Browsing by master's degree program "Translationaalisen lääketieteen maisteriohjelma"
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(2019)Sleep difficulties have been on the rise for the past decade. Insomnia and sleep difficulties have associations with an increased risk of overall mortality, as well as with a diverse array of complex diseases, such as coronary heart disease, major depressive disorder, fibromyalgia and Alzheimer’s disease. Epigenomics provides information on how environmental factors influence the genome via epigenetic mechanisms, such as DNA methylation. Thus far, epigenome-wide association studies looking at the effects of sleep disturbances on the methylome have provided evidence of distinctive methylation patterns in insufficient sleep, involving biological processes related to neuroplasticity and neurodegeneration. However, more knowledge is needed to determine how the severity of sleeping difficulties influence the methylome. This thesis investigates the effects of increasing sleep difficulties on DNA methylation with an epigenome-wide association study. The study sample is derived from the Health 2000 general population survey. Subjects were divided into three different groups by their self-reported level of sleeping difficulty, and methylation measurements performed from whole blood samples utilizing the Illumina Infinium MethylationEPIC kit, encompassing >850,000 CpG sites. To identify differentially methylated sites, a multivariable regression model was used with age, gender, smoking, alcohol use, cell type distribution and plate and array data as covariates. None of the differentially methylated CpG sites identified remained significant after multiple testing correction. To gain more information regarding which biological processes the methylated sites may be part of, those CpG sites with an uncorrected p-value of <0.0005 were subjected to pathway analysis. Notable significant pathways included oxytocin- and serotonin receptor-mediated signalling pathways and Alzheimer’s disease-amyloid secretase pathway. Altogether, six pathways remained significant after multiple testing correction, with a total of 12 different genes appearing in them. Furthermore, a post-hoc regression analysis was conducted between these 12 genes and their corresponding CpG sites, and health-related quality of life questionnaire responses. Significant results included associations between sleep, and discomfort and symptoms (including pain). As an additional analysis, a database search was conducted to learn more about the genes’ functionality at the level of phenotype. Results included some variant trait associations to sleep, Alzheimer’s disease and cognitive performance. The associations to Alzheimer’s disease and cognitive performance warrant further research with a similar additive model, perhaps with a larger sample.
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(2019)Liver Kinase B1 (LKB1), also known as STK11, is a well-known tumor suppressor and a metabolic regulator, mutated in Peutz-Jeghers syndrome (PJS) and other sporadic cancers. LKB1 regulates several cellular functions: metabolism, polarity, cytoskeleton organization, differentiation, and proliferation by activating 14 AMPK-related downstream substrates. In a recent study, LKB1 was found to maintain intestinal homeostasis by repressing ATOH1 with the involvement of pyruvate dehydrogenase kinase 4 (PDK4). ATOH1 is a transcription factor and master regulator of secretory lineage in the intestine. It has been reported that ATOH1 is epigenetically regulated in inner hair cells and intestinal epithelium via Polycomb repressive complex 2 (PRC2). However, the repression mechanism of ATOH1 by LKB1 is currently unknown. This study aimed to determine the molecular mechanism of ATOH1 repression by LKB1. In this study, Ls174t, a human colorectal adenocarcinoma cell line, was used to investigate ATOH1 induction by LKB1 signaling from two angles: First, involvement of LKB1 downstream substrates was investigated using shRNA mediated knockdown screen. Interestingly, silencing of either MARK4 or SIK3 alone was found to induce ATOH1 and thus mimic silencing of LKB1 unlike other LKB1 substrates including AMPK kinases. A second angle explored possible epigenetic mechanism in ATOH1 repression pathway, using dichloroacetate (DCA, a PDK4 inhibitor) and GSK126 (a PRC2 inhibitor). DCA treatment resulted in no change on the global levels of histone modifications tested. In addition, GSK126 caused the downregulation of ATOH1 in both control and LKB1 depleted condition. Thus, this study concludes that LKB1 represses ATOH1 through MARK4 and SIK3 and that global changes in the histone modifications investigated are not involved in the mechanism.
