Browsing by discipline "Biotechnology"

Tiivistelmä–Referat–Abstract Background: Cancer is one of the leading causes of death around the world and in Finland. Ambitious research projects have been carried out for decades investigating cancer and how it spreads. Over 35 years ago, the systems that regulate vascular formation were discovered; the vascular endothelial growth factor (VEGF)-VEGF receptor VEGFR system and the more recent Angiopoietin-TIE system. These are the main endothelial growth factor receptor pathways involved in regulation of vessel quiescence and angiogenesis. The VEGF-VEGFR system is the first discovered endothelial cell (EC) specific receptor tyrosine kinase (RTK) signaling system. VEGF is a major growth factor involved in proangiogenic activity and vascular permeability when bound to its receptor, VEGFR2. Tumor cells take advantage of the VEGF-VEGFR2 system by secreting VEGF to stimulate angiogenesis in surrounding tissue to create new blood vessels allowing for greater access to nutrients and oxygen for tumor growth. The Angiopoietin-TIE system is the second EC specific RTK signaling system that was discovered. Angiopoietin-1 (ANG1) is the ligand for the TIE2 RTK. ANG1 is an obligatory TIE2 agonist and its effects on intracellular signaling, cell cytoskeleton, and junction-related molecules allows ANG1 to restrict the amount and size of gaps that are formed at EC junctions in inflamed vessels, increasing barrier function and decreasing vascular permeability. Angiopoietin-2 (ANG2) is an autocrine context-dependent TIE2 agonist/antagonist which is implicated in stimulating pathological angiogenesis, inflammation and vascular permeability. Integrins are important cell surface receptors that all cells use to communicate with their environment. Recently, it has been discovered that ANG2 is capable of inducing pathological angiogenesis, and can destabilize ECs when bound to integrin, specifically β1-integrin, via ANG2 N-terminal region. Objectives: The general aim of this study was to discover which part of angiopoiten-2’s N-terminus region was responsible for integrin activation. Materials and Methods: Fibronectin fragment containing type III 7-10 domains was produced and fluorescently labeled with Alexa 647. Integrin activation was measured using the fluorescently labeled Fibronectin III 7-10 and angiopoietins. Fluorescence activated cell sorting (FACS) was used to collect the results, which were analyzed using excel. Chimeric angiopoietins were produced using retroviral vectors and used for FACS experiments. A cell internalization assay was performed in Hela cells using CellTracker™ Orange CMRA and angiopoietin proteins, stained with secondary antibody anti-human Alexa 488 and Texas Red Phallodin. Results: Optimization of the FACS assay defined the minimum number of cells required to reliably measure integrin activation and showed that BD Accuri FACS machine was better suited than Guarva FACS machine for the assay and that the amount of integrin varied between cell passages used for the assay. In addition, it was essential to ensure a homogenous mix of cells and recombinant proteins during the assay and the quality of the produced FN III 7-10 was critical for the success of the assay. Results from the FACS assay confirmed that ANG2 is capable of activating integrin. In addition, chimeric angiopoietins that were expressed and secreted from CHO cells, were capable of activating integrins to a variable degree. The results confirmed the importance of ANG2 N-terminus in integrin activation. Cell internalization assay visually demonstrated angiopoietin binding to Hela cells. ANG2 was internalized by the cell and resistant to the acid wash, while the majority of ANG1 bound to the cell surface was washed away by acid wash. Conclusions: In this thesis work, integrin activation assays were optimized and carried out, along with cell internalization assays, to determine which specific part of ANG2 is responsible for inducing integrin activation. The findings from this work confirmed that ANG2 is capable of activating integrin. Several chimeric constructs were successfully expressed in CHO cells, and the cell supernatants were used to activate integrins. However, more studies are needed to determine which specific region of ANG2 is responsible for integrin activation. Investigating angiopoietin induced integrin activation would allow for a better understanding of the angiopoietin signaling pathway with potential translational significance.

Tiivistelmä – Referat – Abstract Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) provide a good model to study their function in human context. The hiPSC technology allows to create patient-specific cell lines. The cell lines can further be differentiated into any cell type retaining the same genetic information as the donor. Heart diseases such as Long QT syndrome (LQTS) and Non-compaction cardiomyopathy are caused due to mutations in ion channels in cardiac cells. The genetic abnormalities in these channels can cause life threatening arrhythmias. However, the diseases remain undiagnosed until later stage of the disease and the first sign could be sudden cardiac death. Hence, understanding the disease mechanism at a cellular level is crucial. The aim of the thesis is to study the functionality of cardiomyocytes from patient specific cells to analyse if the clinical representation of the diseases can be seen at the cellular level. An additional aim is to compare and study the Ca2+ transients in patient-derived and control cells. One way to study the functionality of cardiomyocytes is by Ca2+ imaging. The method involves live cell imaging of differentiated cardiomyocytes using fluorescent dyes (Fluo4 AM and Cal520). The Ca2+ transients of diseases such as LQTS and non-compaction cardiomyopathy are recorded and further analysed by Clampfit software. The main findings of this study include a) different Ca2+ dyes (Cal520 and Fluo4) had different profiles in control cardiomyocytes b) a significantly different profiles were observed between the SCN5A-mutant and control cardiomyocytes c) the effect of adrenaline was most significant at low doses and when exposed for a short time; prolonged exposure to adrenaline leads to adrenaline receptor desensitization. Moreover, different types of arrhythmias have been detected in cell lines such as oscillations, plateau abnormality, multiple peaks etc. In conclusion, detection of Ca2+ transients may be an indicator of heart diseases due to genetic mutations which will further help in taking preventive measures.

Psychrotrophic lactic acid bacteria (LAB) play a versatile role in research, food, farming and medicinal applications, but also play a role as a source of food spoilage. The effects of temperature changes has yet to be studied in depth. In this study, to analyze in a transcriptome level, cold and heat shock stress to spoilage lactic acid bacteria, a time-dependent RNA-seq for Lactococcus piscium with a temperature of 0 °C and 28 °C was conducted. The data of protein regulation during the experiment shows that Lactococcus piscium has the essential machinery to survive against different types of environmental stress. I observed known heat shock related genes and stress related genes to be present in the regulated response of both temperature extremes. Cold shock upregulation is observed after 35 minutes, which could indicate that the metabolic response at cold temperatures is related to growth rate. With a clear downregulation of pathways of energy consumption and an adaptation in terms of RNA being more prominent that at 28 ºC. Certain surface, cell wall and transport proteins are noticeable more upregulated at 0 ºC in addition to a downregulation of energy related proteins, which in correlation with its growth curve, we can assume it is part of a mechanism of protection against cold environments, in comparison with the regulation of proteins at 28 ºC. This gives an insight of a well controlled preservation mechanism that Lactococcus piscium exhibits that can be linked to its environment. The findings of this research offer new understandings into the survival skills of Lactococcus piscium to a cold and heat shock. Future studies should focus on analyzing the regulation and function of possible new genes for the response to heat stress, as well as the specific function of proteins related to temperature stress in the events where change of temperature plays a constant role in the environment.