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Browsing by study line "Genetiikka ja genomiikka"

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  • Borgman, Vesa Petteri (2023)
    Intestinal stem cells maintain the regenerative potential of intestinal epithelium, which needs to be renewed constantly. Dysregulation of intestinal stem cell proliferation is associated with aging and intestinal diseases. The midgut of Drosophila melanogaster is a valuable model for studying intestinal stem cell driven tissue regeneration. It is similar in function to the mammalian small intestine but has a simpler cellular structure. Drosophila midgut is divided into five major regions with specialized physiological functions, characteristic morphological features and distinctive gene expression profiles. The midgut consists of a monolayer of absorptive enterocytes, small secreting enteroendocrine cells, intestinal stem cells and enteroblasts. Intestinal stem cells differentiate into enterocytes through a transient enteroblast phase. 5-hydroxytryptamine has been shown to activate proliferation of intestinal epithelium in mammals, but its mechanism of action is still unknown. Also, sex-specific differences in 5-hydroxytryptamine signalling have been recorded in mammals. 5-hydroxytryptamine signalling pathway has several downstream targets that have diverse downstream effect. Thus, 5-hydroxytryptamine signalling creates a complex and versatile regulatory network. The aim of my thesis is to study the effects of 5-hydroxytryptamine signalling on intestinal stem cell proliferation and cellular turnover in Drosophila midgut in both sexes. The effects of excessive amounts of 5-hydroxytryptamine are first studied by feeding Drosophila with 5-hydroxytryptophan, a product of the rate-limiting step in the 5-hydroxytryptamine synthesis pathway. The effects of 5-hydroxytryptamine signalling are further studied by knocking down and overexpressing a component of the 5-hydroxytryptamine signalling pathway in intestinal stem cells and enteroblasts. Dissected midguts are immunofluorescently stained, imaged and analysed both visually and with bioinformatics tools. The results indicate that 5-hydroxytryptamine signalling has both regional and sex-specific functions that affect intestinal stem cell proliferation and cellular turnover in Drosophila midgut. The most dramatic effects are seen in cellular turnover, which indicates that 5-hydroxytryptamine signalling plays a role in enteroblast differentiation. Furthermore, the results suggest that bidirectional signalling between enteroblasts and dying enterocytes facilitates cellular turnover in the midgut. As 5-hydroxytryptamine signalling is indicated in inflammatory bowel diseases such as Crohn’s disease, my results might help in the development of treatments for such conditions.
  • 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.
  • Veltman, Laurens (2024)
    Cell-based immunotherapies offer highly targeted treatment, potentially leading to higher response rates and reduced long-term side effects compared to chemotherapy. In the clinics, chimeric antigen receptor T-cell therapies are already being used as neo-adjuvant therapy for certain types of cancer, however, they can have significant drawbacks. Natural killer (NK) cells have emerged as a promising alternative option for targeting various hematological and solid tumors. Unlike T-cells, NK-cells do not need prior sensitization to the target and have the potential to be used as a allogeneic, ‘Off-the-shelf’, product. Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Chemotherapy and small molecule drugs are typically used for the treatment of AML. However, the prognosis for relapsed or refractory AML patients remains unsatisfactory. Therefore, a collaborative project between the Helsinki Central Hospital and the Advanced Cell Therapy Center from the Finnish Red Cross, Blood Service was initiated to develop clinical-NK products targeting high-risk AML patients in Finland. A reliable and suitable in vitro cytotoxicity assay was required to assess the efficacy of NK cells before they could be given to AML patients. A luminescence-based method utilizing cellular ATP, was found to be the best performing method considering constraints such as the limited amount of patient material. AML blasts were successfully isolated from the whole blood of patients using CD33 microbeads and maintained in a composition of cytokines mix in RPMI media. The cytotoxicity assay paired with statistical analysis was able to identify significant differences in cytotoxic efficacy between NK cells from different donors. Additionally, results indicate improved cytotoxic efficacy in activated NK cells compared to non-activated NK cells, highlighting the usefulness of activated NK cells for use in the clinics.
  • 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.
