Browsing by Subject "Genetiikan ja genomiikan opintosuunta"

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  • Kuitunen, Essi (Helsingin yliopisto, 2019)
    Glutamine, the conditionally essential amino acid, is a major carbon and nitrogen carrier required for a range of cell functions, such as protein synthesis and maintaining redox balance. While healthy cells adjust their activities in response to glutamine availability, tumor cells display deregulated glutamine uptake and metabolism allowing quick proliferation and survival in cellular stress conditions. Hence, further knowledge of the glutamine sensing network is of interest. Utilizing Drosophila melanogaster, the roles of formerly identified glutamine sensing regulator candidates, Forkhead box O (FoxO), Super sex combs (Sxc), Spalt major (Salm) and Spalt-related (Salr), were explored. Drosophila is an efficient model organism for analyzing gene regulatory mechanisms, with its simple genome but conserved genes and metabolic pathways. Loss-of function and gain-of-function mutants of the candidates were cultured with/without glutamine, and their physiological response and gene expression changes were analyzed. The results show the glutamine intolerant phenotype of FoxO and Sxc deficiency, not dependent on altered food intake levels of larvae. However, glutamine intolerance of Salr and Salm deficiency was not observed. Moreover, we aimed to gain further insight to the roles of FoxO and Sxc in glutamine metabolism. Since amino acid catabolism produces ammonia, and glutamine metabolism plays a vital role in ammonia detoxification, we performed a pH-based measurement of foxo and sxc mutant larvae hemolymph on food with/without glutamine. However, we could not associate FoxO or Sxc with regulation of glutamine-derived ammonia clearance. In addition, we explored FoxO downstream regulator candidates. Putative promoter areas of Paics, Uro, Sesn, salr, Prat2 and Gdh were cloned into reporter vectors and the luciferase activity was analyzed under the expression of foxo. The results indicate that FoxO is a regulator of all of the 6 genes. Next we could utilize the here constructed plasmids to see whether the FoxO-mediated regulation is affected by altered glutamine levels in cell culture.
  • Peltola, Sanni (Helsingin yliopisto, 2019)
    In recent decades, ancient DNA recovered from old and degraded samples, such as bones and fossils, has presented novel prospects in the fields of genetics, archaeology and anthropology. In Finland, ancient DNA research is constrained by the poor preservation of bones: they are quickly degraded by acidic soils, limiting the age of DNA that can be recovered from physical remains. However, some soil components can bind DNA and thus protect the molecules from degradation. Ancient DNA from soils and sediments has previously been used to reconstruct paleoenvironments, to study ancient parasites and diet and to demonstrate the presence of a species at a given site, even when there are no visible fossils present. In this pilot study, I explored the potential of archaeological sediments as an alternative source of ancient human DNA. I collected sediment samples from five Finnish Neolithic Stone Age (6,000–4,000 years ago) settlement sites, located in woodland. In addition, I analysed a lakebed sample from a submerged Mesolithic (10,000–7,000 years ago) settlement site, and a soil sample from an Iron Age burial with bones present to compare DNA yields between the two materials. Soil samples were converted into Illumina sequencing libraries and enriched for human mtDNA. I analysed the sequencing data with a customised metagenomics-based bioinformatic analysis workflow. I also tested program performance with simulated data. The results suggested that human DNA preservation in Finnish archaeological sediments may be poor or very localised. I detected small amounts of human mtDNA in three Stone Age woodland settlement sites and a submerged Mesolithic settlement site. One Stone Age sample exhibited terminal damage patterns suggestive of DNA decay, but the time of deposition is difficult to estimate. Interestingly, no human DNA was recovered from the Iron Age burial soil, suggesting that body decomposition may not serve as a significant source of sedimentary ancient DNA. Additional complications may arise from the high inhibitor content of the soil and the abundance of microbial and other non-human DNA present in environmental samples. In the future, a more refined sampling approach, such as targeting microscopic bone fragments, could be a strategy worth trialling.
