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Browsing by study line "Genetics and genomics"

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  • Koistinen, Ville (2024)
    Silver birch (Betula pendula) is both the third most commercially important, as well as abundant, tree in Finland. Faster growth is of interest from an industry, as well as climate change, perspective. In addition to higher biomass yield from a commercial point of view, faster growth in trees leads to absorption of more carbon dioxide, thus increasing their effect as a carbon sink to lessen the impact of climate change. By conducting a genome-wide association study, the aim of this thesis was to investigate the genetical background of B. pendula on three first-year growth traits: maximum height, maximum growth rate and time to cessation. Whole-genome sequencing data from 405 mother trees was used to provide a high-resolution genome-wide map for assessing genes associated with the three growth traits. A high-quality genotypic dataset of 5,727,473 single-nucleotide polymorphisms (SNP), which span across the whole genome, was established through rigorous filtering. Phenotypic data was estimated with a mixed linear model by best linear unbiased prediction from mother trees’ 7,266 seedlings, which were grown in a greenhouse under controlled conditions to monitor their height trajectories. Association testing was done as both a uni- and multivariate mixed linear model with the GEMMA algorithm. Overall, 16 unique suggestively significant SNPs were discovered among the three traits. Genes putatively associated with the SNPs were related to metabolic regulation and cell transportation processes, as well as biotic and abiotic stress. On each of the three traits, SNPs with the largest effects had around 5% deviation against the average phenotypic value, which could be considered a major effect for a polygenic trait. Only 4 out of the 16 suggestively associated SNPs were within gene regions, even though the putative genes themselves had also variants present abundantly in the dataset. This could suggest that the associated SNPs could be related to gene expression regulation, or they could be in linkage disequilibrium with multiple different genes, and thus be markers for combined effects of multiple genes on the focal trait. To validate the results, further studies such as gene editing on the associated genes, or a duplicated study on a different population, would be needed.
  • Högel, Caroline (2022)
    The aim for this project is to set up a high-content imaging pipeline for phenotypic analysis of single cells in peripheral blood mononuclear cell (PBMC) samples from healthy blood donors. The blood donors selected for the optimization experiments are known to carry specific allele variants of interest, based on an earlier FinnGen study. The main question is whether these genetic differences result in phenotypic changes in the PBMCs that can be identified by microscopic imaging and AI-guided image analysis. In this Pro Gradu work, I have optimized the pipeline of PBMC sample handling, immunostaining, and phenotypic imaging. PBMCs were gathered from healthy donors at the Blood Service Biobank. The frozen PBMC samples were thawed, and cells were plated on 384-well plates prior to immediate fixation with paraformaldehyde. The cells were then stained with fluorescent cell markers based on the Cell Painting assay (Bray 2016), followed by wide-field and confocal imaging with Opera Phenix high-content confocal microscope (FIMM High Content Imaging and Analysis unit). Novel deep learning methods are now being developed (Pitkänen group) to automatically learn phenotypes from the collected imaging data and associate them to the donor’s genotypes. We also used in-house tools for cell segmentation and further analysis as well as quality control (Paavolainen group). Primary results based on the features extracted from acquired images showed promising cell type - and donor -type specific clustering.
  • Natraj Gayathri, Swethaa (2024)
    The TTN gene is composed of 364 exons (363 coding) and encodes for titin, the largest protein in nature. Pathogenic TTN variants result in a wide spectrum of skeletal muscle and cardiac disorders known as Titinopathies. These differ in inheritance patterns, onset age, disease course and severity. The biological mechanisms underlying disease-causing variants specific to titinopathy patients are still elusive. Investigating gene signatures causing the biological pathomechanisms is crucial for understanding genotype-phenotype corelations. RNA-sequencing emerges as a valuable technique for analysing transcriptomic data and exploring gene expression profiles of patient and control samples. To elucidate common pathomechanisms in titinopathies, including adult tibial muscular dystrophy (TMD) due to heterozygous FINmaj variant, and biallelic recessive titinopathies, an extensive differential gene expression (DGE) analysis was conducted using three RNA cohorts from human muscle biopsies. The cohorts involved two polyA enriched and one rRNA depleted batch-corrected cohort. Human DGE analysis identified 265 commonly upregulated genes and 147 commonly downregulated genes in the titinopathy cohorts. A significant downregulation of TTN expression levels was observed in one of the cohorts. To validate and understand the biological significance of these findings, data from a mouse model was incorporated with homozygous Ttn FINmaj variants. Common genes among all cohorts accounted for the structural integrity of the extracellular matrix. This study indicates the pathomechanisms for a skeletal muscle pathology and discusses the future steps in efficiently performing RNA-Seq for titinopathies.
