Browsing by study line "Genetiikka ja genomiikka"

Human induced pluripotent stem cells (iPSCs) are an important in vitro model of disease and development. iPSCs can be differentiated in culture into cell types which are difficult to access from patients, such as neurons. Applying iPSC-derived cellular models to disease studies requires a thorough characterization of the derived cell types, as well as assessing reproducibility across cell lines or differentiation batches. With the aim of providing such a comprehensive molecular characterization at an early stage of cortical neuronal differentiation in vitro, six iPSC lines from four donors were differentiated to cortical neural progenitors using a modification of an established protocol (Shi et al., 2012a). The protocol successfully produced neural progenitors, with over 75% of the differentiated cells aligning with a cortical identity, as confirmed via qPCR and immunocytochemistry of established markers such as PAX6, NES and SOX1. To further classify the cell types produced as well as identify potential differences between cell lines, gene expression of the obtained cells was profiled with single cell RNA sequencing of ~22,000 cells, which uncovered the heterogeneity of neural progenitors produced. Further, although two differentiation batches produced similar cell-type compositions on a whole, a fraction of the lines showed inter-individual differences in cell type composition, which correlated with expression variability of known marker genes. Additionally, the cell types produced in vitro were compared to those produced in vivo by mapping our dataset to a reference fetal brain dataset (Polioudakis et al., 2019). It was observed that the in vitro dataset represented a subset of the cell types present at mid-gestation. Overall, the single cell characterization of differentiated cells allowed greater resolution in understanding cell-type heterogeneity of cortical neurogenesis, which is of key relevance for future applications such as disease modeling.

The wide distribution of Scots pine (Pinus sylvestris L.) in boreal forests and the outstanding properties of its wood have made it an economically significant resource at the forest sector. The highly valued chemical and mechanical properties of Scots pine wood are related to heartwood, a specialized tissue forming the innermost part of a mature trunk. Decay resistance of Scots pine wood is largely defined by heartwood extractives of which the stilbene pinosylvin has the highest quality trait breeding interest. Pinosylvin concentration is a high-heritability trait that positively correlates with the heartwood decay resistance. Pinosylvin biosynthesis pathway is upregulated both developmentally at the mature tree transition zone between sapwood and heartwood and as stress response in various tissues of young trees. Identification of the regulators of pinosylvin synthase could speed up quality trait breeding providing a basis for variant screening in the natural populations and for analysing functional properties of the variants. Early genotyping would enable selection of the desired quality individuals before the start of developmental pinosylvin production and significantly accelerate breeding programs. Scots pine pinosylvin synthase PST-1 is proposed to be both stress-induced and developmentally regulated. Previous studies have identified several MYELOBLASTOSIS (MYB) domain transcription factors (TFs) that co-regulate with stilbene pathway transcripts under pinosylvin production inducing conditions or that have promising homologs in other species. In this study, eight Scots pine MYB TFs were examined in PST-1 promoter interaction studies using quantitative luciferase assay and yeast one-hybrid assay. This study aimed to clone the MYB coding sequences and confirm the integrity and MYB character of the proteins they encode, and to verify whether any of the MYB TFs are direct regulators of PST-1, and to characterize the regulatory functions of the MYB TFs as activators or repressors. This study identified one MYB TF as a direct regulator of PST-1 whereas the other studied MYB TFs did not bind the most promising MYB target elements in the promoter. The discovery of a direct regulator of pinosylvin synthase provides a potential marker for early selection making the finding highly valuable for quality trait breeding efforts. Additionally, another MYB TF was detected as a potential indirect regulator of pinosylvin biosynthetic pathway or as a regulator of neighbouring pathways suggesting that it would also be an interesting target for further studies. The MYB TFs were successfully cloned and seven out of eight MYB TFs were classified into MYB subfamilies. Tentative characterizations for the MYB TFs were presented based on the sequence analysis. The Gateway compatible vectors generated in this study will facilitate future experiments. The MYB coding sequences were incorporated in the verified entry clones ready-to-use in generation of other types of expression vectors. The MYB TF plant vectors could be directly used in Arabidopsis, as well. Two multisite Gateway compatible entry clones for N-terminal fusions to VP16 and SRDX transcriptional regulatory domains were generated for the plant expression vectors. The protocol developed for the 3’ fusion entry clones comprises of sequential polymerase chain reactions easily applicable for other cloning purposes. The yeast one-hybrid prey vectors could be utilized not only in another one-hybrid but also in two-hybrid studies. Several of the MYB TFs, including the PST-1 direct regulator, were hypothesized to interact with other types of TFs. The protein – protein interaction studies would detect possible co-factors involved in the MYB TF mediated regulation of Scots pine pinosylvin synthase. Identification of each member in the regulatory complexes would enable targeting the quality trait breeding efforts most effectively

