Browsing by study line "Genetiikka ja genomiikka"
Now showing items 21-40 of 62
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(2020)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.
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(2022)Every year in the western world 3–5% of newborns suffer permanent damages due to prenatal alcohol exposure. Alcohol causes the symptoms of Fetal Alcohol Spectrum Disorders (FASD), which consist of various structural, cognitive, and behavioral neurological defects and distinctive craniofacial features, although in many cases the condition is undiagnosed. The frequency, amount, and timing of alcohol consumption during pregnancy critically influence the symptoms and their severity. Despite the serious consequences and frequent incidence, there is still no clear information on the etiology of FASD symptoms or the timing specific effects of alcohol. However, it has been hypothesized that the early pregnancy is especially susceptible to environmental exposures, such as alcohol, because there is rapid cell proliferation, cell differentiation, and epigenetic reprogramming taking place in the embryo. Gastrulation is a crucial developmental stage in early embryonic development where the three germ layers, endoderm, mesoderm, and ectoderm form and create a foundation for all further development. The aims of this thesis are to study how alcohol affects the gene expression in undifferentiated human embryonic stem cells (hESCs) compared to cells differentiating into the germ layers, and how the gene expression in each of the germ layers is affected. To study the differentiation in gastrulation, hESCs were differentiated in vitro under alcohol exposure to endoderm, mesoderm, and ectoderm with STEMdiff™ Trilineage Differentiation Kit. Gene expression in differentiated germ layers and undifferentiated hESCs was analyzed with 3’mRNA sequencing. The results show that the number of genes with alcohol-induced differential expression is considerably higher in hESCs than in the germ layers indicating that undifferentiated hESCs are more susceptible to alcohol than differentiating cells, which is in agreement with findings from previous studies. In the germ layers, alcohol affected the expression of many genes involved in developmentally important signaling pathways such as FGF, Wnt, and TGF-β. Each of the germ layers have different gene expression profiles and accordingly, they exhibit a unique response to alcohol. Furthermore, the differentially expressed genes reveal intriguing connections to the FASD phenotype, notably, in ectodermal cells alcohol caused differential expression in many genes related to neurodevelopment.
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(2023)The intestinal stem cells (ISCs) adapt in response to environmental factors and continually proliferate to renew the mammalian intestinal epithelium due to its rapid turnover. Overall, intestinal homeostasis is maintained by the differentiation and self-renewal of ISCs, which are regulated by different mechanisms, including epigenetic histone modifications. Earlier studies in the host laboratory have shown that the histone methyltransferase Su(var)3-9 is essential in the nutrient-induced activation of intestinal stem cells. Su(var)3-9 specifically trimethylates histone H3 on lysine 9 (H3K9me3), which is a repressive histone mark, responsible for transcriptional silencing at heterochromatin regions. It influences stem cell maturation, lineage specification, and many other cellular processes. However, the precise mechanisms behind its function in ISCs remain unknown – that knowledge is important for understanding the development of many diseases, including cancer and metabolic disorders. This thesis aimed to investigate the distribution of the heterochromatin mark H3K9me3 in the intestine with an emphasis on ISCs, using the Drosophila midgut and mouse intestinal organoids as models. Confocal microscopy was used together with cell-type-specific fluorescent staining, to obtain the expression of the H3K9me3 specific histone methyltransferase Su(var)3-9, in the midgut. An antibody was used for the detection of H3K9me3 distribution along the anterior/posterior axis in Su(var)3-9 overexpressed flies. Additionally, DNA adenine methyltransferase identification (DamID) was applied in order to find target genes of the H3K9me3 regulation in the genome with the specific chromo domain of M-phase phosphoprotein 8 (MPHOSPH8) that binds to H3K9me3. The number of lineage-labeled differentiated enterocytes was shown to be locally higher in the Su(var)3-9 overexpressed flies compared with the control, although the flies were on starvation without nutrient-induced activation. Moreover, the number of lineage-labeled progenitor cells was not remarkably altered between the samples. However, the intensity of H3K9me3 was significantly higher throughout the whole midguts in the Su(var)3-9 overexpressed flies in comparison to the control. According to one replicate, the DamID in mouse intestinal organoids revealed that the peaks of H3K9me3 were divergent between the samples grown in different conditions. The first sample was assumed to contain more ISCs, whereas the other one was assumed to contain more differentiated intestinal cells. According to my results, the Su(var)3-9 overexpression drives the stem cells against the differentiation of enterocytes. Furthermore, the MPHOSPH8 chromo domain in the organoids was successfully applied in DamID; thus, more replicates should be prepared for additional analysis, because I found several potential target genes of H3K9me3. In the future, it is important to further study the epigenetic regulation of ISCs, for applying the epigenetic marks as targets for the treatment of many human pathophysiological conditions, such as cancer, obesity, and metabolic disorders.
