Browsing by study line "Genetik och genomik"

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.

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.

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.

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.

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.

Multiple myeloma (MM) is a heterogeneous plasma cell cancer that results from the excessive proliferation of mutated B cells in the bone marrow and the accumulation of ineffective antibodies, monoclonal proteins, in the blood. Despite recent advances in research and novel therapeutics, MM remains incurable, mainly due to the mechanisms underlying disease progression and drug resistance. Therefore, novel biomarkers and therapeutics for the treatment of relapsed and refractory MM are urgently needed. MicroRNAs (miRNAs), short non-coding RNA molecules that play a key role in post-transcriptional gene regulation, have been found to be associated with different hallmarks of MM. Previous studies have indicated that abnormally functioning miRNA-mediated gene regulation followed by oncogene activation and tumor suppressor gene silencing results in drastic alterations in cell proliferation, apoptosis, growth, and metabolism. These changes in cellular functions have been indicated to be associated with the pathogenesis, progression, and formation of drug resistance in MM. Therefore, the role and potential of miRNAs to act as biomarkers to predict MM progression and drug sensitivity should be further investigated to ultimately improve the survival rates of patients. The aim of this master’s thesis was to investigate the relationships between drug sensitivity, disease progression and miRNA regulation in MM patients. Bioinformatically predicted miRNAs identified to be associated with sensitivity to panobinostat, a novel histone deacetylase inhibitor, and MM progression were validated in MM patient samples by using real-time quantitative reverse transcription PCR (RT-qPCR). In addition, the specific gene targets of miRNAs involved in the regulation of drug responses and MM progression were predicted by identifying statistically significant, negatively correlated interactions between the miRNA and RNA sequencing data of 45 MM patients in pairwise comparative correlation analysis. Finally, the predicted miRNA targets genes were validated in MM patient samples using RT-qPCR. Based on the bioinformatic analyses and RT-qPCR validation, mir-424 expression was significantly increased in relapsed MM patients as compared to respective patient samples taken at diagnosis, suggesting a potential role of mir-424 in MM progression. Similarly, mir-4433b expression was significantly elevated in panobinostat-resistant patients compared to sensitive patients, suggesting a potential effect of mir-4433b on the regulation of panobinostat drug response in MM patients. In addition, the RT-qPCR validation demonstrated that the disease progression and drug sensitivity associated mir-92b, mir-363 and mir-221, would potentially regulate the expression of FGF2, MFF, and TMEM248, respectively, providing novel insights into the functional roles of miRNAs in MM pathways.

The European rabbit (Oryctolagus cuniculus) is a small mammal native to the Iberian Peninsula, but introduced by humans to all continents except Antarctica. The rabbit has been a remarkably successful invasive species due to its generalist nature and fast reproduction. Its spreading has mostly been destructive to the local nature, and humans have used fatal rabbit diseases such as rabbit haemorrhagic disease (RHD) to control harmful populations. The rabbit population in Helsinki is one of the most northern annually surviving rabbit populations in the world. It is believed to have originated from escaped pet rabbits in the late 1980s, and in the early 2000s, the rabbits spread rapidly around the Helsinki area. RHD spread unintentionally to Finland in 2016, and the disease caused a significant reduction in the Helsinki rabbit population. Rabbit population genetics has previously been studied in several countries, but never before in Finland. The aim of the thesis was to examine the genetic diversity and population structure of the Helsinki rabbit population before and after the RHD epidemic, and to compare the results to similar preceding rabbit population genetic studies. Rabbit populations have previously been found to recover from major population crashes without a notable loss in genetic diversity using DNA microsatellite markers. The recent RHD epidemic in Helsinki provided an opportunity to study, whether a rabbit population can recover from a population crash even in a harsher environment without losing genetic diversity. To conduct genetic analysis, fourteen DNA microsatellite loci were genotyped from individuals caught during two distinct time periods, in 2008-2009 (n=130) and in 2019-2020 (n=59). Population structure was observed in both temporal rabbit populations with small but significant FST values. The 2019-2020 population was more diverse than the 2008-2009 population in terms of allele numbers and expected heterozygosity. This result was unexpected considering the recent RHD-epidemic but could be explained by gene flow from new escaped rabbits. Compared to other wild rabbit populations around the world, the Helsinki area rabbits exhibit significantly lower genetic diversity. Bottleneck tests showed a significant signal separately in both temporal populations, but the RHD bottleneck cannot be distinguished based on the tests. The results could be biased by new gene flow, or the initial bottleneck caused by the founder effect of only a few pet rabbits. The rabbits have demonstrated their adaptation and survival skills in the cold climate of Helsinki. The population has significantly lower genetic diversity compared to other wild populations, yet recovered from a major RHD epidemic without reduction in genetic diversity under these more extreme environmental conditions. It has been proven again; the rabbit is a thriving invasive species.

