumor histology followed the usual distribution. All the carriers had a HER2 negative tumor and all except one case were both ER and PR positive. About half of the carriers expressed the cellular proliferation marker Ki67. As a conclusion, the results from this study do not suggest Serpina3 c.918-1G>C as a breast cancer risk variant at least in the Southern Finland population.
  • Kuncheva, Ema (2022)
    Mutations in the KCNQ1 gene have been implicated in the onset of hypopituitarism. Regulating KCNQ1 expression would therefore enable future clinical research on the mechanism of the disease. CRISPR offers a flexible toolset for controlling genetic expression via knockout, knock-in, knockdown, and gene activation. Of these approaches, CRISPR activation (CRISPRa) is distinguished by its ability to induce gene overexpression in a cell’s native context, making it a valuable tool in the interrogation of genetic disorder pathogenesis. This thesis therefore tested the efficacy of a CRISPRa subsystem in increasing KCNQ1 expression. The CRISPRa subsystem, VPR, was chosen because of its high activation efficiency and the ease of controlling the activation system of its doxycycline-inducible mode of action. The cell line used for the experiment, HEK293, was similarly chosen because of its ease of culture and transfection. To validate the proper functioning of the activation system, expression rates of the related genes ASCL1 and GHRH were measured as positive controls. The activation system successfully upregulated the expression rates of the two genes. As the dCas9-VPR system is dependent on the Tet-ON operator for inducing activation in a controllable manner, a test for dCas9 leakage was conducted. RT-qPCR analysis showed the upregulation of ASCL1 expression in the uninduced state of the system, confirming the presence of dCas9-VPR leakage. The dCas9-VPR system finally aimed to test the expression rate of KCNQ1. Although one novel guide RNA successfully upregulated KCNQ1 expression, it did so inefficiently and its success was not shared by any of the other tested guide RNAs. Altogether, the dCas9-VPR system was successfully established in HEK293 cells, and the leakage of the inducible system was confirmed, however, KCNQ1 activation by CRISPRa requires further optimization.
  • Juntunen, Valtteri (2021)
    Adeno-Associated Viruses (AAVs) are quickly becoming one of the most applied vectors for gene therapy applications. In the recent years three new AAV-based gene therapies have been approved by U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). The regulatory bodies require accurate and reliable characterisation of the clinical grade viral vectors during and after production. Analytic methods measuring the purity, potency and safety of the product support the up-stream and down-stream processes during the production and are used for final-drug substance characterisation. Median Tissue Culture Infectious Dose (TCID50) is a well-established method for measuring the infectious titer of a virus. Here, an assay for determining the infectious titer of AAVs, which has previously been used to characterise the existing AAV2 Reference Standard Material (AAV2RSM) was set up and optimised for research use at Kuopio Center for Gene and Cell Therapy (KCT). The assay utilizes the HeRC32-cell line, a HeLa clone, that stably expresses AAV Rep and Cap -proteins and in presence of adenovirus, enables the replication of recombinant AAV-vectors. The cells were grown in 96-well plates and infected with 10-fold dilution series of AAV vectors (AAV2 and AAV6) using human adenovirus type 5 as the co-infector. 72 hours post infection the vector genome replication of AAV was detected with quantitative PCR (qPCR). Thresholds for qPCR determined copy number and cycle threshold (Ct) were set and used for the determination of infection-positive wells. The 50-percent endpoint was observed and used to calculate the infectious titer according to the Spearman-Kärber method. The assay was set up and optimised with the AAV2 Reference standard material (AAV2RSM) using two different primer-probe sets (targeted sequences were; SV40 polyadenylation signal and AAV inverted terminal repeats (ITRs)). Plates infected with AAV2RSM were analysed separately with both primers resulting in mean infectious titers of 8.07 ± 3.13 x 108 TCID50 Infectious Units (IU) / mL (n = 9) and 1.27 ± 0.464 x 109 TCID50 IU/mL (n = 9) for SV40 and ITR, respectively. After the assay was set up with the AAV2RSM, an in-house AAV6 product was analysed with the ITR primers yielding 6.09 ± 3.94 x 109 TCID50 (IU) / mL (n = 5). The assay protocol was successfully set up for research use at the KCT laboratory. Improvements were added to the original protocol to increase assay robustness, accuracy and precision as well as to minimize the possibility of over-estimation of the infectious titer. The assay can be further optimised for a particular therapeutic AAV product in the research laboratory or technology transferred to a production facility for optimisation and validation for the analytics needs of a production pipeline.
