Browsing by Subject "RNA-sekvensointi"
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(2019)Human umbilical vein endothelial cells are responsible for maintaining and forming new vessels from existing ones, in a biological process called sprouting angiogenesis. Sprouting angiogenesis is a crucial mechanism for the resolution of hypoxia and normal development of tissues. It also plays a key role in internal plague hemorrhages, which can lead to embolisms and other cardiovascular complications. Angiogenesis is also crucial for cancer development. Sprouting angiogenesis is initiated by hypoxic tissue excreted vascular endothelial growth factor gradient, which induces normal endothelial cells into either a proliferative stalk cell or a signal sensing tip cell phenotype. Both of these cell types depend on the rapid flow of lipids to their plasma membrane, either to form plasma membrane protrusions in tip cells or as new plasma membrane material in dividing stalk cells. This flow is envisioned to involve both vesicle-mediated and non-vesicular mechanisms. A major non-vesicular route of lipid transfer occurs at membrane contact sites via lipid transport proteins. Furthermore, lipids can be transported to the plasma membrane by the direct fusion of vesicles or endosomes with the plasma membrane This thesis set out to explore the role of two membrane contact site proteins, oxysterol-binding protein- related protein 2 and protrudin, in angiogenesis and lipid transfer. Their role was examined by RNA-sequencing transient knock-down samples of these proteins in HUVECs. The RNA-sequencing data was examined by differential expression, gene ontology overrepresentation and gene set enrichment analyses. Gene expression analysis provided almost 10 000 significantly changed transcripts (adjusted p-values < 0.05), in each silenced cell type. The distribution of differentially expressed genes in oxysterol-binding protein- related protein 2 silenced cells, is skewed toward negative fold changes, whereas the distribution of differentially expressed genes in protrudin silenced samples is normally distributed. The results also show significant changes in gene ontologies related to proliferation, cell cycle, angiogenesis as well as hypoxia in both sample types. Gene set enrichment analysis showed upregulation in angiogenesis related pathways, such as the PI3K-Akt and MAPK pathways, in both samples. Significant downregulation was present in cell cycle related pathways and cholesterol biosynthesis pathway in both ORP2 and protrudin silenced samples.
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(2014)RNA-sequencing is used to measure gene expression levels, characterize alternative splicing, identify SNPs and to study fusion genes. By using this information it is possible to understand function of different genetic elements and to understand embryogenesis as well as disease. The aim of this study was to compare new strand specific RNA sample preparation methods for next generation sequencing (NGS). The new method should have for example the following features: quick library preparation, smaller amount of starting material and strand specificity. Strand specificity is essential if there is no reference genome available or for example when the overlapping antisense transcripts are studied. Two different samples were prepared exactly according to each of four protocols that were tested. One of the samples was an RNA sample extracted from human blood and the other one was a BT474 cell line sample. The methods that were tested were NEXTflex Directional RNA-Seq Kit (Bioo Scientific), NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs), ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) and TotalScript RNA-Seq Kit (Epicentre). Laboratory’s non-strand specific method, Nextera (Illumina, New England BioLabs), which is custom modified to be applied for RNA, was used as a control method. Prepared RNA libraries were sequenced and the data compared for instance by using dendograms, strandedness and read coverage. After the comparison of the results and the usability of the methods it was found out that the ScriptSeq v2 RNA-Seq Library Preparation Kit was the most suitable of the methods for the laboratory’s usage.
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(2017)Endophytic bacterial strain Serratia plymuthica A30 has been shown to be a good bio-control candidate against pathogen Dickeya solani that is causing soft-rot of potato (Solanum tuberosum) also in Finland. The aim of this master’s thesis was to analyze previously produced RNA sequencing data and to understand which hormone signaling pathways were mainly activated in potato tubers treated by antagonist Serratia plymuthica A30 and Dickeya solani Ds 432-1. The main objective was to compare transcriptional differences of the genes related to jasmonate/ethylene signaling pathways and salicylic acid signaling pathway in pathogen/antagonist treated potato and pathogen treated potato at 24 hour time point after treatments. The additional objective was to identify potential differences between gene groups related to plant defense responses. RNA sequencing data was verified by RT-qPCR method. According to the RNA sequencing, the most active hormonal signaling pathways were jasmonate, ethylene and brassinosteroid signaling pathways after simultanous treatment with pathogen and antagonist. The least active pathways were salicylic acid and gibberellin signaling pathways. However, salicylic and gibberellic acid pathways were the most active in pathogen treated potato tubers and jasmonate, ethylene, auxin and brassinosteroid signaling pathways showed less activity. Based on the results of this thesis, it would appear that the high efficiency of Serratia plymuthica A30 against Dickeya solani pathogen in potato would be based on its direct inhibiting effect against the pathogen but also on the possible activation of jasmonate/ethylene signalling pathway which may lead to an efficient defense response against necrotrophic pathogens.
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