  • Laukkanen, Ida (2024)
    Epigenetics is the study of changes in gene expression without alterations in the DNA sequence. Epigenetic modifications, of which DNA methylation (DNAm) is the most known, are crucial for many biological events, especially for normal development and genomic imprinting. In imprinted genes, only one of the parental alleles is consistently monoallelically expressed. The epigenome can be altered by various environmental factors such as diet and chemicals. Moreover, evidence indicates that assisted reproductive technology (ART) is associated with distinct DNAm patterns. Still, it is unclear whether these alterations are a consequence of ART procedures themselves or the underlying subfertility. This thesis aimed to study whether ART procedures and subfertility are associated with aberrant DNAm at the imprinted DLK1-DIO3 locus in the human placenta. The genes encoded from this paternally imprinted locus are essential in mammals' fetal and placental development. Moreover, recent evidence suggests that downregulation of the DLK1 gene is associated with ART and subfertility. Therefore, two gene regions, the IG-DMR and the DLK1 promoter, were chosen as study objects. The IG-DMR acts as the main regulator of gene expression at the whole DLK1-DIO3 locus, and promoter regions are considered important regulators of the expression of their corresponding genes. Eight ART, four subfertility, and six control samples of human placental DNA were studied. The subfertility group consisted of couples who were committed to initiating fertility treatments but eventually got pregnant spontaneously. The study was performed using traditional bisulfite sequencing, after which the differences in DNAm levels between study groups were statistically analyzed. As expected, significantly decreased DNAm level was observed in the placentas of subfertile couples compared to controls. Surprisingly, no significant differences were addressed between ART and control groups in either region. The partly unexpected results are explained by the fact that aberrant methylation at distinct imprinted DMRs, including the IG-DMR, is caused by the in vitro culture media. Moreover, the results indicate that the regulation of DLK1 expression is more complicated than that solely by the IG-DMR and the DLK1 promoter. Further research should be dedicated to differentiating the impacts of ART and subfertility on the epigenome and phenotype to better understand the health implications of ART.
  • 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.
  • Sirjala, Janika (2023)
    Human induced pluripotent stem cells (hiPSCs) are derived from adult differentiated somatic cells and reprogrammed to an embryonic-like state. Pluripotent stem cells can be differentiated into almost any somatic cell type by using directed differentiation methods, but the differentiation efficiency often varies depending on the cell type. hiPSCs and cells differentiated from them can be used as a disease model carrying the patient’s phenotype and genotype. Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease where both upper and lower motor neurons degenerate, leading to paralysis. There is no curative treatment for ALS, and it leads to the death of the patient in 3 to 5 years on average from the first symptoms. The most common genetic cause of familial ALS is a hexanucleotide repeat expansion in C9orf72-gene. ALS pathology is strongly linked to endoplasmic reticulum (ER) stress, which affects cell homeostasis and proteostasis, and leads to apoptosis when prolonged. The primary aim of this research is to characterize the differentiation of four hiPSCs lines towards lower motor neurons and to study the neuroprotective effects of cerebral dopamine neurotrophic factor (CDNF) and CDNF-derived peptide on ER stress and cell viability. This experiment used two control cell lines from two healthy donors and two patient cell lines from two different ALS patients carrying the C9orf72-mutation. To evaluate the efficiency of the differentiation towards motor neurons, molecular markers for pluripotent and neural progenitor cells as well as for maturated motor neurons were analyzed. Relative gene expression levels were measured from weekly time points with qPCR. Immunocytochemical (ICC) antibody staining was performed during differentiation. Endogenic CDNF levels were analyzed from differentiating cells at weekly time points and the effect of CDNF on Thapsigargin (TG) -induced ER stress in motor neurons was analyzed. In addition, cell viability was analyzed in TG-CDNF treatment. All pluripotent and progenitor markers were downregulated in differentiated cells, and the expression of the mature motor neuron markers was upregulated. Mature motor neuron markers were also expressed at the protein level. The endogenous CDNF levels were highest at the progenitor cell stage. The ER stress response was upregulated in TG-treated cells, and there were no differences between treatments against ER stress. Furthermore, TG and growth factor treatments differentially affected the viability of the control and patient cell lines. Treatment decreased viability in control cell lines and increased viability in patient cell lines. Pluripotent stem cells were successfully differentiated toward motor neurons. The differentiation was performed twice, and the results were similar on both individual biological repeats. Analysis of endogenous CDNF expression levels was performed for the first time on hiPSCs lines. In this study, CDNF or its derivate didn’t reduce ER stress but it influenced cell viability, especially in patient cell lines with growth factor treatment. In the future, TG-treatment could be optimized regarding timing and growth factor treatment, or the toxin could be changed to another ER-stress inducing toxin. In addition, the C9orf72 pathology should be identified in order to use differentiated motor neurons as a pre-clinical disease model.