  • Oshin, Afsana (2021)
    Tropomyosins (Tpm) are coiled-coil proteins, which wind around actin filaments to form head-to-tail oligomers. Tpms control actin filament growth, movement and interactions with other actin-binding proteins like myosins and cofilin. Tpms play essential role in the construction and stabilization of complex three-dimensional actomyosin contractile structures called stress fibers, because Tpms regulate structural and functional attributes of actin filament populations. In mammals, there are 4 TPM genes encoding above 40 TPM variants, giving rise to many functional Tpm protein isoforms. They are responsible for several physiological mechanisms in cell such as morphogenesis, cytokinesis, vesicle transportation, metabolism, motility, organ development, and signaling. Even though several studies have been conducted to determine structures and functions of various Tpms, many questions are still to be answered about Tpm2.1 and its significance in cells. So far, Tpm2.1 isoform has been a difficult protein to study due to poor success rate at achieving its complete depletion from the cell. Its involvement in cytokinesis, cell movement, cancer progression, and association with mechanosensing ability of cells were recently reported, and this raised the interest of researchers to focus on unveiling its precise cell biological function. Conditional deletion, degradation or inactivation of a protein helps to determine its function in cells. A new revised Auxin Inducible Degron version-2 (AID2) approach employs the cell´s own ubiquitin mediated protein degradation process, ensuring efficient and rapid depletion of target protein by the help of expressing OsTIR1(F74G) auxin-receptor mutant in presence of 5-phenyl-indole-3 acetic acid (5-Ph-IAA) ligand. In this study, we established a pipeline to identify Tpm2.1’s functions using AID2 technology by integrating OsTIR1(F74G) mutant at AAVS1 locus of the homozygous knock-in U2OS clones, containing mAID-msGFP2-TPM2.1 fusion insert at their endogenous TPM2.1 locus. We aimed to deplete Tpm2.1 from cells using this approach by inducing with 5-Ph-IAA and observe the direct, immediate phenotypes during Tpm2.1 degradation. We succeeded in achieving almost complete Tpm2.1 depletion. By this approach, we revealed that Tpm2.1 controls actin reorganization, stress fiber stability, and maturation of focal adhesions in cultured cells. To our knowledge, Tpm2.1 is the first actin-binding protein to be studied using AID2 approach, and the promising outcome brings hope to study other complicated actin-regulating proteins with this approach.
  • Suonto, Lotta (2019)
    Amelogenesis Imperfecta (AI) is a group of hereditary diseases where tooth enamel is abnormally formed. This disease has been found in Lancashire Heeler dogs. However, none of the mutations known to cause AI in canine, explain the disease in the Lancashire Heeler population. A research study was initiated to investigate the genetic background of the disease in the breed. Based on the pedigree data the disease was suggested to be inherited as an autosomal recessive disease. Whole-exome sequencing of two affected dogs was performed which revealed a plausible candidate variant in a gene regulating intracellular calcium concentration. The aim of this study was to examine whether the mutation in the candidate gene causes AI. The DNA were extracted from blood samples of 249 Lancashire Heelers. A genome-wide SNP genotyping was conducted on four cases and a homozygosity mapping was performed. The candidate variant was genotyped in a large cohort of Lancashire Heelers (n=249) and dogs from related breeds (n=91) to confirm the association with the disease. The expression of the candidate gene in lymphocytes was studied with RT-PCR. Clinical studies were performed to evaluate the clinical features and serum calcium levels were measured. The segregation of genotypes and phenotypes of the dogs was complete. The identified variant is predicted to cause a premature STOP codon, which, if translated, leads to a truncated protein lacking protein transmembrane domains. According to the results acquired from RT-PCR, it is possible that the protein will complete the translation despite the mutation. Clinical studies did not reveal other clinical features than enamel defects and serum calcium levels were normal. According to the results, we suggest that the identified variant is causal for AI in Lancashire Heelers. The results have significant impact because no other genetic connection between the gene and AI has been found before. Consequently, the development of a gene test is possible, creating multiple opportunities for researchers and veterinary medicine. The results are applicable also to human medicine. However, we need more research to achieve a complete understanding of the mutation and its effects.
  • Kuitunen, Essi (2019)
    Glutamine, the conditionally essential amino acid, is a major carbon and nitrogen carrier required for a range of cell functions, such as protein synthesis and maintaining redox balance. While healthy cells adjust their activities in response to glutamine availability, tumor cells display deregulated glutamine uptake and metabolism allowing quick proliferation and survival in cellular stress conditions. Hence, further knowledge of the glutamine sensing network is of interest. Utilizing Drosophila melanogaster, the roles of formerly identified glutamine sensing regulator candidates, Forkhead box O (FoxO), Super sex combs (Sxc), Spalt major (Salm) and Spalt-related (Salr), were explored. Drosophila is an efficient model organism for analyzing gene regulatory mechanisms, with its simple genome but conserved genes and metabolic pathways. Loss-of function and gain-of-function mutants of the candidates were cultured with/without glutamine, and their physiological response and gene expression changes were analyzed. The results show the glutamine intolerant phenotype of FoxO and Sxc deficiency, not dependent on altered food intake levels of larvae. However, glutamine intolerance of Salr and Salm deficiency was not observed. Moreover, we aimed to gain further insight to the roles of FoxO and Sxc in glutamine metabolism. Since amino acid catabolism produces ammonia, and glutamine metabolism plays a vital role in ammonia detoxification, we performed a pH-based measurement of foxo and sxc mutant larvae hemolymph on food with/without glutamine. However, we could not associate FoxO or Sxc with regulation of glutamine-derived ammonia clearance. In addition, we explored FoxO downstream regulator candidates. Putative promoter areas of Paics, Uro, Sesn, salr, Prat2 and Gdh were cloned into reporter vectors and the luciferase activity was analyzed under the expression of foxo. The results indicate that FoxO is a regulator of all of the 6 genes. Next we could utilize the here constructed plasmids to see whether the FoxO-mediated regulation is affected by altered glutamine levels in cell culture.