Bacteraemia, the presence of bacteria in the bloodstream, may lead to severe and costly health issues. Sepsis, a serious complication of bacteraemia, is one of the top causes of mortality globally. Early and specific diagnostics as well as fast acting are essential in successful treatment. However, current diagnosis relies mainly on time-consuming blood culturing and clinical symptoms, which are unspecific for the causative agent. With the advanced technology and decreasing cost, state-of-art sequencing-based (Next generation sequencing) methods provide a new way to investigate the bacteria present. Metagenomics, which means sequencing and studying all DNA extracted from a microbial community sample, is widely used, but it only describes the genetic potential of a community and does not differentiate live from dead microbes. Metatranscriptomics, in which essentially all RNA from a sample is sequenced, provides information about expression and activity together with identification of viable bacteria, However, the high amounts of host cells and host RNA complicate the detection of bacterial transcripts from complex host-microbe samples. In this thesis, I investigated solutions for the efficient isolation and enrichment of bacterial RNA from whole blood to be used in sequencing and metatranscriptomics analysis. Firstly, I tested the capability of bacterial cell lysis of two commercial blood sampling tubes with Escherichia coli and Staphylococcus epidermidis suspensions. Both tubes, Tempus and RNAgard, were able to lyse gram-negative E. coli cells and good-quality RNA was extracted in measurable quantities with their respective RNA extraction methods. With Tempus tubes the RNA yield was clearly higher. With gram-positive S. epidermidis, RNA quantities from both extractions were below the measurement limits indicating insufficient lysis and need for further optimization. Secondly, I investigated the depletion of polyadenylated (poly-A) transcripts in order to reduce the host transcripts and thus to enrich the bacterial transcripts prior to costly sequencing step. I evaluated the performance of a previously designed in-house protocol, based on the capture of poly-A -transcripts with oligo-dT -beads, and tested different parameters to see whether the depletion efficiency could be enhanced. Most significantly, the amount of oligo-dT -bead suspension was reduced to half from the original protocol. In-house protocols were also compared to a commercial solution, which they clearly outperformed. Depletion performances were tested with a RT-qPCR and dot blot assay, which I designed along this thesis work. Finally, to make the poly-A depletion better suited for blood samples infested with globin transcripts (representing up to 80% of all poly-A transcripts extracted from whole blood), I tested and successfully pipelined the leading commercial method for depleting globin transcripts with the in-house poly-A depletion protocol. The optimized sample preparation protocol provides a platform for further bloodstream infection and sepsis studies. Next steps of the process, such as sequencing and testing with clinical samples, are already ongoing with promising preliminary results. In the future, the metatranscriptomics approach can be utilized in fast and specific identification of the pathogens and their antibiotic susceptibilities. In addition, infection mechanisms and host-pathogen interactions may be studied possibly providing novel insights for sepsis diagnostics and treatment.

In the central nervous system, GABAergic neurons serve as the primary source of inhibitory signals. The study focuses on the GABAergic neurons located in the anterior brainstem, which play a pivotal role in modulating monoaminergic circuits critical for mood, motivation, and movement regulation. The development of the anterior brainstem GABAergic neurons relies on the activation of the Tal1 transcription factor (TF) in the neuronal precursors located in the ventrolateral part of rhombomere 1 (rV2 progenitor domain). The aim of the study was to investigate the regulatory mechanisms governing Tal1 expression. Specifically, the co-expression of potential upstream regulators of Tal1 in differentiating GABAergic rV2 neurons was validated using RNAscope® In Situ Hybridization. Findings demonstrated that the early post-mitotic TFs Sox4, Insm1, Ebf1, and E2f1 are not only co-expressed with Tal1 but also precede the activation of Tal1 expression. This supports a potential role for these genes in activating Tal1 expression and therefore influencing the acquisition of the GABAergic identity in the neuronal progenitors of the anterior brainstem. This research contributes to the understanding of the development and differentiation of the anterior brainstem GABAergic neurons.