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(2019)Creatine is a crucial metabolite for chordates, with critical roles in energy transport and buffering, as well as in brain function. The creatine transporter (CRT) is the ubiquitous symporter of creatine in the cell membrane, allowing for the biosynthesis and trafficking of creatine between cells and tissues. Mutations in SLC6A8, the human gene encoding CRT, can cause an X-chromosome linked form of creatine deficiency syndrome, commonly leading to intellectual disability, developmental delay, and seizures. This study details the clinical report and molecular confirmation of a novel mutation in the SLC6A8 gene, Tyr553Asp, in a male patient with metabolic encephalopathy. It is the first mutation of the gene discovered in Finland, leading to a typical creatine deficiency syndrome. The genetic and biochemical confirmation of the mutation pathogenesis is followed up by the in-silico homology modelling of the CRT structure in different conformations of its transport cycle, and an interpretation of the mutation’s predicted structural and functional consequences. These interpretations then prompted a proposed model for the function of the extracellular loops in CRT. The results implicate the understudied extracellular loop 6, the locus of the mutation, as being involved in substrate luring and transient binding as the protein releases its previous substrate into the cytoplasm. These findings shed light on a previously unknown mechanism in creatine transport and elucidate potential therapeutic targets.
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(2017)Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. It arises from skeletal muscle stem cells, which fail to differentiate. Multimodal treatment approach has improved the outcome of RMS during the recent years. However, in case of relapsed or metastatic disease, the prognosis is still very poor. This indicates a high demand for novel targeted therapy approach for RMS. Prox1 transcription factor has been shown to regulate myoblast differentiation into skeletal muscle fibers. Our unpublished observations indicate that Prox1 is highly expressed in RMS tumors and that it is essential for RMS cell growth. The aim of this study was to find novel drug candidates for RMS treatment depending on Prox1 and/or its downstream signaling. High-throughput drug screening with 528 oncology compounds was tested on wild-type and Prox1 silenced rhabdomyosarcoma cells (RD cell line). Gene silencing was performed via lentiviral vectors encoding shRNA for Prox1. In the screening results, we focused on the drugs, which were more potent in Prox1 silenced cells with nano- or micromolar concentrations compared to the wild-type cells. The results revealed 7 potential groups of inhibitors, which had superior suppressive effect on RMS cell viability specifically when Prox1 was silenced. In vitro validation of high-throughput screening results by MTT and luciferase assays confirmed the results. Based on the magnitude of their inhibitory effect and information available on these compounds, three drugs were chosen for further investigation. Two of these compounds also potently inhibited the growth of patient-derived primary RMS cells, which we obtained from the Helsinki University Hospital and named KLHEL1. These drugs were also less toxic to healthy myoblasts. In addition, these two compounds significantly decreased Prox1 mRNA and protein levels in wild-type cells, and completely inhibited the ability of both RD and KLHEL1 cells to form colonies. Combinational exposure to these inhibitors further enhanced the effect compared to a single agent treatment. The present findings demonstrate a potential for repurposing of these drugs for targeted treatment in rhabdomyosarcoma expressing high Prox1 levels.
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(2019)Lipid droplets (LDs) are ubiquitous intracellular storage organelles, consisting of a core of energy rich neutral lipids surrounded by a phospholipid monolayer. Research in the past decade has expanded the view on LDs from simple, passive cytosolic inclusions to dynamic organelles which play an important role in many cellular processes. Furthermore, there is mounting evidence for links between LD biology and human pathologies, such as metabolic disorders, non-alcoholic fatty liver disease and cardiovascular diseases. Thus, understanding the basic biology of LD formation is crucial. LD biogenesis is thought to occur in the microdomains of endoplasmic reticulum (ER), due to the accumulation of neutral lipids between the two leaflets of the ER bilayer before budding into the cytosol. Many proteins are involved in this early formation, but no single indispensable protein has been discovered. After assembly, these early LDs grow through lipid deposition from the ER, and with lipid synthesis on the droplet monolayer. During LD growth, LDs are thought to retain connection to the ER. A protein important for LD biogenesis is seipin. This oligomeric ER protein has been found to localize at contact sites between the ER and LDs. Mutations in seipin give rise to three distinct diseases in humans; BSCL2, seipinopathy and Celia’s encephalopathy. The role of seipin in the formation of LDs and the pathogenesis of these diseases is still unknown. Work from numerous model systems has shown seipin to be important for LD biogenesis and adipocyte differentiation. LD formation is a complex process which is still poorly understood, and seipin likely collaborates with other proteins during LD assembly. In this thesis, APEX2-mediated proteome mapping combined with LC-MS/MS, is set up to identify proteins involved in LD biogenesis. In this technology, an engineered ascorbate peroxidase, APEX2, is genetically inserted to the intracellular region of interest where it rapidly biotinylates nearby endogenous proteins upon exposure to biotin-phenol and hydrogen peroxide. Biotinylated proteins can then be enriched by using streptavidin beads and identified with a mass spectrometry. The aim using this technology is to unravel new interaction partners of seipin and proteins important for LD formation, which is a crucial step for understanding LD formation and diseases related to it.
Now showing items 1-5 of 5