  • Butkovic, Rebeka (2020)
    Autophagy is a cellular recycling and quality control process that eliminates cellular material in a non-selective or selective fashion. Macroautophagy is non-selective, and degrades macromolecules or damaged organelles to sustain cellular homeostasis. The selective autophagy of dysfunctional or excess mitochondria is known as mitophagy. The clinical importance of functional degradation is exemplified by the lysosomal storage disorders (LSDs), where lysosomal hydrolytic enzymes are absent or dysfunctional. Previous investigations of a rare infantile LSD indicated a change in autophagy and decreased mitochondrial content. The aim of this MSc thesis was to quantitatively compare macroautophagy and mitophagy in a cellular model of this rare LSD, by generating fluorescent macroautophagy and mitophagy reporter-expressing cell lines from patient material. Fibroblasts derived from patients diagnosed with a rare infantile LSD were transduced with lentiviruses carrying either mCherry-GFP-LC3 or mito-QC reporters, for the microscopic analysis of autophagy and mitophagy, respectively. I also monitored autophagic flux by traditional biochemistry in untreated and starved cells, in the presence or absence of lysosomal inhibitors (bafilomycin A1). Basal and iron-depletion induced mitophagy was profiled using confocal microscopy, quantitative cell biology and biochemistry. My findings suggest differential autophagic turnover in LSD patient-derived fibroblasts, with a marked accumulation of non-acidified autophagic structures. Basal mitophagy was elevated in two out of three LSD patient cell lines compared to unaffected controls. LSD patient cells exhibited altered mitochondrial content and network architecture compared to controls. These phenotypes were accompanied by distinct changes in the endo-lysosomal system and increased cell size. The patient-derived cells exhibit a profound accumulation of lysosomes and autophagic structures. My findings are in accordance with previous research in the field, suggesting perturbed macroautophagy in this rare LSD. The observations of altered mitochondrial homeostasis in this LSD provide a basis for future investigation. The reporter-expressing cells, generated as part of this MSc thesis project, will enable future studies of mechanisms that underlie phenotypic changes, and will complement essential in vivo work in this area.
  • Peltola, Sanni (2019)
    In recent decades, ancient DNA recovered from old and degraded samples, such as bones and fossils, has presented novel prospects in the fields of genetics, archaeology and anthropology. In Finland, ancient DNA research is constrained by the poor preservation of bones: they are quickly degraded by acidic soils, limiting the age of DNA that can be recovered from physical remains. However, some soil components can bind DNA and thus protect the molecules from degradation. Ancient DNA from soils and sediments has previously been used to reconstruct paleoenvironments, to study ancient parasites and diet and to demonstrate the presence of a species at a given site, even when there are no visible fossils present. In this pilot study, I explored the potential of archaeological sediments as an alternative source of ancient human DNA. I collected sediment samples from five Finnish Neolithic Stone Age (6,000–4,000 years ago) settlement sites, located in woodland. In addition, I analysed a lakebed sample from a submerged Mesolithic (10,000–7,000 years ago) settlement site, and a soil sample from an Iron Age burial with bones present to compare DNA yields between the two materials. Soil samples were converted into Illumina sequencing libraries and enriched for human mtDNA. I analysed the sequencing data with a customised metagenomics-based bioinformatic analysis workflow. I also tested program performance with simulated data. The results suggested that human DNA preservation in Finnish archaeological sediments may be poor or very localised. I detected small amounts of human mtDNA in three Stone Age woodland settlement sites and a submerged Mesolithic settlement site. One Stone Age sample exhibited terminal damage patterns suggestive of DNA decay, but the time of deposition is difficult to estimate. Interestingly, no human DNA was recovered from the Iron Age burial soil, suggesting that body decomposition may not serve as a significant source of sedimentary ancient DNA. Additional complications may arise from the high inhibitor content of the soil and the abundance of microbial and other non-human DNA present in environmental samples. In the future, a more refined sampling approach, such as targeting microscopic bone fragments, could be a strategy worth trialling.
  • Preussner, Annina (2021)
    The Y chromosome has an essential role in the genetic sex determination in humans and other mammals. It contains a male-specific region (MSY) which escapes recombination and is inherited exclusively through the male line. The genetic variations inherited together on the MSY can be used in classifying Y chromosomes into haplogroups. Y-chromosomal haplogroups are highly informative of genetic ancestry, thus Y chromosomes have been widely used in tracing human population history. However, given the peculiar biology and analytical challenges specific to the Y chromosome, the chromosome is routinely excluded from genetic association studies. Consequently, potential impacts of Y-chromosomal variation on complex disease remain largely uncharacterized. Lately the access to large-scale biobank data has enabled to extend the Y-chromosomal genetic association studies. A recent UK Biobank study suggested links between Y-chromosomal haplogroup I1 and coronary artery disease (CAD) in the British population, but this result has not been validated in other datasets. Since Finland harbours a notable frequency of Y-chromosomal haplogroup I1, the relationship between haplogroup I1 and CAD can further be inferred in the Finnish population using data from the FinnGen project. The first aim of this thesis was to determine the prevalence of Y-chromosomal haplogroups in Finland and characterize their geographical distributions using genotyping array data from the FinnGen project. The second aim was to assess the role between Finnish Y-chromosomal haplogroups and coronary artery disease (CAD) by logistic regression. This thesis characterized the Y-chromosomal haplogroups in Finland for 24 160 males and evaluated the association between Y-chromosomal haplogroups and CAD in Finland. The dataset used in this study was extensive, providing an opportunity to study the Y-chromosomal variation geographically in Finland and its role in complex disease more accurately compared to previous studies. The geographical distribution of the Y-chromosomal haplogroups was characterized on 20 birth regions, and between eastern and western areas of Finland. Consistent with previous studies, the results demonstrated that two major Finnish Y-chromosomal haplogroup lineages, N1c1 and I1, displayed differing distributions within regions, especially between eastern and western Finland. Results from logistic regression analysis between CAD and Y-chromosomal haplogroups suggested no significant association between haplogroup I1 and CAD. Instead, the major Finnish Y-chromosomal haplogroup N1c1 displayed a decreased risk for CAD in the association analysis when compared against other haplogroups. Moreover, this thesis also demonstrated that the association results were not straightforwardly comparable between populations. For instance, haplogroup I1 displayed a decreased risk for CAD in the FinnGen dataset when compared against haplogroup R1b, whereas the same association was reported as risk increasing for CAD in the UK Biobank. Overall, this thesis demonstrates the possibility to study the genetics of Y chromosome using data from the FinnGen project, and highlights the value of including this part of the genome in the future complex disease studies.