  • Turku, Teemu (2024)
    Distal myopathies are a group of rare progressive genetic muscle disorders that are extremely varied both genetically and clinically. Typical symptoms include weakness and atrophy limited to the skeletal muscles of distal extremities in hands and legs. The age of onset ranges from early childhood to late adulthood depending on the disease. Currently around 30 genes have been associated with distal myopathies, most of them causing a dominant disease. The objective of the thesis was to identify the disease-causing variant in a family affected by autosomal dominant distal myopathy with early adulthood onset. Affected family members expressed weakness and atrophy in muscles of both hands and legs. To narrow down the chromosomal location of the disease-causing variant, linkage analysis was conducted with genome-wide single nucleotide polymorphism data of family members. Because of the progressive nature of the disease and uncertain disease status of one family member, linkage analysis had to be repeated a few different times with different settings. Both disease statuses and pedigree size were altered to account for the possibility of presymptomatic carriers or incomplete penetrance. Analyses with different parameters led to discovery of multiple possible co-segregating regions. Rare co-segregating small-scale and structural variants as well as repeat expansions in these regions were examined from next-generation sequencing data with multiple bioinformatic detection tools. The segregation of possible candidate variants was validated with Sanger sequencing and PCR. Ultimately, no likely rare co-segregating variant of any type of genetic variation with a likelihood to cause a disease such as distal myopathy was identified by any detection method used. Lack of potential disease-causing variant could be due to incomplete penetrance of the variant or if it was in non-coding regions, such as a deep intronic splicing variant in a gene currently not known to be connected to muscles.
  • Müller, Linda Helena (2022)
    Puberty initiation is a crucial physiological process in human development. A group of hypothalamic neurons secreting the gonadotropin-releasing hormone (GnRH) and expressing the kisspeptin receptor (KISS1R) plays a key role in launching puberty. Furthermore, cellular KISS1R signaling has been shown to regulate GnRH expression and secretion. Although the in vitro differentiation of human pluripotent stem cells into GnRH-secreting neurons has been successful, it is of high interest to generate KISS1R expressing GnRH neurons. By utilizing the CRISPR activation technology, this study aimed to establish a conditional KISS1R-activation cell line using H9 human embryonic stem cells. Through controlling dCas9VP192 abundance using the Tet-On system combined with the dihydrofolate reductase destabilizing domain, the transcriptional activation of KISS1R was temporally regulated by the addition of two antibiotic drugs - doxycycline and trimethoprim. KISS1R expression was primarily assessed by qPCR and verified by immunocytochemistry and the use of a KISS1R-GFP reporter cell line. The main finding of this study is the achievement of a 6217 ± 2286 fold change in KISS1R transcription by introducing two guide RNAs (N = 3). Nevertheless, leaky gene activation was observed without drug treatment (fold change of 63 ± 51). Concludingly, this study successfully led to the generation of a KISS1R-activation cell line. After further characterization and refinement of the activation protocol, the established cell line will enable to investigate whether KISS1R upregulation modulates in vitro GnRH neuron differentiation, electrophysiology, hormone expression, and secretion in the future. Respective outcomes may lead to advances in understanding and treating pubertal disorders.