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(2022)Schizophrenia is a debilitating psychiatric disorder associated with reduced life expectancy. The biological mechanism of schizophrenia is nebulous; however, many findings point to the central nervous system and neurons, where a reduction in dendritic spines has been indicated by previous research. The genetic findings support the involvement of synapses in the pathogenesis of schizophrenia. To study the biological properties stemming from genetics, relevant model systems and efficient methods are needed. Induced pluripotent stem cell (iPSC) technology offers a robust method for modeling the biological processes underlying schizophrenia. Somatic cells, e.g. fibroblasts, can be reprogrammed back to a pluripotent state resembling embryonic stem cells, and further differentiated into any cell type of the body, which might not be otherwise accessible. This allows establishing and characterizing neuronal cultures from patient and control cell lines, potentially revealing biological differences associated to the disease phenotype. The field of schizophrenia research has adopted iPSC technology and multiple studies have been conducted. These include assessments of synaptic density in the produced neuronal cultures, many of which reported decreased density associated with schizophrenia. In this thesis, a modified version of Nehme et al. (2018) protocol was used to differentiate iPSCs into neurons in co-cultures with human iPSC-derived astrocytes. The overarching aim was to construct an immunocytochemistry (ICC) -based assay to measure synaptic density in the produced co-cultures. First, suitable markers for characterization by ICC were tested and selected. The markers were selected to inform about neuronal identity, maturity, and synapses of the differentiated neurons. Next, the culturing conditions were optimized regarding the cell density and coating of the culturing wells. Finally, to estimate the utility of the assay, a pilot study was performed with three cell lines derived from a healthy control and a monozygotic twin pair discordant for schizophrenia. iPSCs from these cell lines were differentiated into neurons in co-cultures with astrocytes, and then characterized with ICC using selected markers and image analysis software. The synaptic density was quantified for each cell line. The performance of the assay was evaluated with analysis of variance (ANOVA) and restricted maximum likelihood model (RELM). An assay to quantify synaptic structures in mature neurons was established. The average synaptic density for all cell lines was approximately 1 synapse per 100μm of neurite. Analysis of the data produced with the assay revealed a notable batch effect and technical variation. This suggests that further optimization is needed to reduce variance from undesired sources. The pilot data suggests that the differences in synaptic density between cases and controls may be modest, further highlighting the need for minimizing noise in the assay to improve signal to noise ratio. However, indicated by power analysis, large sample sizes are needed to identify meaningful differences between cases and controls. In light of these results, more attention should be drawn to the methodology in the field of iPSC-based studies, as the principals of the assay constructed here were similar to other synaptic assays used in previous publications.