  • Nykänen, Sonja (2019)
    Colorectal cancer (CRC) kills more than half a million people a year worldwide. Usually the disease develops over several years via multiple steps which involve both genetic and epigenetic alterations. CRC is often diagnosed at late stage, when the cancer has already metastasized, and the prognosis is relatively poor. Several studies suggest that the first changes towards colorectal cancer occur and can be detected in histologically normal tissue before the appearance of any detectable lesion. The precancerous cells harbouring those changes may form a field of tissue, which is predisposed to malignant transformation. The study of pre-cancerous tissue might reveal the earliest changes in CRC development, which can be used as biomarkers for early detection and prevention of CRC. The aim of this thesis was to revise and investigate whether the aberrant expression of the six chromosomal segregation genes, Bub1, Mis18a, Pms2, Rad9a, Tpx2, and Mlh1, would signal carcinogenesis in mouse colon mucosa. Altogether fourteen mice, of which six had a proximal colon carcinoma, were selected for the study. The expression analysis was performed to histologically normal colon mucosa collected from the proximal and distal colon of each mice in order to investigate whether the possible pre-cancerous changes are found exclusively in the close proximity to the carcinoma. The expression was quantified with reverse transcription quantitative polymerase chain reaction (RTqPCR). No statistically significant gene expression differences were found between the carcinoma and control mice, indicating that the studied mice did not display cancer-preceding expression changes of the six studied genes in the carcinoma adjacent histologically normal colon mucosa. The results differed from the previously reported results, where the expressions of the six genes were found to be downregulated in the carcinoma adjacent mucosa. Here, the sample size was presumably not large enough to reveal statistically significant clustering of the expression patterns. However, Bub1 seemed to have a downregulated trend in the carcinoma adjacent mucosa, which supports the previously suggested role of Bub1 alterations in CRC initiation.
  • Mantela, Fanni (2021)
    There are no comprehensive research data on Finnish matriculation examinations in biology. This type of data is needed, because evaluation guides what and how students learn and what they consider important. Genetics is one the most challenging topics in biology, and in the opinion of teachers it will continue to be an important discipline in the future. The importance of studying genetics can also be justified with philosophical, social and health reasons. This is why the present study focused on the genetics component of the matriculation exam in biology. The aim of the study was to provide information on the challenges and contents of past matriculation examinations in biology and how they have aligned with high school curricula. The results of the study could be used to evaluate this alignment in relation to genetics questions in the biology exam, and could help in designing new matriculation examinations that align better with the existing high or new high school curricula and their aims. The research questions were: 1. What knowledge and cognitive dimensions are measured with the genetics-related questions in matriculation examinations in biology? 2. How do knowledge and cognitive dimensions in genetics-related questions in biology matriculation examinations relate to high school curriculum aims? The data comprised matriculation examination papers in biology from spring 2011 to autumn 2020 (20 exams) and the aims of the Finnish national High School Curriculum in 2003 and in 2015. Qualitative content analysis was performed on the knowledge dimensions (factual, conceptual or procedural knowledge) and the cognitive process dimensions (remembering, understanding, applying, analyzing, evaluating or creating). The basis of this qualitative content analysis was Bloom’s revised taxonomy. The analysis was conducted on genetics-related matriculation examination questions and on the aims of the high school curriculum. The test questions and the aims were compared to determine whether they aligned. Classified questions were divided into two subcategories depending on which high school curricula they corresponded to. Genetics-related questions from spring 2011 to autumn 2017 corresponded to the High School Curriculum in 2003 and questions from spring 2018 to autumn 2020 corresponded to the High School Curriculum in 2015. Questions from the previous period were divided into all knowledge dimensions. All questions, except one, incorporated lower cognitive dimensions (remembering, understanding and applying). The main combined class was understanding conceptual knowledge. Questions from the later time period were also divided into all knowledge dimensions. Mostly lower cognitive dimensions were incorporated into the questions, but a few subquestions addressed higher cognitive dimensions (analyzing, evaluating and creating). The main combined class was understanding conceptual knowledge. All the aims were classified into conceptual or procedural knowledge classes. The aims were also divided between all cognitive dimensions, except remembering. Using constructive alignment as the basis for matching aims with questions, two aims in the High School Curriculum of 2003 and six aims in the High School Curriculum of 2015 had no questions that matched them. These aims mostly measured the cognitive dimension of creating. Several aims appeared to incorporate higher cognitive dimensions, but the questions were less well aligned with the aims than with those incorporating lower cognitive dimensions. The results concerning knowledge and cognitive dimensions were mostly as expected. Lower cognitive dimensions were highlighted in genetics-related matriculation examination questions in biology. The challenge of interpretation brought ambiguity to the aims and cumulative levels of cognitive dimensions when aligning questions with aims, as some of the questions aligned with aims did not assess such high cognitive dimensions as would be expected based on the aims, but were nonetheless aligned with them. Furthermore, there may be several reasons behind the absence of the creating dimension in matriculation examination questions. The alignment of questions and aims would be important to consider in the future, because evaluation has a considerable impact on studying.