  • Reinikka, Siiri (Helsingin yliopisto, 2020)
    Endometrial polyps are one of the most common benign uterine lesions, affecting approximately 10% of all adult women. While endometrial polyps have a high prevalence, their molecular pathogenesis and genetic background are largely undefined. Accordingly, the aim of this thesis was to characterize the somatic mutational landscape of endometrial polyps – to identify mutations in cancer-associated genes, and to identify mutational signatures contributing towards the somatic mutational spectrum. The present study was conducted using whole exome sequencing of 23 endometrial polyps and 18 matching normal blood samples. Mutational signature analysis was conducted using MutationalPatterns and SigProfiler. Endometrial polyps were found to carry varying number of somatic mutations in their exomes, most of them present at a low allelic fraction. Moreover, 43% (10/23) of the polyps were identified to carry one to four cancer-associated mutations, including mutations in genes such as PIK3CA 17% (4/23), KRAS 13% (3/23) and ERBB1 9% (2/23), which are well-established cancer driver genes. Cancer-associated mutational signatures do not have a notable contribution towards the somatic mutational spectrum of endometrial polyps. However, a novel signature, ‘signature B’, characterized by T>G mutations, was found to affect a subset of polyp samples. To conclude, the whole exome sequencing of endometrial polyps revealed several mutations in cancer-associated genes and a novel mutational signature, which may contribute to the development of these benign tumours. However, further research is required to confirm and validate the novel signature, and to define the genetic alterations leading to the polyp pathogenesis.
  • Elomaa, Ellinoora Juulia (Helsingin yliopisto, 2020)
    The human cerebral cortex is characteristically large and folded, which can be majorly attributed to the high number and variety of neural progenitors during embryonic development. Radial glial cells are essential neural progenitors during neurogenesis. In addition to giving rise to new cell types, they also provide scaffold for migrating newborn neurons. Radial glia are known to portray peculiar characteristics in their cell division process, including unique migratory behavior as well as specifically regulated cleavage furrow orientation. While these processes of radial glial division have been studied extensively, the underlying molecular mechanisms are still largely unknown. ABBA (actin-bundling protein with BAIAP2 homology) and NEDD9 (neural precursor cell expressed, developmentally downregulated 9) are proteins, which are both known to be expressed in certain radial glia progenitors during embryonic development, while they are mainly absent in neurons. ABBA has a defined role of regulating plasma membrane deformation and actin polymerization in radial glia, while NEDD9 expression levels are a known factor in the correct progression from mitosis to cytokinesis. An interaction between ABBA and NEDD9 has previously been identified in a yeast two-hybrid screen done for the embryonic mouse brain. The aim of this thesis was to validate the interaction between ABBA and NEDD9 biochemically. First, their interaction was evaluated by doing co-immunoprecipitation assays on the endogenous proteins from C6 cells. The second approach was to test, whether their interaction is directly mediated by the N-terminal SH3-domain of NEDD9 and the proline-rich C-terminal portion of ABBA. This was done by doing biochemical binding assays using purified proteins and domains of interest. While co-immunoprecipitation of the two proteins gave results indicating an interaction, I could show that there is no direct binding between NEDD9 SH3-domain and ABBA, suggesting that the interaction might require other domains or be indirect. Together, these results provide valuable information that will help characterize what roles of ABBA and NEDD9 play in cortical development and beyond.
  • Ukwattage, Sanjeevi (Helsingin yliopisto, 2019)
    Background- Colorectal cancer (CRC) is the third most common epithelial carcinoma. There is an increased risk of colorectal cancer in people with longstanding inflammation in the large intestine, including individuals with ulcerative colitis (UC). Epigenetic changes in CRC such as aberrant DNA methylation alterations are common changes in human cancer. The aim of this study is to identify the DNA methylation alterations of selected inflammation related genes in UC-CRC vs. Lynch syndrome (LS). Method- DNA was extracted from archival tissue specimens from normal and tumor samples from UC-CRC (n= 31), and LS-CRC (n=29). Methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) assays were used to detect CIMP status and CpG promoter methylation status of seven inflammation related genes. Microsatellite instability analysis was carried out using two mononucleotide repeat markers BAT25 and BAT26. Results- Increased hypermethylation frequencies in carcinoma vs. normal colonic mucosa were detected for all the inflammatory marker genes in specimens of UC-CRC patients. Statistically significant differences for methylation frequencies were observed in the NTSR1 gene (p value =0.008) and SOCS2 gene (p value =0.04) in specimens of UC-CRC patients. NTSR1 gene showed significantly increased methylation of normal colonic mucosae from UC-CRC vs. LS patients (p value=0.01). Conclusion- UC-CRC and LS tumor specimens revealed varying frequencies of hypermethylation in all the inflammatory genes. Methylation of the NTSR1 in the normal colonic mucosa suggests a possible field defect in UC-CRC, and could thus be used as an early biomarker to detect increased UC-CRC risk in non-neoplastic epithelium.