  • Vallo, Tuuli (2024)
    The proper differentiation of cells in early human development is essential for the success of a pregnancy. The first cell-fate decision occurs when totipotent cells differentiate into inner cell mass and trophectoderm, forming a blastocyst. Trophoblast cells differentiate from the trophectoderm and form the placenta. Defects in trophoblast development can lead to several pathologies, such as preeclampsia, miscarriage and intrauterine growth restriction. TFAP2C is a gene that is known to have an important role in the differentiation of trophoblast, although its function is not completely understood. Recent studies have suggested that TFAP2C has a promoter that has not been previously annotated, and it is not yet known what functions different TFAP2C promoters have. The aim of this thesis is to characterise how the activation of this novel TFAP2C promoter or the consensus promoter affects TFAP2C expression and whether the activation of different promoters changes how human pluripotent stem cells differentiate. In addition, this thesis aims to detect whether different TFAP2C protein variants, which are produced by these promoters, explain the possible variation in differentiation. TFAP2C promoters were activated with CRISPR activation, and different TFAP2C variants were expressed as transgenes in pluripotent stem cells. Gene expression was studied with immunocytochemistry and quantitative reverse transcription PCR. The activation of the consensus promoter increased TFAP2C expression more than the activation of the novel promoter. However, activation of both and expression of the protein variants produced from them made cells differentiate into trophoblast-like cells and express trophoblast markers. Nonetheless, the novel promoter and protein variant seemed to differentiate cells into trophoblast more efficiently. Because the activation of the promoters and the expression of their corresponding protein variants led to similar results, it seems likely that the alteration in differentiation is caused by the different TFAP2C protein variants. The functional difference between variants might be affected by the presence of a SUMOylation motif in the wild-type TFAP2C. These results may help to better understand the role of TFAP2C in human embryonic development and create applications for assisted reproduction and infertility treatments, as well as facilitate the development of cell models that can be used in research and therapy.
  • Larmala, Saara (2023)
    The field of gene technology, which falls under the umbrella of biotechnology, presents challenges in business development and commercialisation. Understanding the field characteristics is crucial for successful commercialisation, as it can significantly impact the available strategies for bringing products or services to market, ultimately shaping the business model. This study aims to investigate and understand the challenges associated with commercializing gene technology, including identifying any typical challenge profiles specific to the field and possibly arising from the biological material. The research involves semi-structured interviews with multiple companies, venture capitals, and experts in the field to gain a comprehensive understanding of the challenges. The collected data is then analysed to identify common characteristics and business practices against a commercialisation model frame. The motivation behind this study is to provide researchers and other stakeholders with insights into the challenges they may face while commercializing gene technologies, with the aim of lowering the threshold for business creation. The findings reveal that there are two major groups of companies, each with their specific challenges. The challenges for the major group revolve around business know-how, HR, and sales, while the minor group faces challenges related to technology and regulation. However, a common theme is the limited market awareness among customers, which requires significant efforts in sales, marketing, and communications. The study provides guidance to company founders on the different challenges they should be prepared for and offers insights to society on how to harness the value of gene technologies.
  • 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.