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(2023)The group has identified two rare, previously uncharacterized missense variants in the YBX3 gene in a Finnish patient presenting with an unusual form of nemaline myopathy. The patient also inherited two biallelic TPM3 variants, one RYR1 variant from the father and one SRPK3 variant from the mother. TPM3 and RYR1 are known nemaline myopathy causing genes and the other variants identified in the patients, including the YBX3 variants, are thought to have a modifying effect on the phenotype. YBX3 encodes Y-box binding protein 3 (YB-3) and, YB-3 is a member of the Y-box binding (YB) protein family, that in addition to YB-3 consists of YB-1 and YB-2. The YB-proteins have mainly been studied in the context of cancer, with most studies focusing on YB-1. Studies indicate the ability of YB-proteins to compensate for the loss of one homolog suggesting functional redundancy between YB-3 and YB-1, and YB-3 and YB-2. Compared to its homologs, YB-3 is highly expressed in skeletal muscle. The aim of this thesis was to try out a new cell culturing method when investigating the role of YB-3 in the differentiation of myoblasts into myotubes. MSY-3 is the murine orthologue of YB-3. MSY3-knockdown mouse C2C12 myoblast lines were established using GIPZ lentiviral short hairpin constructs and by selection with puromycin. The success of transfection was determined using qPCR. The myoblasts were differentiated for 20 days on a gelatin hydrogel surface to support long-term culture and to provide phenotypes of higher physiological relevance with improved contractile maturity. Myoblasts cultured on coverslips were immunofluorescently stained for MSY-3. HeLa cells were transfected with a construct encoding N-terminally FLAG-tagged human YB-3 in a pcDNA-vector. YB-3-FLAG was purified using anti-FLAG magnetic beads. The eluated immunoprecipitation sample was sent to N-terminal sequencing to obtain information on post-translational modifications, to support further experiments regarding the post-translational cleavage of YB-3. N-terminal sequencing revealed an enrichment of YB-3 and YB-1 in the immunoprecipitation sample but not of YB-2, and previously undescribed post-translational modifications were identified. The MSY3-knockdown myotubes exhibited no spontaneous twitching on the hydrogel, while the control C2C12 myotubes twitched frequently. Misalignment of the MSY3-knockdown myotubes and changes in morphology was also observed in one of the MSY3-knockdown cell lines. This suggests that differentiating myoblasts on gelatin hydrogel is a potential strategy for studying the functions of YB-3 in myoblast differentiation and to elucidate its role in skeletal muscle.
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(2020)Leaf senescence is a developmental and physiological phase in plants to end leaf development. Environment factors such as drought stress, extreme temperature, and pathogen threat and internal factors including age and reactive oxygen species induce leaf senescence. Some phytohormones such as jasmonic acid and salicylic acid play a key function in cell death in plants. WRKY transcription factors is known as one of the largest transcription factor family in plants which regulates a variety of plants processes. WRKY75 which belong to WRKY transcription factors has shown multiple functions in plant development like regulation of Pi starvation responses and root development and flowering. In my thesis, I focused on the role of WRKY75 in senescence and stress responses. WRKY75 was identified as a positive regulator of cell death in Arabidopsis. WRKY75 can promote salicylic acid biosynthesis by promote transcript levels of SID2 and also cause hydrogen peroxide accumulation by suppressing the transcription of CAT2. Hydrogen peroxide and salicylic acid can promote WRKY75 transcription at the same time. To evaluate the function of WRKY75 transcription factor in SA signalling and cell death, three lesion mimic mutants acd5, cat2, dnd1 and their corresponding wrky75 double mutant were used. Interestingly, no different phenotypes were found between acd5, cat2, dnd1 and their corresponding wrky75 double mutants in cell death and hydrogen peroxide accumulation detection in Arabidopsis leaves. Meanwhile, marker genes transcription levels were not different in both short day and long day growth condition. However, different phenotypes were observed in botrytis infection. Based on these results, we formed a hypothesis that gene redundancy could influence genetic characterization of WRKY75. To overcome this problem, SRDX-WRKY75 chimeric repressor transgenic lines were generated. The SRDX domain act as a dominant negative regulator to suppress WRKY75 target genes. In future research, these new lines can be used to test transcript levels for putative WRKY75 target genes.
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(2021)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.