  • Rahnasto, Johanna (Helsingin yliopisto, 2019)
    Preeclampsia is a vascular pregnancy disorder characterized by new-onset hypertension and proteinuria and/or new-onset preeclampsia associated symptoms during the second half of pregnancy. The pathophysiology of the disorder is not fully understood, but incomplete placentation and maternal tolerance towards fetal tissue are known to play a part in the disease pathogenesis. Predisposing factors include nulliparity, obesity, diabetes, chronic hypertension and autoimmune diseases. Furthermore, women who have experienced preeclampsia are more susceptible to cardiovascular disease later in life. One established biomarker for preeclampsia is the increased concentration of the soluble Fms-like tyrosine kinase 1 (sFlt1) in the maternal serum. sFlt1 is frequently overexpressed in preeclampsia and it is linked with angiogenic imbalance and endothelial dysfunction, although its role in the disorder is not completely clear. Preeclampsia has a genetic background. There are protective and predisposing variants in and near the Fms related tyrosine kinase 1 gene (FLT1; coding for sFlt1) that have been associated with preeclampsia either in the mother or in the fetus. In this study, five genetic polymorphisms over a 2.3 kb region in the 3’ untranslated region of FLT1 were genotyped by Sanger sequencing and fragment analysis in altogether 1200 individuals consisting of case and control mother–child pairs of the Finnish Genetics of Pre-eclampsia Consortium (FINNPEC) cohort. These polymorphisms were tested for association with various preeclampsia-related phenotypes by Fisher’s exact test. In the maternal genome, the minor alleles of rs17086497 and rs57760154 were associated with extreme hypertension (systolic blood pressure >180 mmHg) (p=0.004, OR=1.77) and obesity (p=0.023, OR=1.63). Homozygosity for these minor alleles was associated with pregnancy complications in general (p=0.026, OR=2.53) and the early-onset form of preeclampsia (p=0.004, OR=3.34). Additionally, the minor alleles of rs9554314, rs3138582 and rs149279513 were associated with extreme hypertension (p=0.045, OR=1.63) and obesity (p=0.023, OR=1.78). Moreover, a suggestive association to severe proteinuria (> 5 g/24h) was found in the maternal genome. In the fetal genome, significant negative associations were reached for rs17086497 and rs57760154 in terms of the serum concentration of sFlt1 in the preeclampsia group (p=0.008, OR=0.23). Overall, the results seem to link the studied region in the maternal genome to preeclampsia with severe features. This supports the idea of preeclampsia as a heterogeneous disorder with varying etiology and mechanisms and thus highlights the importance of differentiating between the various sub-phenotypes. For example, the association of the same allele in the fetal genome with lower maternal sFlt1 levels and in the maternal genome with severe symptoms of preeclampsia suggests that the sFlt1 level might not be a good measure in all patients. Additionally, the observed associations with extreme hypertension and obesity point to the possibility that this region might be relevant for the endothelial damage that is thought to be a central factor in creating the later-in-life disease susceptibility.