Gerbera hybrida, a common ornamental plant, has natural resistance to fungal diseases. While there may be several bioactive compounds behind this trait, this master’s thesis focuses on two of them: gerberin and parasorboside. The gerberin/parasorboside biosynthesis has been profoundly investigated by Gerbera Laboratory at University of Helsinki. Gerberin and parasorboside are polyketide derivatives, a vast group of bioactive metabolites. The pathway that produces these compounds involves several enzymes: a polyketide synthase (PKS), two polyketide reductases (PKRs) and a glycosyl transferase. For gerberin to be synthesized, it requires presence of three enzymes: (1) G2PS1 (Gerbera 2-pyrone synthase 1) which initiates the chain by synthesizing the carbon backbone, and (2) an unknown first acting reductase, that continues the chain by reduction, lactonization, and (3) addition of a sugar molecule. Parasorboside production requires also a fourth enzyme, a reductase described by Zhu et al. (2022). This master’s thesis delves into post-modification of gerberin and parasorboside intermediates by the PKRs, which have not yet been verified. Until now, there has only been PKR-candidates for this position, derived from gerbera transcriptome library. The genes behind the proposed reductases were known from previous research. The objective of this master’s thesis is to demonstrate which of the proposed reductases coexpressed along with the G2PS1-reductase will produce the desired compounds (gerberin and parasorboside) in a model plant tobacco (Nicotiana tabacum in stable transformation and Nicotiana benthamiana in transient expression). The gene combinations were constructed into plasmids via PCR and Golden Gate cloning. Enzyme production was analysed by western blot and the secondary metabolites by HPLC. The hypothesis for this work was to produce the aglycones of gerbera’s natural products gerberin and parasorboside in the model plant tobacco via constructing the plasmids carrying our genes of interest. This master's thesis documents the successful construction of double and triple fusion plasmids, their integration into agrobacteria, and subsequent transformation into stably transgenic tobacco. Gerberin production in tobacco was observed with certain gene combinations, parasorboside production was not analysed yet in this work. Through meticulous work, the desired combination of genes for gerberin synthesis was identified in both agroinfiltrated plants with co-expressed genes and stable transgenic tobacco lines expressing genes from a single transcript cleaved by 2A-peptides.

Bartonella species are facultative intracellular bacteria causing variety of diseases in humans and also infects endothelial cells and erythrocytes. Some Bartonella species utilize VirB/VirD4-type IV secretion system (T4SS) in order to secret Bartonella effector protein A (BepA) which infects endothelial host cells by inhibiting the apoptosis. But the enterotoxin homolog in Bartonella gene A (EhbA) and the enterotoxin homolog in Bartonella gene B (EhbB) are found in the non-BepA Bartonella strains. In my Master’s thesis, I study the host cell binding activity and identify host cell surface receptor of EhbB in Bartonella. In my thesis, the cell adhesion of multimeric B proteins of enterotoxin homologue in Bartonella (Ehb) have been analyzed with cell adhesion assay using HEK293T, HeLa 229, Ea.hy926, and CHO-K1 cells. The assay was conducted with EhbB1 and EhbB 1-1C proteins from Bartonella Bovis strain Bermond and Bartonella strain spp 1-1C and the experiment indicated the cell adhesion activity of both EhbB proteins compared to the controls used in the experiment. Moreover, the binding activity of EhbB1 with Ea.hy926 was studied at several incubation time points, such as; 30 min, 2 hours, 4 hours, 6 hours, and 8 hours. Several incubation period of EhbB1 and EhbB 1-1C with Ea.hy926 cells did not enhance cell surface adhesion because the same absorbance compared to controls. The interaction of EhbB1 with cell membrane HEK293T was studied by using western blot on cell membrane preparation from Ea.hy926 cells which was used to identify possible protein receptor of EhbB1. The experiment suggests that EbB1 is binding to receptors present on the cell membrane of HEK293T which could be protein. The cell adhesion activity of HEK293T cell membrane with EhbB1 was analyzed by inhibition assay. This experiment indicated that EhbB1 protein attached to cell surface receptors present on the HEK29T cell membrane, which inhibited EhbB1 protein to attach to Ea.hy926 cells. This also indicate that the cell surface receptor for EhbB1 could be protein but requires further study.