  • Wei, Xiaodong (2022)
    The composition and dynamics of the early life gut microbiota plays a major role in establishing neonatal immunity and is suggested to have multiple impacts on the child’s long-term health. Meanwhile, the composition of the infant gut microbiome has been shown to be affected by the birth mode, infant health and diet. However, the characterization of the infant gut microbiome and its impact on the host’s health is still challenging as the contribution and importance of multiple co-factors on the early microbiome during infant growth is still poorly understood and characterized. The Health and Early-life microbiota (HELMi) is a cohort of more than 1000 healthy Finnish infants currently followed from birth to 4-5 years old. By now, the HELMi dataset comprises more than 400 whole genome shotgun metagenomes obtained from stool samples from 80 infants and parents, but also an in-depth characterization of the families’ lifestyle, environment, health and nutrition, allowing for a precise and cutting-edge characterization of the early gut microbiota. Based on the datasets from the HELMi, this project used Metaphlan3, Kraken and Braken to determine the best computational approach for the taxonomic profiling of the metagenomic reads. Then a PERMANOVA test was performed to evaluate and determine the factors significantly associated with the compositional microbiota variation within the infant gut metagenomes. This study first identified technical factors introducing bias in taxonomic profiling (e.g., DNA extraction batch), which served as confounders in the analysis of environmental and host variables. The investigation of these biological factors indicates that pre-natal and peri-natal variables such as the mode of delivery significantly impact the infant gut microbiota, while we did not identify any significant impact of breastfeeding habits and medication exposures in this study.
  • Abbas, Salma Magdy Hussein Jr (2024)
    Elevated low-density lipoprotein cholesterol (LDL-C), hypercholesterolemia, is characterized by complex and poorly understood genetic contributions. Cellular LDL uptake mediated by the LDL receptor is pivotal to disease progression. After LDL internalization LDLR is recycled to the plasma membrane. Genetic mutations are known to exist in factors driving LDLR recycling but their contribution to hypercholesterolemia is not known. SNX17 has been postulated to be important for LDLR recycling. The goal of this study was to investigate the effect of SNX17 on cellular LDL uptake and to evaluate whether functional characterization of SNX17 gene variants can be performed. At the same time, adjusting an existing semi-automated analysis pipeline to generate expression constructs for SNX17 genetic variants. In this study, using an SNX17 knock-out cell line and an SNX17 rescue cell line (SNX17 knock-out cells transfected with GFP-SNX17 construct), it was shown that SNX17 might have a role in LDL uptake. The semi-automated workflow for generating genetic variants was successfully adopted to SNX17, warranting further experiments to define the optimal conditions for the functional characterization of SNX17 gene variants. This thesis sets the foundation for a deeper understanding of SNX17 in LDLR recycling and provides first insights into the potential regulation of this pathway, while also initiating the way for the later characterization of SNX17 variants. Hence, functional genomic studies together with the functional characterization of genetic variants in LDLR recycling factors can improve our understanding of how genetic variation contributes to disease progression and develop better risk assessment tools.
  • Uriona Egia, Garazi (2023)
    The ends of eukaryotic chromosomes are formed by a special heterochromatic structure, the telomere, which is essential to guarantee chromosome stability. Telomeres protect chromosomic ends from DNA degradation, repair, and recombination events. However, they are difficult to replicate due to their repetitive and heterochromatic nature, which hinder DNA replication fork progression. In yeast, Mph1 helicase promotes replication fork regression, cross-over suppression during homologous recombination (HR), and telomere maintenance. Moreover, Mte1 is a D-loop binding protein involved in response to DNA damage and maintenance of telomere length, which interacts with Mph1, thereby stimulating its regression capacity as a helicase and fork. Thus, the Mte1-Mph1 complex is recruited to stressed telomeres. Mte1 also shares a domain of unknown function, DUF2439, with Rad51 and Rdh54. Additionally, Esc2 protein is involved in the regulation of DNA damage through template switch (TS) recombination, preventing HR events caused by Mph1. This thesis aimed to uncover the potential roles and interactions of proteins involved in telomere maintenance, such as Mph1, Mte1, Esc2 and Rdh54, for which two main assays were conducted: (1) Telomere Stability assay, consisting of Tus/Ter barrier based on the high-affinity binding of the E. coli protein, Tus, to specific DNA sequence called Ter; (2) Template Switching assay, focused on the capability of the proteins in reconstructing a functional LYS2 gene by TS. The obtained results demonstrated that (1) the absence of Rdh54 enhances replication fork regression, (2) Mte1 and Esc2 show opposite roles in telomere maintenance, (3) the interaction between Mte1 and Rad51 plays a crucial role in ensuring telomere stability and nuclear foci formation, (4) Mph1 and Mte1 promote cell survival through the break-induced replication (BIR) pathway. Further studies should assess the plausible interaction between Mph1 and Rdh54 proteins and characterize the function and interplay of the proteins involved in TS.