The intestinal stem cells (ISC) are responsible for the regeneration of the intestine epithelial barrier after acute injury and for the replenishment of its cells overall. How the ISC activation and resulting proliferation is controlled is complex and still under study. The ISCs of the midgut, which is the functional analogue to mammalian small intestine, are also highly responsive to changes in nutrition, and with proper methodologies it is possible to study the effects of diet on stem cell activation. The metabolic flux of the nutritional components of the diet can then shed light on which metabolic pathways are necessary for nutrient-dependent proliferation. One nutrient that has garnered interest is glutamine (Gln). It is well established that glutamine supplementation can in parenterally fed patients diminish intestinal barrier atrophy, extend the time the patient can be kept under the regime, and increase survivability of critically ill patients. Consequently, glutamine or its downstream metabolites may have stem cell activating characteristics. However, the exact regulatory mechanisms and specific effects of Gln are not well known, and studies have found contradictory results on the beneficial effects of Gln supplementation. Glutamine itself is a conditionally essential amino acid that has a variety of functions: it is an important source of nitrogen and cellular energy and contributes carbon into the tricarboxylic acid cycle (TCA) and is involved in protein and nucleotide synthesis. In this thesis, the effects of Gln supplementation on the cell populations of D. melanogaster were studied via microscopy and computational analysis. Cross-breeds of fruit fly were established to lineage label the ISC with a GAL4/UAS driver system. Confocal microscope was used to image the midguts which were then analysed with Imaris software. A novel analysis method was developed to study population changes and varying features of the cells in the midgut in an unprecedented region-by-region bulk analysis. Earlier studies into nutrient control of ISC have had limited focus within the midgut and might have consequently given a restricted view of ISC activation. This new Longitudinal Analysis of Midgut (LAM) can be utilized in a diverse set of further studies to describe conditional variation within midgut, and possibly other tissues. Gln was found to increase total cell numbers to comparable levels with well-fed midguts, and to drive limited endoreplication in enterocytes. Lineage labelled cell population grew primarily in the R3 and R4 regions of the midgut. Additionally, enteroendocrine cells (EE) were greatly increased in the posterior part of R3 but had conceivable minor increases along the whole length of the midgut. Improved nutrition was also found to affect the proportions of the midgut, presenting itself as elongated posterior and stunted anterior. Overall, the pipeline and analysis method established during this study enable more expeditious research of effects of other nutritional components and allows for study of effects of other mechanisms, for example how gene knock-downs or altered gene activities affect cell populations of the midgut.

Antimicrobial resistance is an emerging concern at the global scale, threatening the effectiveness of antibiotics in treating bacterial infections. Among anthropogenically impacted environments, wastewater treatment plants have been indicated as possible reservoirs of antibiotic resistance genes, putative hotspots for their horizontal gene transfer, and a source of their dissemination to the environment. Generally, the abundance of antibiotic resistance genes is reduced during the wastewater treatment process. However, some genes were shown to be enriched in purified effluent water and dried sludge, which are then released to the environment, compared to influent water. Also, the taxonomy of the hosts carrying antibiotic resistance genes could change as a result of horizontal gene transfer events. The aim of this study was to analyse and compare the host range of a series of antibiotic resistance genes in influent water, effluent water and dried sludge collected from the Viikinmäki wastewater treatment plant in Helsinki, Finland, by applying Emulsion, Paired Isolation and Concatenation PCR (epicPCR). EpicPCR is a method that can link a gene of interest to the 16S rRNA gene from the genome of the host bacterium, without any cultivation step. The abundance of the hosts was also evaluated by sequencing the 16S rRNA gene from the whole bacterial community. In several cases, the target antibiotic resistance genes (blaIMP, blaNDM, ermB, ermF, sul1 and strB) were carried in effluent water and dried sludge by taxa that were not hosting them in influent water, suggesting that horizontal gene transfer events might have occurred during the treatment. All the examined genes were detected both in abundant and in rare taxa, including genera that also comprise pathogenic species, such as Arcobacter and Acinetobacter. Some of the detected hosts were not previously known to show resistant phenotypes, namely members of the family Methylophilaceae. These results corroborate the idea that wastewater treatment plants might be hotspots for the horizontal gene transfer of resistance determinants, and potentially disseminate antibiotic resistant pathogens to the environment. However, in order to ensure the accuracy of the results, the limits of epicPCR as a method need to be identified and addressed.

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