  • Viitanen, Arto I. (Helsingin yliopisto, 2019)
    The intestinal stem cells (ISC) are responsible for the regeneration of the intestine epithelial barrier after acute injury and for the replenishment of its cells overall. How the ISC activation and resulting proliferation is controlled is complex and still under study. The ISCs of the midgut, which is the functional analogue to mammalian small intestine, are also highly responsive to changes in nutrition, and with proper methodologies it is possible to study the effects of diet on stem cell activation. The metabolic flux of the nutritional components of the diet can then shed light on which metabolic pathways are necessary for nutrient-dependent proliferation. One nutrient that has garnered interest is glutamine (Gln). It is well established that glutamine supplementation can in parenterally fed patients diminish intestinal barrier atrophy, extend the time the patient can be kept under the regime, and increase survivability of critically ill patients. Consequently, glutamine or its downstream metabolites may have stem cell activating characteristics. However, the exact regulatory mechanisms and specific effects of Gln are not well known, and studies have found contradictory results on the beneficial effects of Gln supplementation. Glutamine itself is a conditionally essential amino acid that has a variety of functions: it is an important source of nitrogen and cellular energy and contributes carbon into the tricarboxylic acid cycle (TCA) and is involved in protein and nucleotide synthesis. In this thesis, the effects of Gln supplementation on the cell populations of D. melanogaster were studied via microscopy and computational analysis. Cross-breeds of fruit fly were established to lineage label the ISC with a GAL4/UAS driver system. Confocal microscope was used to image the midguts which were then analysed with Imaris software. A novel analysis method was developed to study population changes and varying features of the cells in the midgut in an unprecedented region-by-region bulk analysis. Earlier studies into nutrient control of ISC have had limited focus within the midgut and might have consequently given a restricted view of ISC activation. This new Longitudinal Analysis of Midgut (LAM) can be utilized in a diverse set of further studies to describe conditional variation within midgut, and possibly other tissues. Gln was found to increase total cell numbers to comparable levels with well-fed midguts, and to drive limited endoreplication in enterocytes. Lineage labelled cell population grew primarily in the R3 and R4 regions of the midgut. Additionally, enteroendocrine cells (EE) were greatly increased in the posterior part of R3 but had conceivable minor increases along the whole length of the midgut. Improved nutrition was also found to affect the proportions of the midgut, presenting itself as elongated posterior and stunted anterior. Overall, the pipeline and analysis method established during this study enable more expeditious research of effects of other nutritional components and allows for study of effects of other mechanisms, for example how gene knock-downs or altered gene activities affect cell populations of the midgut.
  • Pezzutto, Denise (Helsingin yliopisto, 2019)
    Antimicrobial resistance is an emerging concern at the global scale, threatening the effectiveness of antibiotics in treating bacterial infections. Among anthropogenically impacted environments, wastewater treatment plants have been indicated as possible reservoirs of antibiotic resistance genes, putative hotspots for their horizontal gene transfer, and a source of their dissemination to the environment. Generally, the abundance of antibiotic resistance genes is reduced during the wastewater treatment process. However, some genes were shown to be enriched in purified effluent water and dried sludge, which are then released to the environment, compared to influent water. Also, the taxonomy of the hosts carrying antibiotic resistance genes could change as a result of horizontal gene transfer events. The aim of this study was to analyse and compare the host range of a series of antibiotic resistance genes in influent water, effluent water and dried sludge collected from the Viikinmäki wastewater treatment plant in Helsinki, Finland, by applying Emulsion, Paired Isolation and Concatenation PCR (epicPCR). EpicPCR is a method that can link a gene of interest to the 16S rRNA gene from the genome of the host bacterium, without any cultivation step. The abundance of the hosts was also evaluated by sequencing the 16S rRNA gene from the whole bacterial community. In several cases, the target antibiotic resistance genes (blaIMP, blaNDM, ermB, ermF, sul1 and strB) were carried in effluent water and dried sludge by taxa that were not hosting them in influent water, suggesting that horizontal gene transfer events might have occurred during the treatment. All the examined genes were detected both in abundant and in rare taxa, including genera that also comprise pathogenic species, such as Arcobacter and Acinetobacter. Some of the detected hosts were not previously known to show resistant phenotypes, namely members of the family Methylophilaceae. These results corroborate the idea that wastewater treatment plants might be hotspots for the horizontal gene transfer of resistance determinants, and potentially disseminate antibiotic resistant pathogens to the environment. However, in order to ensure the accuracy of the results, the limits of epicPCR as a method need to be identified and addressed.