  • Lukander, Volter (2022)
    Spinal muscular atrophy of Jokela type (SMAJ) is an autosomal dominant motor-neuron disease caused by a missense mutation c.197G>T, p.G66V in the gene CHCHD10. Coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10) is a nuclear-encoded mitochondrial protein located in the intermembrane space (IMS) of mitochondria with an unknown exact function and disease-causing mechanism. In this project, the overarching aim was to correct a heterozygous SMAJ-causing mutation in patient myoblast cells with CRISPR-Cas9 genome editing. The goal was to create a genetically identical, isogenic, cell line to study only the effects of the mutation on cellular phenotype in vitro. Human myoblast cells isolated from patient biopsies provide the most pertinent experimental model to study neuromuscular atrophy-associated mutations in their natural genomic environment. More specific aims included genome editing optimization with myoblast cells, since it is not as widely conducted as with some other cell types, such as iPSCs. CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology-directed repair (HDR) in the genome of patient-derived myoblast cells and correct the mutation. After optimization of electroporation conditions for myoblast cells, guide RNAs were designed and transfected into patient myoblasts. Clonal cell lines were made by utilizing techniques such as fluorescence adjusted cell sorting (FACS) and manual colony picking. The success and precision of genome editing were analyzed by Sanger sequencing, comparing the performance of the different guide RNAs with restriction enzyme analysis and Synthego ICE CRISPR web tool, and screening regions of potential off-target genome editing. A genome-edited myoblast cell line with the CHCHD10 c.197G>T mutation corrected, was successfully generated to provide an isogenic control for the patient myoblast cell line. Optimization of myoblast electroporation was successful and conditions used proved to be effective. Clonal cell line creation proved to be challenging with myoblast cells and work is still needed to improve the viability of single-cell clones after FACS. Nevertheless, the advances taken here regarding myoblast genome editing with CRISPR-Cas9 offer a fertile avenue for future research of myoblasts genome manipulation, myogenic disorders, and the role of CHCHD10 in skeletal muscle and SMAJ. Comparing the CHCHD10 protein level and mRNA expression between patient cells, corrected myoblasts, and differentiated myotubes is an area of future research. Future work also includes measuring the mitochondrial integrated stress response in both cell lines and co-culturing myotubes and iPSC derived motor neurons to study the effects of p.G66V on neuromuscular junction (NMJ) formation.
  • Keskinen, Timo (2020)
    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited autosomal dominant disease that leads to cognitive impairment, vascular dementia and ischemic strokes. In CADASIL, vascular smooth muscle cells (VSMCs) degrade gradually and are replaced by connective tissue in the small and mid-sized arteries in the brain. Extracellular granular osmiophilic material (GOM) that surround the VSMCs are a unique feature in CADASIL. The causal gene behind CADASIL is Notch3, which encodes a transmembrane protein with a signaling function. There are over 200 cysteine-altering mutations that cause CADASIL in Notch3. The potential pathology causing mechanism is still unclear, but most likely the mechanism is linked to the aggregation of GOM deposits that are potentially toxic to VSMCs. This thesis project aimed to correct CADASIL causing c.475C>T mutation in Notch3 in different CADASIL cell lines with different CRISPR base editor systems. Another aim was to create induced pluripotent stem cell (iPSC) lines from a CADASIL patient-derived skin biopsy sample to be used in the creation of an in vitro disease model for CADASIL. RNA-based ABEmax base editor system was used to correct immortalized- and primary- CADASIL cell lines. DNA-based ABEmax base editor system was used as a positive control. Simultaneous pluripotent reprogramming and pathogenic CADASIL mutation correction were done in the same transfection during this project. The editing efficiencies were evaluated by Sanger sequencing the genomic target region before and after the transfection. The editing efficiencies were good in general compared to literature. They ranged from 27 % to 73 % target base editing efficiency depending on the editing system-, guide-RNAs - and electroporation parameters used. Confirmed proximal off-target effects were not detected, and distal off-target effects were not evaluated.
  • Forsén, Robin (2024)
    ASH1L is a Histone lysine methyltransferase belonging to the KMT family, which plays an important role in epigenetic gene regulation during development, and has been linked to neurodevelopmental disorders (NDDs). Mutations in ASH1L have been linked to NDDs including intellectual disability, autism spectrum disorder and Tourette’s syndrome. Induced pluripotent stem cell (iPSC) based models in combination with CRISPR/Cas9 gene editing provide powerful tools for studying the genetic causes of NDDs. The broad aim of this thesis was the creation of genetically modified iPSC lines for modelling NDDs linked to ASH1L. Patient and healthy cell lines were obtained from the Northern Finland Intellectual Disability cohort. With the long-term goal of generating a model by which to understand the impact of genetic background on reported causative mutations, CRISPR/Cas9-based genetic engineering was employed to correct the mutation in a patient cell line, and conversely, to generate a patient mutation in a healthy line. iPSC lines are known to be intrinsically variable and require thorough characterization of their genetic stability and pluripotency before use. Therefore, the secondary aim of this thesis was to subject newly reprogrammed iPSC lines to a battery of assays to first determine their suitability for downstream applications. Single-guide RNAs (sgRNAs) were designed to target a site ≤16 bp from the edit site. Single-stranded oligodeoxynucleotides (ssODNs) were used as HDR templates, incorporating the mutation of interest and 3-4 silent mutations to prevent binding by sgRNA after successful HDR. The Cas9-sgRNA complex and HDR template were introduced into the cell by nucleofection. Both mutations are frameshift mutations and are predicted to cause loss of function. Editing efficiency was evaluated with a T7E1 assay after nucleofection. Individual clones were isolated and MiSeq was used to sequence the region to a read depth of >1000reads per clone around the edit site to identify successful edits in these clones that can be used in downstream NDD modelling applications. Edit efficiencies were found to vary between sgRNAs and cell lines. In the correction attempts, guides were found to be almost entirely ineffective, producing only a single successfully edited clone among the combined 192 isolated clones. In the knock-in lines, both guides were effective at producing edited clones. The knock-in guide with the highest predicted efficiency and the shortest edit distance predictably produced the highest number of edits, but also a higher number of homozygotic knock-ins.