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(2023)Progressive retinal atrophy or PRA is a collective term for a group of hereditary degenerative retinal diseases in dogs. PRA affects the photoreceptor cells of the eye ultimately progressing into complete vision loss. Documented in over 100 breeds, it is the most common type of canine retinal diseases. PRA is considered a homologous disease to human retinitis pigmentosa, thus providing a large animal model for studying retinal biology and genetic aetiology of its diseases. The objective of this thesis was to study the genetic cause of a novel form of PRA in young Finnish Lapphunds. Analysis built upon a combination of gene mapping methods and analysis of next generation sequencing data. Gene mapping was performed with two analysis methods, genome-wide association study and homozygosity mapping, utilising single nucleotide polymorphism microarray based genotype data. Identifying a clinical phenotype from the canine biobank at the University of Helsinki resulted in a study cohort of six case and 10 control dogs. Combined with pedigree information, this early-onset PRA was most likely a new autosomal recessive condition in the breed. Genome-wide analyses resulted in the discovery of a disease-associated locus on chromosome 27. Findings of single nucleotide variant filtering of one whole-genome sequenced affected dog led to the prioritisation of an intronic substitution variant (T > C) in SOX5 gene as a potential cause of PRA. Genetic validation of the variant with 23 dogs showed promising results. Four out of five affected dogs were homozygous for the variant, while controls were either wild-type or heterozygotes. As a result, a previously unknown disease locus was successfully identified, suggesting a possible new spontaneous canine model of retinitis pigmentosa. By better understanding the pathophysiological processes of disease, improved diagnostics and marker-based testing as well as novel therapies can be developed for both dog and man. However, further studies are needed to understand the underlying molecular mechanism of the candidate disease variant.
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(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.
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(2021)Henoch-Schölein purpura (HSP) is a vasculitis of small vessels and its characteristics include abnormal accumulation of IgA immunocomplexes on vessel walls as well as abnormal glycosylation patterns of IgA. HSP is an autoimmune disease like inflammatory bowel diseases (IBD). The genetic background of HSP has not been studied in Finnish population before, and only one genome-wide association study has been conducted for HSP before. Therefore investigating the Finnish genetic associations of HSP on a genome-wide level is of value. In this study the genetic background of HSP is studied with genome-wide association analyses performed on 424,041 genotyped SNPs passing quality control, HLA alleles imputed from the SNPs, and for their allele-level HLA protein sequences with the aim of replicating previous HSP associations in a Finnish cohort. There were 46 HSP individuals and 18,757 controls (216 bone marrow donors and 18,541 blood donors) passing quality control and included in the study. R package HIBAG was used for HLA imputation, and SPAtest package was used for the association analyses. In the association analyses, a region in chromosome 6 passed genome-wide significance (SNP with the smallest p-value: p 6,57 x 10-10, OR 0.14[0.1-0.2]) and the region contained both predisposing and protective associations. Of HLA alleles, DQB1*05:01, DQA1*01:01 ja DRB1*01:01 surpassed genome-wide significance level (p values 4,99 x 10-9, 1,04 x 10-8 and 2,37 x 10-8, respectively) and were positively associated with HSP. Five amino acid positions were significantly associated with HSP (p-values 3,9 x 10-10, 7,37 x 10-9, 1,26 x 10-8, 1,69 x 10-8 and 2,41 x 10-8), being both protective and predisposing to HSP. In addition, the genetic background of HSP was compared with that of IBD by comparing their GWAS results of genotyped SNPs, HLA alleles and their protein sequences. There were 49 IBD patients after quality control, and the same controls as for HSP (18,541 individuals) were included in the association analyses of IBD. The diseases seem to share some of their genetic background. According to the results, HSP seems to associate primarily with HLA class 2 and the result is also compatible with previous studies linking HSP to this region. The results also replicate previous GWAS findings in HLA class 2. According to this it is likely that the same HLA alleles are notable genetic factors in both Finnish and Spanish populations. The connection between HSP and IBD could potentially have to do with intestinal microbes aiding the onset of autoimmune diseases in genetically susceptible hosts.
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(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 et.al. 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.
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(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.
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Identifying the disease-causing variant in a large family with a late-onset dominant distal myopathy (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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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Modeling early neuronal development in Kabuki Syndrome using human induced pluripotent stem cells. (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.
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