  • Jäntti, Maija (2020)
    Uterine leiomyomas are benign tumors originating in the smooth muscle cells of the uterine wall. Leiomyomas represent one of the most common tumor types in women affecting up to 80% of pre-menopausal women. Besides having extensive implications on women´s health through the numerous symptoms they cause, leiomyomas are a cause of remarkable financial burden worldwide. Bivalent promoters are defined by the co-occurrence of two histone modifications with opposite functions: trimethylation of lysine 4 on histone 3 (H3K4me3) and trimethylation of lysine 27 on histone 3 (H3K27me3). H3K4me3 is associated with promoters of actively expressed genes, whereas H3K27me3 is frequently found at promoters of silenced genes. The genes controlled by the bivalent promoters are reversibly silenced or expressed at low levels and remain poised for fast activation or full repression as a response to external cues. Bivalent chromatin is gaining more and more importance as new roles are identified in tumorigenesis and cell differentiation. Despite this, the vast majority of data available was obtained from cell lines, and not from human tissue. The aim of this thesis work was to map the genomic location of bivalent promoters in uterine leiomyoma and myometrium tissue, and to characterize the functions of bivalently-controlled genes in differentiated tissue. This would provide novel information about bivalent promoters’ distribution in human tissues and also their potential role in myomagenesis. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) against H3K4me3 and H3K27me3 was performed on fresh frozen tissue samples of uterine leiomyomas and corresponding myometrium. A promoter was defined as bivalent, if it showed overlap between H3K4me3 and H3K27me3 peaks within a 2 kb region of a gene’s transcription start site in all samples. Altogether 951 bivalent promoters were found in myometrium and leiomyoma. Strikingly, only 231 (24.3%) promoters were present in both tissue types, most bivalent promoters being tissue-specific. These findings indicated bivalent promoters regulating a substantial number of genes also in differentiated tissue and the presence of extensive alterations in bivalent promoter distribution during myomagenesis. Gene ontology analyses of the bivalently-controlled genes in myometrium revealed the highest score for developmental processes. Instead, for leiomyomas, the highest enrichment was detected in stem cell fate specification-related processes. The data presented in this thesis suggests that bivalent chromatin plays an important role during myomagenesis, as it undergoes a significant reorganization during the process. Future experiments will provide novel insights about the role for these changes, i.e.: if they underlie the process.
  • Larkiala, Taru (2020)
    Calsyntenin-3 is a type I transmembrane protein, that is mainly expressed on the post-synaptic cell membranes. It belongs to the calsyntenin family that is part of the cadherin superfamily. Calsyntenin-3 consists of a cytosolic C-terminal region, a transmembrane domain and an extracellular N-terminal part, that consists of a laminin G-like domain (LNS) and two cadherin domains (CAD). Calsyntenin-3 is mainly expressed in the brain, but it can also be found in the heart, liver, pancreas, lung, skeletal muscle and placenta. Calsyntenin-3 has an effect on neurogenesis by affecting the development of excitatory and inhibitory synapses. It might also play a role in Alzheimer’s disease, as it has been found to be able to bind β-amyloid peptide, that is known to play a key role in the development of Alzheimer’s disease. Calsyntenin-3 acts as a synaptic adhesion protein, that binds to the post-synaptic neurexins with its extracellular region. However, the previous studies have contradicting results regarding the calsyntenin-3 domains that mediate the interaction between the calsyntenin-3 and neurexins. There is also disagreement whether calsyntenin-3 binds neurexin-α, neurexin-β or both. Because of these discrepancies, the aim of this master’s thesis study was to produce the calsyntenin-3 ectodomain constructs that contained either the two CAD domains, the LNS domain or all three domains, using baculovirus mediated protein production in insect cell cultures. These purified protein constructs were meant to be used for the determination of the binding domains. Unfortunately, only the purification of the calsyntenin-3 LNS domain was successful and the purification of the constructs, containing the CAD domains, was unsuccessful. A SEC-MALLS experiment, that was performed for the calsyntenin-3 LNS domain, revealed that it forms dimers in a solution, which is consistent with experiments performed with the LNS domain of human sex hormone‐binding globulin. The second aim of this master’s thesis study was to express the calsyntenin-3 ectodomain constructs on the surface of HEK293T cells and to test the binding between calsyntenin-3 and neurexins in a cell surface binding assay. The results of the cell surface binding assay indicated that the binding is mediated by the calsyntenin-3 CAD domains and that calsyntenin-3 binds to neurexin-α, but the binding to neurexin-β was not detected. However, the results from the cell surface binding assay were conflicting: the binding between the calsyntenin-3 full ectodomain construct and neurexin-α was not detected, but the binding was detected between calsyntenin-3 CAD ectodomain construct and neurexin-α. Therefore, the cell surface binding assay cannot be considered entirely reliable and should be repeated before making further conclusions.