  • Alajoki, Reetta (2023)
    Uterine leiomyomas (ULs) are common benign tumors that originate from the smooth muscle cells of the uterine wall known as the myometrium. Around 70% of pre-menopausal women are affected by these tumors. The high prevalence of ULs is a significant public health issue and ULs are the leading cause for hysterectomy. Many tumors remain asymptomatic, but 15-30% of affected women develop symptoms ranging from pain and heavy menstrual bleeding to pregnancy complications and infertility. Despite their common occurrence, the underlying mechanisms of UL genesis are still largely unknown. Based on mutually exclusive recurring genetic alterations, ULs can be divided into molecular subclasses. Three main molecular subclasses have been established: MED12 mutated tumors, HMGA2 overexpressing tumors and tumors with biallelic FH inactivation. Combined, these three subclasses represent around 90% of ULs, indicating that additional smaller molecular subclasses also exist. Recently, novel mutations associated with ULs have been identified in genes encoding for subunits of the SRCAP chromatin remodeling complex that deposits histone variant H2A.Z onto chromatin. These included loss-of-function mutations in YEATS4, DMAP1 and ZNHIT1, and resulted in deficient H2A.Z loading in the tumors. The detailed functional consequences of these driver mutations need to be further investigated to fully understand their significance in UL genesis. This work aimed to elucidate the effects of YEATS4 mutations by characterizing previously established CRISPR-Cas9 edited immortalized human myometrial cell models carrying heterozygous mutations in YEATS4 using various molecular biology methods. Subcellular fractionation and western blot analysis was used to detect chromatin bound H2A.Z from cell lysates. Quantitative PCR was performed to determine relative YEATS4 expression levels in mutated and wild-type cells. No significant reduction of chromatin bound H2A.Z or YEATS4 expression was observed in the studied heterozygous mutants when compared to wild-type immortalized myometrial smooth muscle cells. Additional myometrial cell models were created by CRISPR-Cas9 gene editing. Objective was to achieve homozygous YEATS4 mutations to better reflect the changes previously reported in ULs. One homozygous YEATS4 mutant cell line was achieved. Understanding the detailed molecular mechanisms behind UL genesis will be instrumental for developing curative non-invasive therapies in the future. Insight into dysregulated pathways and identification of UL biomarkers could improve diagnostic accuracy and help design personalized targeted therapies effective for specific UL subclasses. Characterization of each molecular subclass offers a unique opportunity to understand UL genesis.