Diabetic ovarian cancer patients who take metformin as part of their anti-diabetic medication generally respond better to DNA-damaging cancer treatment. The molecular mechanisms of the anti-cancer effects of metformin are currently being investigated, but they remain poorly elucidated. Not much is understood about the metformin effect on DNA damage in ovarian cancer cells, where it is of particular importance. When chemotherapy-induced double-stranded DNA breaks are unrepaired, cells reach a point when they cannot tolerate the accumulated DNA damage and die. However, some ovarian cancer cells efficiently employ DNA repair mechanisms, the most prominent being homologous recombination (HR), to overcome DNA damage. Efficient HR causes chemoresistance. An important question is whether metformin has the ability to induce the HR-deficient state in cancer cells, thereby sensitizing them to treatment. This study did not examine HR directly, but it assessed HR indirectly by observing the effect of metformin on recovery from DNA damage in two ovarian cancer cell lines: OVCAR4 (HR-proficient) and Kuramochi (HR-deficient). Additionally, this study evaluated the metformin effect on cell proliferation and apoptosis. OVCAR4 and Kuramochi cells were exposed to varying metformin concentrations (0,5 mM, 5 mM, 10 mM, 15 mM, 20 mM and 25 mM) and for varying durations (24 hours and 48 hours). This study also tested how metformin pretreatment affected the cells’ ability to repair externally (ionizing irradiation) induced DNA damage. The cells were imaged with a high-content imaging system, and percentages of nuclei that were positive for markers for different cellular processes (i.e., DNA damage, proliferation, and apoptosis) were calculated. The study found that only high metformin concentrations, such as 20 mM were able to increase DNA damage and reduce cell proliferation in HR-proficient OVCAR4 cells, both non-irradiated and irradiated. The HR-deficient Kuramochi cell line was generally more sensitive to metformin, particularly with regards to DNA damage, which increased using metformin concentrations < 20 mM. However, 20 mM concentration resulted in the most significant effects. Similarly, only high metformin concentration (25 mM) increased apoptosis, although data were obtained only for a limited number of Kuramochi cells. More experiments on apoptosis would be beneficial. Also, more extensive experiments for the irradiation part are needed to validate these preliminary findings, as well as examining whether high metformin concentrations (> 20 mM) affect specifically the HR-mediated DNA repair pathway.

Colorectal cancer (CRC), which refers to the cancer of the colon and the rectum currently ranks as the second leading cause of cancer related death worldwide and as the third most common form of cancer in both males and females. The latest reports show that approximately 10% of all new cancer cases globally are diagnosed as CRC annually. Initiation of sporadic CRC is commonly caused by somatic mutations causing the loss of function of the tumor suppressor gene APC. This leads to aberrant activation of the canonical Wnt signalling pathway. The ApcMin/+ mice model the progression of CRC as they carry a constitutive heterozygous nonsense mutation in Apc allele and develop intestinal adenomas. TCF/LEF transcription factor family members are best known as the main downstream effectors of canonical Wnt signalling. In the presence of nuclear β-catenin, TCF/LEF proteins bind to it through their β-catenin-binding domain and activate the transcription of Wnt target genes. The TCF7 gene encodes several isoforms of TCF1 protein, which are traditionally divided into long and short isoforms, transcribed from different promoters. Previously, it has been shown that Tcf7 deletion (Tcf7-/-) in ApcMin/+ mice increases the formation of adenomas. The aim of my study is to better understand the role of Tcf7 and its isoforms in CRC tumorigenesis. To study the Tcf7 deletion in intestinal adenoma development, ApcMin/+; Tcf7mut/mut; Villin CreERT2 mouse strain was established. The expression of the full-length isoforms (p45) is constitutively prevented in the Tcf7mut/mut mice. Moreover, tamoxifen administration to these mice led to the deletion of all isoforms in the intestinal epithelium. The number of intestinal tumors, their sizes and the survival of the Tcf7 deleted ApcMin/+ mice were analyzed and compared to ApcMin/+ mice. Intestinal tissues of the mice were collected after euthanasia. The tissue samples were preserved in paraffin, and later cut into sections for IHC, stained and imaged. The deletion of Tcf7 was confirmed at the RNA level by qPCR, and at the protein level by immunohistochemistry (IHC). IHC and single-cell RNA sequencing was used to further analyze the effect of Tcf7 deletion in mouse intestinal adenomas. The deletion of all Tcf7 isoforms or only the p45 isoforms in ApcMin/+ mice increased robustly the numbers of intestinal tumors. IHC analysis of the intestinal adenomas showed that the deletion of p45 isoforms was sufficient to cause a dramatic decrease in total Tcf1 expression in the adenoma cells. These results were supported by the qPCR results. In summary, our results lead us to believe that the deletion of p45 isoforms causes an acceleration of tumorigenesis in the adenoma model. Without the Apc mutation, the mice did not develop intestinal adenomas. Interestingly, the expression of the Wnt-target gene Prox1 in intestinal adenomas was decreased when Tcf7 was deleted. We next aim to optimize our protocol for single cell dissociation of adenomas and re-run the single-cell RNA sequencing analysis for further analysis of the mechanisms behind the increased tumorigenesis.