  • Savelius, Mariel (Helsingin yliopisto, 2020)
    Breast cancer remains as the leading cause of cancer deaths among women. Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes and lacks targetable receptors, consequently, cannot be treated with current hormone of anti-HER2 targeting therapies. Thus, there is a need for discovering novel and well-tolerated therapies. MYC is a proto-oncogene and a transcription factor, that is frequently amplified and overexpressed in breast cancers. MYC is involved in many cellular processes promoting cell proliferation, however, overexpression of MYC can also sensitize cells to replicative stress and apoptotic cell death. In our previous studies we have shown that pharmacological activation of AMPK, a cellular energy sensor, synergises with Bcl-2 family inhibitors, such as navitoclax and venetoclax, and activates MYC-dependent apoptosis in breast cancer cell lines, transgenic mouse models of MYC-dependent mammary tumorigenesis and in MYC-high patient-derived explant cultures (PDECs). In subsequent study we observed, that indirect AMPK activator metformin alone inhibited tumor growth in vivo, but did not induce apoptosis in mouse tumors or in PDECs. Metformin, a type II diabetes mellitus drug, has shown anti-cancer effects in some population studies and is under investigation for a cancer therapies, however the whole mechanism of action in cancer is still not well-known. To elucidate metformin’s effects on MYC overexpressing triple-negative breast cancer cells, I will present, that metformin has anti-proliferative effects and show that long term metformin treatment induces senescence biomarkers in MYC-high TNBC breast cancer cell lines. To study metformin's short and long-term anti-proliferative activity, cell proliferation during and after drug treatment was investigated, which showed, that metformin’s effects do not seem to persist long after drug withdrawal. In conclusion, the key observation of this thesis was, that metformin does inhibit the proliferation of MYC overexpressing cancer cells and presents a senescence phenotype that possibly can be exploited to find new targeted therapies for triple-negative breast cancer patients.
  • Wanne, Vilma (Helsingin yliopisto, 2019)
    GRACILE (Growth Retardation, Aminoaciduria, Cholestasis, Iron Overload, Lactic Acidosis, and Early death) syndrome (Fellman disease, MIM603358) is a mitochondrial disorder that belongs to the Finnish disease heritage and follows an autosomal recessive inheritance pattern. It is a lethal neonatal disease and the affected infants usually survive only a couple of days, but in some cases up to four months. The disease is very rare affecting about 1 in 47,000 infants in Finland. GRACILE syndrome is caused by a homozygous Finnish founder mutation (c.A232G) in the BCS1L gene, which encodes an assembly factor for the mitochondrial respiratory chain complex III. The missense mutation (c.A232G) causes an amino acid change (p.S78G) in the BCS1L protein. Alternative oxidase (AOX) is a terminal oxidase that is not naturally present in mammals. It enables the respiratory chain electron flow to bypass complexes III and IV. The AOX pathway works parallel with the respiratory chain and gets activated under stress conditions in plants and lower animals. In mitochondrial diseases with a complex III deficiency, such as GRACILE syndrome, AOX expression could alleviate the symptoms caused by the complex III dysfunction. The aim of this study was to investigate the effects of AOX expression on early-onset manifestations of the disease in the Bcs1lc.A232G mouse model of GRACILE syndrome. The mice used in this study have a genetic background with a short survival to P35-40. The respiratory chain function in freshly isolated mitochondria from the liver and kidney was studied, as well as complex III activity, mitochondrial mass and liver and kidney histology. The findings of this study suggest that AOX has a strong beneficial effect on both liver and kidney histology and it is able to restore the glycogen stores to some extent, thus alleviating the glycogen depletion seen in the mice. The Bcs1lc.A232G mice also had an improved body weight in the presence of AOX, suggesting a less severe energy deficiency due to the activity of the AOX transgene. CI- and CII-linked respiration was also robustly improved in the liver. Overall, the condition of the Bcs1lc.A232G mice was improved by the presence of the AOX transgene compared to the Bcs1lc.A232G mice not expressing AOX. These results are highly encouraging for further studies on the rescue effects that AOX seems to have on this disease model.