  • Kyriacou, Mikael Sakarias (2021)
    MLH1 is a gene that codes for one of the four mismatch repair (MMR) proteins alongside MSH2, MSH6, and PMS2. The main function of the MMR proteins is to recognize base mismatches and insertion-deletion loops formed during DNA replication and aid in their excision. Inherited heterozygous pathogenic variants in any of the four MMR genes lead to Lynch syndrome, an inherited cancer syndrome that predisposes to multiple different cancer types, most notably colorectal cancer. Loss of the expression of an MMR gene causes MMR-deficiency, which leads to microsatellite instability, the accumulation of mutations in microsatellite regions of the DNA. The higher mutational burden caused by MMR-deficiency is thought to be the main driving force of genomic instability and tumorigenesis in MMR-deficient cells. In addition to MMR, MLH1 and the MMR machinery have roles in other anticarcinogenic cellular processes, such as DNA damage signaling and DNA double-strand break repair. Recently, MLH1 has also been shown to have a significant role in regulating mitochondrial metabolism and oxidative stress responses. The identification of MMR-proficient tumors in Lynch syndrome patients begs the question whether the lower amount of functional MLH1 observed in MLH1 mutation carriers could cause problems with these functions and pose alternative routes to tumorigenesis. In line with this, it has been shown that the role of MLH1 in cell cycle regulation in DNA damage signaling is notably more sensitive to decreased amount of the protein compared to its role in MMR. The main goal of the thesis was to study the effects of decreased MLH1 expression on gene expression, cellular functions, and possible alternative tumorigenic pathways. In order to achieve this, the coding transcriptome of human fibroblast cell lines expressing MLH1 at different levels was sequenced and the resulting data analyzed. The study revealed that decreased MLH1 expression affects cellular functions associated with mitochondrial function and oxidative stress responses in cells with functional MMR. Particularly NRF2-controlled cytoprotective defence systems were observed to be downregulated. Decreased MLH1 expression was also observed to affect several cellular functions associated with reorganization of the cytoskeleton and interactions with the extracellular matrix. These results strengthen the recently made notions that MLH1 has a role in controlling the function of mitochondria and in mitigating oxidative stress, and that these two functions are connected. The study also brings to light new information on the possible role of MLH1 in controlling the organization of the cytoskeleton, which has previously received little attention. Dysfunction of mitochondria, increased oxidative stress, and reorganization of the cytoskeleton, as a result of decreased MLH1 expression, could pose events that facilitate malignant transformation of cells prior to the total loss of MMR function.
  • Weiss, Johanna (2024)
    Drought events will increase in the future due to climate change and thereby threaten agricultural production. The growing world population needs stable yields nonetheless thus, solutions need to be found. One way to do so is to explore mechanisms that allow plants to withstand harsh conditions and recover from them. Autophagy is such a mechanism. It is a bulk degradation pathwaythat allows plants to recycle cell components. During biotic and abiotic stress autophagy pathways are enhanced. Even though this mechanism is very useful and is proven to enhance abiotic stress tolerance, very little is known about it in plants. This thesis aimed at defining the informative time points for drought and autophagy response during drought and recovery in barley. To do so barley plants were subjected to gradual drought by withholding irrigation. They were kept at a level of severe drought for 4 days and were then rewatered. The tip of the third leaf was sampled for RNA extraction followed by qPCR. The genes this thesis focused on were GST and HSP17 as indicators of drought stress and ATG6 as indicator for autophagy. Additionally, phenotypic data was collected via RGB imaging for monitoring wilting and thermal imaging for stomata closure. GST was upregulated during the onset of drought, which seems to make it an early response gene, while HSP17 was upregulated during severe drought. ATG6 had a high transcription rate during all stages of drought and only decreased during recovery. However, because the primer efficiencies calculated in this experiment appear to be very low, and data points were missing due to pipetting practice, the results are not reliable and need to be repeated. Thus, no prediction about the informative time points can be made. The thermal data showed good results and the expected pattern of stomatal closing during drought could be observed. But because the greenhouse is a variable environment a reference would make the data even more precise.
  • Korkiakoski, Satu (2024)
    The RNA splicing process is an important part of gene expression in which the introns are removed from the pre-mRNA so that the mature mRNA only contains protein coding exons. The splicing process is executed by the spliceosome in two subsequent transesterification reactions that occur partly co-transcriptionally. In the first step an intron lariat is formed between the exons. This is followed by splicing of the intron lariat and ligating the exons together. Genetic variants that affect the splicing of a particular gene are called splicing variants and they may disrupt the normal splicing process. Splicing variants can be both exonic or intronic and have effects on splice site recognition, activate cryptic splice sites or create new splice sites. These changes can lead to for example exon skipping or intron retention in the transcript. Diagnosing splicing variants is challenging because of the unknown functional effects of the variants. Splicing prediction tools can help predict the possible effects of variants. Different sequencing methods enable the detection of aberrant splicing transcripts and thus may help in variant interpretation. The aim of this master’s thesis was to develop a detection method suitable for the diagnostic laboratory for RNA splicing variants in congenital disorders. The methods that were tested included RNA and Sanger sequencing. First, the patient selection was performed using splicing predictions and previous research on the variants. Secondly, after receiving patient samples, RNA was extracted, and its integrity measured. The laboratory work was then divided into two parts, the other leading to the RNA sequencing and the other to Sanger sequencing. Before Sanger sequencing primer design, RT-PCR, PCR and analysis of the PCR fragment sizes was performed. RNA sequencing was preceded by RNA library preparation. The studied variants in this thesis were BRCA2 c.476-3C>A, MSH2 c.2005+3A>T and CYLD c.2350+5G>A. The PCR fragment analysis and Sanger sequencing was able to detect an aberrant splicing transcript with exon skipping on two patients caused by a variant in the CYLD gene. The RNA sequencing results confirmed the aberrant splicing transcript. In addition, fragment analysis showed evidence of a possible splicing isoform with skipping of two exons caused by a variant in the BRCA2 gene that was not expressed enough to show on the Sanger sequencing results. The RNA sequencing detected a splicing transcript with exon skipping in two BRCA2 patients. However, this was not the same transcript as interpreted from the fragment analysis results and no results in the RNA sequencing indicated a transcript with skipping of two exons.