  • Perkiö, Anna (2021)
    Long interspersed nuclear element 1 (LINE-1 or L1) belongs to a class of retrotransposons. In other words, it is a DNA element that can copy and paste itself around the genome. There are approximately 500,000 copies present in humans, but only around 5,000 are expected to remain transcriptionally competent. The activity of L1s is generally strongly repressed in normal human tissues, but in many cancers, these elements are reactivated. Both L1 transposition and transcription can have significant effects on cellular function, making it an interesting topic of research from a pathological point-of-view. By studying and understanding more about this transposon, it could be possible to find novel screening methods or even therapeutics for different cancers. One of these cancer types is high-grade serous ovarian carcinoma (HGSOG), which is known for exhibiting L1 upregulation. However, the quantification of L1 transcription has been proven to be very challenging, mostly due to alignment issues caused by the repetitive nature of the element. In addition, a large proportion of L1s reside within genes, meaning that L1 sequence -containing transcripts frequently do not originate from the L1’s own promoter. This thesis aimed to tackle these challenges; I quantified L1 expression at the single-locus level in 11 pre- and post-chemo HGSOC sample pairs, as well as in 5 samples from healthy women, based on single-cell RNA-sequencing. In addition to comparing L1 activity in different sample and cell types, I researched whether L1 activity was associated with any changes in gene expression. The poly(A) site of an L1 is relatively weak, meaning that L1 transcription frequently extends over it. Based on this fact, the utilized approach was to quantify L1 expression based on reads mapping to the 1 kilobase downstream window of each L1 locus, thus minimizing the alignment issues of repetitive elements. Thereafter, the features of the detected loci were carefully assessed to separate false-positive L1s from those with evidence supporting genuine activity, such as tumor sample enriched expression, lack of correlation to host gene, and detection with bulk RNA-sequencing. The activity of the latter loci was then further analyzed to search for differences in L1 expression between pre- and post-chemo samples. In addition, the association between L1-activity and gene expression was examined based on regression models both at the individual gene and molecular signature gene set-level. It was found that L1 expression data is filled with factitiously active loci, highlighting the importance of careful analysis and wet lab validations when studying transposon activity. However, regardless of the issues arising from a sparse and unreliable dataset, I showed that L1 activity was negatively associated with the expression of MYC target genes. MYC has been previously shown to be a transcriptional repressor of the L1, indicating that the obtained results are legitimate. Even though the results obtained from this study appear to be biologically justifiable, they would require further validation to ensure their authenticity. In addition, for the future it would be essential to enhance the sensitivity of the utilized workflow to minimize the sparsity of the data, so that statistical analyses performed would become more reliable. Nevertheless, it was shown that assessing L1 expression at the single-cell level using RNA-sequencing is executable.
  • Dreilinger, Olivia (2023)
    Animal coloration is as striking as it is diverse; however, the transcriptional basis of coloration is not deeply understood. Cichlid fishes are a tractable system for studying coloration as they exhibit a wide range of phenotypic diversity while remaining genetically similar. This facilitates the study of genotype-phenotype correlations and the identification of causative genes. RNA sequencing is a powerful approach to investigate the genes which characterize chromatophores. However, RNA-seq results can be plagued by the high abundance of rRNA in cells. This thesis aims to investigate differential gene expression between differently pigmented regions as well as explore the effects of tissue treatments and rRNA depletion on gene expression. Gene sets acquired with polyA selection, riboPOOL probes optimized for zebrafish, and zebrafish probes complemented with newly designed riboPOOL cichlid probes were compared to assess the functionality of these different rRNA depletion strategies. The use of zebrafish probes complemented with newly designed cichlid probes captured the greatest diversity of genes, many transcripts of which were missing from the other gene sets. Furthermore, as experiments such as scRNA-seq rely on a dissociation step, the effect of dissociation on gene expression was examined and found to promote the expression of stress response genes. The results of this upstream optimization were applied in the analysis of differential gene expression between the vertical stripes of the cichlid Pseudotropheus demasoni to better understand the molecular basis of vertical striping in fish. The dark stripes exhibited upregulation of melanic marker genes and the light, iridescent stripes showed an increase in iridophore marker gene expression. These findings were corroborated with cell count data from FACS to link transcriptional profiles and cell type quantifications. Overall, the study provides insight into the transcriptional basis of coloration in cichlid fishes and underscores the importance of optimizing methods drawing meaningful conclusions.