  • Olgac, Ezgi (2022)
    Background– The BCL-2 protein family members are major regulators of apoptosis, and the anti-apoptotic (pro-survival) members of the family is commonly targeted with BH3 mimetic drugs in haematological cancers. However, these treatments have not been very impactful when administered as single agents and they have long been investigated for combination therapy with other agents. Acute myeloid leukaemia (AML) is one of the difficult-to-cure haematological malignancies. A recently approved therapy for AML consists of the combinatorial administration of venetoclax (a selective BCL-2 inhibitor) and a DNA methyltransferase (DNMT) inhibitor such as azacitidine or decitabine. Although this novel therapy has shown promising clinical results, the majority of the patients still relapse under this treatment. These relapsed patients typically become highly resistant to treatment and have poor prognosis, emphasising the need for new effective drug combinations. Apart from BCL-2, other family members like BCL-xL and MCL1 are also common targets of BH3-mimetic drugs. This project thus aims to understand and characterise the resistance against BH3-mimetics and investigate new therapeutic approaches to overcome the challenges of resistance. Aims– This study aims (i) to characterise BH3-resistant AML cell lines for uncovering the mechanisms of drug resistance, and (ii) to identify possible combination treatment options for overcoming drug-resistance. Methods– Viability assays with Cell Titer Glo® (CTG) and Drug Sensitivity and Resistance Testing (DSRT). The long-term effectiveness of venetoclax, azacitidine and talazoparib (a PARP inhibitor) as single agents, double combinations and triple combination were investigated with Time-to-Progression (TTP) assay. For the resistant cell line models, underlying resistance mechanisms were assessed by checking protein expression of pro- and/or anti-apoptotic members of the BCL-2 family members with western blot (WB). Real-time quantitative PCR (RT-qPCR) and WB were carried out for transcriptional and translational expression analyses of certain DNA damage-associated genes in PARP inhibitor-resistant cell lines. Results– Drug screening with DSRT has revealed promising results for two combination treatments of a BCL-xL inhibitor (A-1331852) (i) with an Aurora kinase A inhibitor (alisertib) and (ii) with an MCL1 inhibitor (S63845) for BCL-xL inhibitor-resistant cells. WB analyses of BCL-2 family members showed translational upregulation of un-inhibited members of the anti-apoptotic proteins in BH3-mimetic-resistant cell lines. A venetoclax-resistant AML cell line showed increased levels of the DNA damage marker P-γ-H2Ax upon treatments containing venetoclax, as well as increased levels of cleaved-PARP1, indicating induction of apoptosis. RT-qPCR analyses revealed increased mRNA expression of PARP1 in two resistant cell lines, whereas no significant expression changes in other DNA repair mechanism genes on the transcriptional level. Conclusions– In BH3-mimetic-resistant AML cell lines, apoptosis is avoided through translational upregulation of un-inhibited anti-apoptotic members of the BCL-2 family, and this resistance can be countered by combination treatment for additional inhibition of the compensatory anti-apoptotic proteins. Venetoclax is still effective on cells resistant to it, by inducing DNA damage and sensitising these cells against inhibitors of the members of DNA repair pathway. The transcriptional upregulation of PARP1 and the increase in its auto-catalytic activity suggests the DNA damage-inducing effects of the triple combination treatment [Ven + Aza + Tal].
  • Reinikka, Siiri (2020)
    Endometrial polyps are one of the most common benign uterine lesions, affecting approximately 10% of all adult women. While endometrial polyps have a high prevalence, their molecular pathogenesis and genetic background are largely undefined. Accordingly, the aim of this thesis was to characterize the somatic mutational landscape of endometrial polyps – to identify mutations in cancer-associated genes, and to identify mutational signatures contributing towards the somatic mutational spectrum. The present study was conducted using whole exome sequencing of 23 endometrial polyps and 18 matching normal blood samples. Mutational signature analysis was conducted using MutationalPatterns and SigProfiler. Endometrial polyps were found to carry varying number of somatic mutations in their exomes, most of them present at a low allelic fraction. Moreover, 43% (10/23) of the polyps were identified to carry one to four cancer-associated mutations, including mutations in genes such as PIK3CA 17% (4/23), KRAS 13% (3/23) and ERBB1 9% (2/23), which are well-established cancer driver genes. Cancer-associated mutational signatures do not have a notable contribution towards the somatic mutational spectrum of endometrial polyps. However, a novel signature, ‘signature B’, characterized by T>G mutations, was found to affect a subset of polyp samples. To conclude, the whole exome sequencing of endometrial polyps revealed several mutations in cancer-associated genes and a novel mutational signature, which may contribute to the development of these benign tumours. However, further research is required to confirm and validate the novel signature, and to define the genetic alterations leading to the polyp pathogenesis.