  • Liu, Jianyin (2022)
    Cytokine release syndrome is a severe systematic inflammatory disease that can be triggered upon pharmaceuticals intake. Evaluating the potential risk levels of novel therapeutics with an optimal assay is therefore essential. In this study, we tried to set up and validate a cytokine release assay from human peripheral blood mononuclear cells (PBMCs) for its application in nonclinical immunotoxicity assessments. Fresh PBMCs were isolated from buffy coats obtained from 11 healthy donors of different characteristics. Freshly isolated PBMCs were treated with LPS, positive control antibodies (anti-CD28, anti-CD3) and their corresponding isotypes (negative control antibodies) in both aqueous and solid formats to assess their abilities to induce cytokine release. Similarly, cryopreserved frozen PBMCs were also incubated with LPS, the positive control antibodies and the negative control antibodies, and compared their cytokine releasing capacities with freshly isolated PBMCs. A nine-cytokine panel (IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNFα, IL-12) was screened to select four cytokines (IFNγ, IL-2, IL-6, TNFα) in the following experimental setup. Freshly isolated PBMCs appeared to have higher sensitivity in response to the treatments as shown by the higher level of cytokine release. However, similar trends of cytokine release were observed between freshly isolated and frozen PBMCs in both aqueous and solid assay formats. LPS and anti-CD3 strongly induced cytokine release in all donors. Conversely, anti-CD28 induced cytokine release in some, but not all donors, possibly due to donor specificity. In summary, we have successfully developed and optimized a cytokine release assay, and it can be used to test the potential risk of immune-modulating drug candidate in the preclinical safety studies.
  • Gómez Sánchez, Celia (2022)
    Kv7.1 is a potassium ion channel comprised of the KCNQ1 protein, which can coassemble with distinct β-subunits modulating the channel functions in different tissues. In 2017, Raivio’s group (from the University of Helsinki) found two missense mutations in the KCNQ1 gene, p.(Arg116Leu) and p.(Pro369Leu), responsible for causing pituitary hormone deficiency and maternally inherited gingival fibromatosis. The facial features and bone structure pointed to a cranial neural crest (CNC)-derived phenotype caused by an alteration in the potassium channel balance, given that these cells form the bone and cartilage of the cranial zone. To understand the implication of the CNC in the KCNQ1 syndrome, I attempted to replicate the CNC differentiation protocol of Suga and Furue (2019) with the aim of obtaining cranial neural crest cells (CNCCs). This would enable future generation of a KCNQ1-related disease model. The differentiation process was carried out thrice, and two BMP4 concentrations (10 and 100 ng/ml) were assayed. The differentiated cells exhibited a CNC-like morphology as well as upregulation of the marker genes (TFAP2A, SOX10, DLX1, MSX1, and DLX2) associated to this cell lineage. However, the gene expression was low according to the qRT-PCR Ct values, which were in most cases higher than 30. Additionally, no differences were found between the two BMP4 treatments. Furthermore, the cells did not express KCNQ1, and thus the impact of the two KCNQ1 mutations was not investigated under this protocol. In conclusion, the protocol had a low efficiency in the generation of CNCCs that was not improved by increasing the BMP4 concentration. Further optimization of the protocol, such as the BMP4 concentration or the cell density of the culture, will be needed to improve its efficiency and obtain an adequate disease model.