  • Iacoviello, Francesco (2022)
    Neurodevelopmental disorders (NDDs) are disabilities in which the formation and development of the central nervous system is altered. NDDs severely impact the quality of life of the individuals that are affected by them, however little is known about the causes or the molecular mechanisms that are behind their onset. For this reason, being able to model them is pivotal to our society since, by understanding the mechanisms underlying such disorders, we could develop possible treatments. Previous research has suggested that disturbances in the early neuronal development could be at the basis of NDDs onset. Therefore, in this work, I have modeled neuronal differentiation in Kabuki syndrome (KS), a known NDD, assaying the expression of key early neurodevelopmental markers at four specific timepoints, using induced pluripotent stem cell (iPSC) technology. By concurrently differentiating three KS patient-derived and three control iPSC lines to neural precursor cells (NPCs) and profiling them with immunocytochemistry (ICC) and quantitative real-time PCR (RT-qPCR), I was able to identify differences in the early developmental trajectories of NPCs between the two conditions. The ICC data suggested that differentiating KS cell lines incur in precocious differentiation when compared to control cell lines, suggesting that the disease-causing mutations could lead to accelerated neuronal maturation of early NPCs. However, RT-qPCR analysis of the expression patterns of key neurogenesis markers was unable to statistically confirm the observed trend between the two phenotypes, likely due to limitations in statistical power. Despite this, the expression of four out of seven NPC markers was higher in early KS cells than in control cell lines, supporting the hypothesis of accelerated neuronal maturation. Taken together, this work highlighted some of the challenges related to iPSC-based disease modelling studies, and the need to further confirm the inferred mechanisms of asynchronous neuronal development observed in this work.
  • Sundaresh, Adithi (2022)
    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.
  • Patrikainen, Linda (2023)
    Breast cancer is globally the leading cause of death in women. ER positive, HER2 negative breast cancer is the most common subgroup, covering two thirds of all breast cancer cases. The different isoforms of ERα, ERα66 and ERα36 are responsible of genomic and non-genomic ER signaling respectively. Tamoxifen is one of the most used drugs in ERα+ breast cancer. As a SERM tamoxifen blocks the activity of ERα66, but plays as an agonist for ERα36, which is associated with tamoxifen resistance. Tamoxifen resistance concerns more than 25% patients with ERα+ breast cancer but the molecular mechanisms that lead to development of resistant disease remain uncovered. Thus, the aim of this thesis was to reveal how two different ERα isoforms are used and regulated in tamoxifen resistance in two commonly used ERα+ breast cancer cell lines MCF7 and T47D. We studied the effect of hormones to tamoxifen sensitivity and to utilization of ERα isoforms. Additionally, we compared the transcriptomics of resistant and parental cells in both cell lines and tested how inhibition of key regulators affect the sensitivity against tamoxifen. In this thesis we report that MCF7 and T47D cell lines obtain different mechanisms of tamoxifen resistance, and that the development of tamoxifen resistance is a parallel process with the cell identity switch from luminal to basal. The EZH2 is involved in maintaining the luminal progenitor type of mammary cells, whereas c-Myc is highly expressed in the resistant cell lines. Hence, EZH2 and c-Myc are key players in development of tamoxifen resistance and could be considered as therapy targets in ERα+ breast cancers.