  • Lamichane, Nicole (2019)
    Over the past years sugar consumption has seen great increases worldwide, together with a rise in the prevalence of metabolic diseases. There is a growing need for a comprehensive characterisation of the genes involved in sugar metabolism, yet the mechanisms by which cells sense and respond to sugars in vivo have remained incompletely understood. Here, I analyse members of a protein family best known for their regulation of differentiation during development with regards to their role in sugar metabolism. The Hairy and Enhancer of Split (HES) protein family are a group of basic helix-loop-helix (bHLH) transcription factors that function as major downstream effectors of the Notch signalling pathway. In mammals, the HES proteins have mostly been studied for their role in cell differentiation, but HES1 has been implicated in metabolic control. Drosophila has several transcription factors belonging to the HES family, including Hairy and seven bHLH transcription factors located in the Enhancer of split complex (E(spl)-C). The E(spl)-C bHLH transcription factors display high homology and are considered to be genetically redundant, and therefore little is known about their individual functions. The other HES family members in Drosophila have not previously been linked to metabolic regulation, but Hairy has been shown to repress the tricarboxylic acid cycle. In light of the findings implicating HES1 and Hairy in the regulation of metabolism, I systematically investigated the role of the HES transcription factors in sugar metabolism. By using the GAL4/UAS system in Drosophila melanogaster, I knocked down gene expression of each of the family members, and raised the flies on diets varying in sugar content to identify possible sugar intolerance phenotypes. Here, I show that knockdown of one of the E(spl)-C bHLH genes led to severe sugar intolerance that affected both survival and organismal growth, but did not alter the levels of circulating carbohydrates and storage lipids as measured with colorimetric assays and lipid staining. Furthermore, I identify the tissues in which this transcription factor functions to provide sugar tolerance. Using analysis of publically available chromatin-immunoprecipitation sequencing data coupled with quantitative RT-PCR, I uncover mTOR target Thor/4E-BP as a putative target gene. Additionally, I show that Hairy is similarly required for complete sugar tolerance, but that the mechanism differs from the E(spl)-C bHLH transcription factor. Hairy binds to and positively regulates expression of genes involved in glycolysis and the pentose phosphate pathway, suggestive of a cooperation with earlier known regulators of sugar sensing. In conclusion, I have shown that only two HES family members are involved in the regulation of sugar metabolism and that their regulatory mechanisms are distinct, implying that the HES family members have more diverse roles than previously assumed.
  • Puutio, Johanna (2020)
    Extracellular vesicles (EVs) are phospholipid bilayer-enclosed nanoparticles that are secreted by eukaryotic and prokaryotic cells. EVs carry macromolecules and signalling molecules to adjacent cells and play an important role in intercellular communication under both pathologic and homeostatic conditions. Therefore, they have become of significant interest for their therapeutic, diagnostic and prognostic potential. EVs are small and highly heterogeneous in size, shape, cargo and membrane composition, posing several challenges for establishing analytical and clinical guidelines. Therefore, EV research requires standardized and robust methods for their separation and characterization. In this study physical and immunochemical methods were employed to characterize human platelet-derived EVs (pEVs) generated from platelets activated with different external biochemical stimuli. The platelet-activating effect of the pro-inflammatory S100A8/A9 protein complex and a combination of thrombin and collagen were studied with nano flow cytometry. The size distribution of pEVs was studied with nanoparticle tracking analysis (NTA) and asymmetrical flow field-flow fractionation (AF4), which represents a newly emerging method on the EV field. Finally, fluorescent labelling and co-localization analysis were employed to characterize membrane marker composition of pEVs and assess its usefulness as an analytic tool for EV research. We succeeded in providing new hints towards meaningful discoveries in platelet biology by characterizing the way platelets respond to inflammatory and hemostatic signals by shedding pEVs. When platelet activation markers are characterized with flow cytometry, the S100A8/A9 protein appeared to cause a shift in membrane activation markers when compared to the thrombin- collagen mix and the baseline control. Increased TLT-1 translocation and decreased integrin αIIbβ3 expression on pEV surfaces suggests that S100A8/A9 induced pEV secretion through differently packed platelet α-granules, rather than from the plasma membrane. An increase in TLT-1 expression compared to decreased P-selectin and αIIbβ3 suggests that S100A8/A9 stimulation shifts platelet phenotype towards secretion rather than aggregation. A protocol for small pEV separation with AF4-MALS was set up. With this method, subtle differences between small pEV populations were seen that were not distinguishable with NTA or flow cytometry. When investigated with AF4-MALS, S100A8/A9 induced pEVs appeared larger than those produced with thrombin- collagen activation. The mean particle sizes of the pEV populations obtained from activated platelets were generally also larger than those produced without an activator. We tested novel methods to detect subtle differences in small EV population sizes that are easily missed with conventional methods due to their technical limitations. A well-optimised AF4 protocol can detect different pEV subpopulations and is a promising tool for EV. In the future, when AF4 is combined with a MALS detector and a fraction collector, nanoimaging of fluorescently labelled EVs could be combined with it as a downstream application to obtain information on their versatile biological functions.
  • Elomaa, Ellinoora Juulia (2020)
    The human cerebral cortex is characteristically large and folded, which can be majorly attributed to the high number and variety of neural progenitors during embryonic development. Radial glial cells are essential neural progenitors during neurogenesis. In addition to giving rise to new cell types, they also provide scaffold for migrating newborn neurons. Radial glia are known to portray peculiar characteristics in their cell division process, including unique migratory behavior as well as specifically regulated cleavage furrow orientation. While these processes of radial glial division have been studied extensively, the underlying molecular mechanisms are still largely unknown. ABBA (actin-bundling protein with BAIAP2 homology) and NEDD9 (neural precursor cell expressed, developmentally downregulated 9) are proteins, which are both known to be expressed in certain radial glia progenitors during embryonic development, while they are mainly absent in neurons. ABBA has a defined role of regulating plasma membrane deformation and actin polymerization in radial glia, while NEDD9 expression levels are a known factor in the correct progression from mitosis to cytokinesis. An interaction between ABBA and NEDD9 has previously been identified in a yeast two-hybrid screen done for the embryonic mouse brain. The aim of this thesis was to validate the interaction between ABBA and NEDD9 biochemically. First, their interaction was evaluated by doing co-immunoprecipitation assays on the endogenous proteins from C6 cells. The second approach was to test, whether their interaction is directly mediated by the N-terminal SH3-domain of NEDD9 and the proline-rich C-terminal portion of ABBA. This was done by doing biochemical binding assays using purified proteins and domains of interest. While co-immunoprecipitation of the two proteins gave results indicating an interaction, I could show that there is no direct binding between NEDD9 SH3-domain and ABBA, suggesting that the interaction might require other domains or be indirect. Together, these results provide valuable information that will help characterize what roles of ABBA and NEDD9 play in cortical development and beyond.