Endometriosis is a common complex disease that affects the quality of life of millions of women worldwide. It is characterized as an inflammatory condition where endometrium-like tissue is found at ectopic sites. The main symptoms are pain and infertility. There is no cure for the disease yet. Diagnosis requires surgery in most cases, the invasiveness is a problem. The costs for societies due to endometriosis are immense. Endometriosis, despite being a benign disease, shares characteristics with malignancies: invasion, proliferation, and angiogenesis. These enigmatic aspects make this disease an interesting subject for research. Endometriosis is shown to have a heritability of 50%. Research on the molecular genetic background is needed for the development of low-invasive diagnostic methods and better treatments for the disease. Genetic research has recently focused on genome-wide association studies of large patient and control cohorts. By design, these studies can only explain a portion of the low-risk genetic variants of common diseases. No causal high-risk gene defects behind endometriosis are found yet. In this study whole exome analysis is utilized for searching a heritable gene defect from a family of four closely related Finnish endometriosis patients in two generations. Two of the patients have a combined phenotype of endometriosis and ovarian or tubal carcinoma. Endometriosis is known to increase the risk for certain types of malignancies, endometriosis-associated ovarian cancers. Four candidate susceptibility genes for endometriosis were identified in this study FGFR4, NALCN, ZNFX1, and NAV2. The findings still need to be validated in patients not related to the study family. The variants found in this study lay a basis for screening additional endometriosis patients and functional analysis of the variants. Subsequent research on these found candidate susceptibility genes may elucidate the pathogenic pathways behind endometriosis or endometriosis-associated ovarian cancer in the future.

Biallelic germline mutations in ERCC6L2 cause bone marrow failure (BMF) and predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The patients often develop varying cytopenias, and underlying hypoplasia in the bone marrow is usually evident. The aim of this thesis was to characterize the transcriptome of patient -derived fibroblasts with biallelic germline ERCC6L2 mutation. Moreover, the aim was to study changes on the gene expression at the RNA level in fibroblasts in different media conditions, ROS levels in ERCC6L2 -mutated fibroblasts, and whether addition of glutamine impacts the ROS levels. Fibroblasts from 16 skin biopsies were cultured; eight samples were from healthy controls and eight samples from patients with known mutations in ERCC6L2. Fibroblasts were cultured in different media conditions, followed by RNA extraction and RNA sequencing. We observed downregulation in base excision repair, nucleotide excision repair, mismatch repair, DNA replication, homologous recombination, and cell cycle in ERCC6L2 -mutated cells. MAPK signaling pathway, p53 signaling pathway, apoptosis, AMPK signaling pathway, and TGF-beta signaling pathway were in turn upregulated in ERCC6L2 -mutated cells. The medium did not affect the gene expression significantly across samples. We suspect that the effect of medium was not detected at the RNA level, but it might affect post-translational modifications. We also detected increased ROS levels in ERCC6L2 samples compared to control and observed decreased ROS levels in ERCC6L2 and control samples with excess glutamine. This study shows that biallelic mutations in ERCC6L2 do not only affect the bone marrow but can also affect tissues outside of the hematopoietic system. The transcriptomic analysis identified important biological processes, which could be studied with more detail in the future to further explore the pathology of the ERCC6L2 disease.