  • Talka, Markus (2022)
    Acute leukemia is a life-threatening disease of blood and bone marrow, which is caused by malignant transformation of immature white blood cells. These malignant white blood cells invade space in bone marrow decreasing its ability to produce normal blood cells, eventually leading to death within weeks after the diagnosis without treatment. The acute leukemia can be broadly divided into its lymphoblastic and myeloid form, based on the affected cell lineage. Furthermore, acute leukemias can be classified based on different genomic features, such as gene fusions. Fusion genes are strong drivers in various cancers such as acute leukemias, and they are formed when two or more original genes join together forming a novel hybrid gene. If the novel hybrid gene is transcribed, it can lead to a translation of an abnormal fusion protein with altered function. The detection of the gene fusions is very important, since it affects to diagnosis and treatment of the patient. Various techniques can be used for fusion gene detection, of which the RNA sequencing is the method of choice, due to its ability to provide an unbiased identification of all known and novel gene fusions from the sample in a single experiment. In this thesis, the overarching aim was to develop an optimal sampling protocol for fusion gene detection using RNA sequencing for acute leukemia diagnostics. First, the whole blood samples in EDTA-tubes were collected from acute leukemia patients based on the findings from routine diagnostics. Next, the RNA was extracted at three different timepoints (0h, 8h, and 32h). The samples were stored at 4°C between the extractions. Finally, the RNA sequencing libraries were constructed, and the RNA sequencing was performed. After the sequencing, the data was analyzed using the FusionCatcher algorithm for fusion gene detection and the EdgeR-package for differential expression analysis. The FusionCatcher detected the same gene fusion in all the four fusion gene positive patients compared to routine diagnostics. However, the FusionCatcher failed to recognize the gene fusion in some of the samples with very low number of fusion breakpoint-spanning reads. These reads were visualized with IGV, suggesting that the detection failure resulted from the very low number of break-point-spanning reads. Furthermore, the sample storage did not affect on gene fusion detection. In addition, FusionCatcher detected PIK3AP::BLNK gene fusion from one of the fusion gene negative patients, suggesting a possibility that the patient truly was fusion gene positive. The differential expression analysis revealed changes in gene expression between the different timepoints. The results showed changes in various pathways related for example to cell death and protein biosynthesis, but also to pathways related to cancer. The results showed that prolonged sample storage alters the gene expression profile thus affecting the results of a gene expression study.
  • Bodington Celma, Silvana (2024)
    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.
  • Owusu, Rafaela (2022)
    High-throughput sequencing techniques make it possible to identify DNA variants at a reasonable cost, representing a first-tier diagnostic test for rare mendelian diseases. However, a substantial number of variants identified through the analysis of sequencing data are frequently classified as variants of uncertain significance (VUS). Accordingly, only 30–60% of individuals receive a conclusive molecular diagnosis depending on the clinical phenotype. Reanalysis of older sequencing data has been encouraged by recently developed and improved methodologies for analysis and more robust bioinformatic pipelines to enhance variant interpretation and raise the diagnostic/detection rate. This study focused on reanalyzing data from a targeted gene panel, MYOcap, a targeted gene panel for patients with neuromuscular disorders. The aims were to find elusive (i.e., previously undetected/misinterpreted) variants in patients still missing a molecular diagnosis and, by using novel bioinformatic tools, focusing on pathogenic and likely pathogenic variants (according to ACMG guidelines) in Varsome as well as on variants affecting the splicing as predicted by SpliceAI. With this setting, the detection rate of solved cases increased by 2,7% in the first cohort and 0,5% in the third. This study suggests that additional data, such as segregation data or transcriptomic and proteomic data are essential for reducing the number of VUS and increase the detection rate. Notably, this study represents an essential first step of a larger reanalysis project, aiming at providing a diagnosis to an increasing number of myopathy patients.
  • Rinas, Anastasia (2024)
    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.