  • Tiusanen, Ville (2021)
    Enhancers are important regulatory elements of DNA, that are bound by transcription factors (TFs) to regulate gene expression. Enhancers control cell type specific gene expression and they can form structures called super-enhancers, that consist of multiple normal enhancers and are bound by high numbers and variety of transcription factors. These super-enhancers are important for defining cell identity and changes in the super-enhancer landscape have been linked to different cancers. In this project, characterization of super-enhancers and their transcription factors composition between primary and cancer cells were studied using genome-wide next-generation sequencing data from multiple assays, such as ChIP-seq, RNA-seq and ATAC-seq. The focus of the project was on the data processing and analysis to identify and characterize the super-enhancers. Analyses included GSEA, heatmap binding analysis, peak and super-enhancer calling and IGV analysis. This project used pancreatic HPDE cell line for primary cells and different cancers with endodermal origin as cancer cell lines. The goal of the thesis was to try show characteristic features of super-enhancers and their features in normal and cancer cells. Data analysis showed that distinct super-enhancers can be identified in cancer cells and defined super-enhancers had typical strong binding for specific transcription factor and histone modification such as histone 3 lysine 27 acetylation (H3K27ac) mark of active enhancers. Super-enhancer regions were located in highly accessible chromatin regions of the genome, and genes that were associated with HPDE super-enhancers could be shown to have association with cell identity. Peak and super-enhancer calling counts varied between cell lines for transcription factors, histone modifications and super-enhancers. Visualization of super-enhancers was successful and could show transcription factor binding and active enhancers that establish the super-enhancer structure. Comprehensive analyses allowed us to characterize typical features of super-enhancers and show differences in the numbers of super-enhancers between primary and cancer cell lines and cancer cell lines of different organ types. Analysis of the transcription factor binding showed unique peaks on some of the super-enhancers, and these peaks might have a role in inducing the super-enhancer structure.
  • Jokinen, Vilja (2021)
    Uterine leiomyomas are benign smooth muscle tumors arising in myometrium. They are very common, and the incidence in women is up to 70% by the age of 50. Usually, leiomyomas are asymptomatic, but some patients suffer from various symptoms, including abnormal uterine bleeding, pelvic pain, urinary frequency, and constipation. Uterine leiomyomas may also cause subfertility. Genetic alterations in the known driver genes MED12, HMGA2, FH, and COL4A5-6 account for about 90 % of all leiomyomas. These initiator mutations result in distinct molecular subtypes of leiomyomas. The majority of whole-genome sequencing (WGS) studies analyzing chromosomal rearrangements have been performed using fresh frozen tissues. One aim of this study was to examine the feasibility of detecting chromosomal rearrangements from WGS data of formalin-fixed paraffin embedded (FFPE) tissue samples. Previous results from 3’RNA-sequencing data revealed a subset of uterine leiomyoma samples that displayed similar gene expression patterns with HMGA2-positive leiomyomas but were previously classified as HMGA2-negative by immunohistochemistry. According to 3’RNA-sequencing, all these tumors overexpressed PLAG1, and some of them overexpressed HMGA2 or HMGA1. Thus, the second aim of this study was to identify driver mutations in these leiomyoma samples using WGS. In this study, WGS was performed for 16 leiomyoma and 4 normal myometrium FFPE samples. The following bioinformatic tools were used to detect somatic alterations at multiple levels: Delly for chromosomal rearrangements, CNVkit for copy-number alterations, and Mutect for point mutations and small insertions and deletions. Sanger sequencing was used to validate findings. The quality of WGS data obtained from FFPE samples was sufficient for detecting chromosomal rearrangements, although the number of calls were quite high. We identified recurrent chromosomal rearrangements affecting HMGA2, HMGA1, and PLAG1, mutually exclusively. One sample did not harbor any of these rearrangements, but a deletion in COL4A5-6 was found. Biallelic loss of DEPDC5 was seen in one sample with an HMGA2 rearrangement and in another sample with an HMGA1 rearrangement. HMGA2 and HMGA1 encode architectural chromatin proteins regulating several transcription factors. It is well-known that HMGA2 upregulates PLAG1 expression. The structure and functionality of HMGA2 and HMGA1 are very similar and conserved, so it might be that HMGA1 may also regulate PLAG1 expression. The results of this study suggest that HMGA2 and HMGA1 drive tumorigenesis by regulating PLAG1, and thus, PLAG1 rearrangements resulting in PLAG1 overexpression can also drive tumorigenesis. A few samples, previously classified as HMGA2-negative by immunohistochemistry, revealed to harbor HMGA2 rearrangements, suggesting that the proportion of HMGA2-positive leiomyomas might be underestimated in previous studies using immunohistochemistry. Only one study has previously reported biallelic inactivation of DEPDC5 in leiomyomas, and the results of this study support the idea that biallelic loss of DEPDC5 is a secondary driver event in uterine leiomyomas.