  • Hiltunen, Antti Olavi (2022)
    Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases and has the worst clinical outcome. Characterizing features of TNBC are high recurrence and mortality rates, and the absence of three commonly targetable breast cancer biomarkers estrogen receptor, progesterone receptor, and HER2, limiting the number of targetable therapy options. Cytotoxic CD8 positive T cells play a crucial role in the anticancer immune response and act as a major component of successful cancer immunotherapies. However, cancer cells can evade T cell-mediated killing by overexpressing programmed death-ligand 1 (PD-L1) resulting in T cell exhaustion and limited immune response via the interaction with programmed death protein 1 (PD-1). Systemic anti-PD-L1/PD-1 therapies aim to prevent this immunosuppressive mechanism, but they are burdened with potentially life-threatening autoimmunity-type adverse effects. Therefore, cancer cell-specific targets to downregulate PD-L1 could offer efficacious and less harmful ways to overcome PD_L1/PD-1 mediated immunosuppression. Serine protease hepsin is commonly overexpressed in many solid tumors where it is responsible for the activation of HGF/MET signaling pathway as well as degradation of desmosomes and hemidesmosomes leading to the loss of epithelial integrity, invasion, and metastasis. Earlier studies have linked hyperactive HGF/MET pathway to the upregulation of immune checkpoint molecule PD-L1. In this thesis, I show how pharmacological inhibition of hepsin leads to decreased MET activity and downregulation of PD-L1 in a panel of TNBC cell lines. My results demonstrate the potential of hepsin-mediated regulation of PD-L1 in tumor immunosuppression, and hint at the potential of hepsin as a therapeutic avenue towards safe and efficacious immunotherapy in the future. These results are part of a larger study addressing the role of hepsin as a regulator of PD-L1 breast cancer.
  • Begum, Sakina (2021)
    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.
  • Halme, Milla Marjaana (2022)
    Colorectal cancer is one of the most common cancers in the world, and in 2020 it was the cause of nearly 1 million deaths. A major reason for the high death rate is treatment resistance; eventually, almost all colorectal cancer patients with metastatic disease stop responding to chemotherapy. The problem of treatment resistance is not specific to this type of cancer, but it is a widespread issue for all cancer treatments. Chemotherapy resistance is the sum of several cellular and non-cellular factors that together enable sustained cell growth despite the treatment. The non-cellular factors are related to the tumor microenvironment, whereas the cellular factors are related to changes in gene expression, which facilitate e.g. the repair of drug-induced damage or lead to changes in drug metabolism. Lately, researchers have turned their interest to translational control in chemotherapy resistance. This is because translational control plays a major role in stress adaptation. During cellular stress, global translation rates are reduced and those messenger RNAs that are most important for cell survival are translated efficiently. Moreover, translation is fine-tuned by transfer RNA (tRNA) modifications. These modifications are chemical groups that are added to the ribose and the nucleobase of the tRNA molecule, and they affect all aspects of tRNA function, ranging from the structure and stability of the molecule to reading frame maintenance and rate of translation. tRNA modifications are dynamic and change in response to the cellular state, thus facilitating adaptation by translational control. Given the major role of translational control and tRNA modifications in cellular stress responses, their role in the chemotherapy response and adaptation should be thoroughly investigated. The aim of this thesis was to study how chemotherapy affects translation and tRNA modifications in a colon adenocarcinoma model. The cell lines SW480 (from a primary colorectal tumor) and SW620 (from a metastasis) were treated with 5-fluorouracil, oxaliplatin, and leucovorin (FOLFOX), a common combination of chemotherapeutics used in colorectal cancer treatment. The cells were subjected to long-term cyclic treatment as well as 24 h pulse treatment. Chemotherapy resistant cell lines were established by increasing the concentration of FOLFOX for each round of treatment. The effect on translation was studied by polysome profiling, which revealed that FOLFOX treatment causes immediate translational stress, as evidenced by the “shoulders” in the polysomal fractions in the profiles of the pulse treated cells. We hypothesized that these shoulders represent halfmers, polyribosomes without the large subunit. No difference was observed between the long-term treated cells and controls, possibly indicating that the cells had adapted to FOLFOX. The resistant cells exhibited slightly reduced translational activity, which might be due to altered function of ribosomes following the exposure to 5-fluorouracil. Changes in tRNA modification levels were quantified by liquid chromatography mass spectrometry. Several anticodon loop modifications exhibited altered levels after the pulse treatment. In addition, 5-FUrd, a metabolite of 5-fluorouracil, was incorporated into the tRNA. The long-term treated or resistant cells exhibited no differences in the modification levels. In conclusion, this study provided insights on the immediate effects of FOLFOX treatment on translation. This constitutes the first step towards understanding how RNA-based regulatory mechanisms may contribute to the effect and possible resistance to chemotherapy.