Browsing by Subject "Biotechnology"

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  • Almusa, Henrikki (Helsingfors universitet, 2013)
    The next-generation sequencing (NGS) platforms create a large amount of sequence in short amount of time, when compared to first generation sequencers. An overview of the NGS platforms is provided with more in-depth look into Illumina Genome Analyzer II as that is used to create the data for the thesis. There were two main aims in this thesis. First, to create a pipeline which can be used to analyse genomic sequencing. Second, to use the pipeline to compare whole human exome capture methods from two manufacturers, Roche Nimblegen and Agilent. The pipeline is describe in detail in material and methods. All the inputs for the pipeline are described and examples shown. In the pipeline the given sequences are first aligned against the reference genome. Then various separate analysis is performed to retrieve variants and coverage of the sequencing. Supplementary results include paired-end anomalies, larger insertion and deletion polymorphisms and assembly of non-aligned sequences. The two capture methods are also described and changes to the manufacturers' recommended protocols are listed. Finally, the section has the options and various inputs used in the pipeline runs of the exome data. The results of the pipeline is a basic level of analysis of the sequencing as well as various graphs showing the quality of the run. All the output files intended for user are described. By using the results of the pipeline, the user can do more in-depth analysis as required by the project. When comparing the two exome capture methods, the Nimblegen capture was shown to be more efficient in capturing the CCDS exome. While the Agilent capture kit provided better one fold coverage over the exome, higher fold coverage (over 10 fold), which is required for reliable variant calling in nextgeneration sequencing, was better reached using the Nimblegen capture kit. Also, significantly fewer false positive paired-end anomalies were observed in the library created by using the Nimblegen capture.
  • Duru, Ilhan Cem (Helsingfors universitet, 2017)
    Lactobacilli are gram-positive lactic acid bacteria with wide beneficial properties for human health and food production. Today most of the fermented products and probiotic foods are produced by lactobacilli species. One of the most using area of lactobacilli species is fermented products especially dairy products. Lactobacilli species can be used as starter or adjunct cultures in dairy products and play important role for preservation and quality, texture and flavor formation. Additionally, probiotic properties of lactobacilli species provide several health effect for human by stimulation of immune system and protection against pathogens. Lactobacillus rhamnosus LC705 is a facultatively heterofermentative type lactobacilli which is used in production of dairy products as adjunct starter and protective culture. The complete and annotated genome sequence of L. rhamnosus strain LC705 published on 2009. Known characteristics of L. rhamnosus strain LC705 are food preservation, toxin removal and health benefits when combined with other probiotic strains. However, molecular mechanism behind these characteristics are not known or not clearly understood. To get further insight on these properties and roles in cheese ripening of strain LC705, we re-annotated genome of the LC705 with updated methods and databases, analyzed metabolic pathways of LC705, and performed RNA-seq experiment to determine gene expression changes of LC705 during warm room (25 °C) and cold room (5 °C) cheese ripening process. Several un-characterized proteins of LC705 were annotated (77) and 1197 enzyme commission (EC) numbers are added to annotation file with re-annotation of genes of LC705. More importantly, re-annotation provided us 72 new pathways of LC705 which is 35% of the entire collection of 201 pathways. Analyzes of pathways showed that genome of LC705 has responsible genes for production of flavor compounds such as acetoin and diacetyl which are provide buttery flavor to dairy products, and hydrogen sulfide which is a volatile sulfur compound that cause unlikeable odor. Additionally to flavor compounds, we defined genes that produce anti-fungus compounds and bacteriocin which provide food preservation characteristic to LC705. Determination of gene expression respond of LC705 during warm room and cold room cheese ripening process with RNA-Seq showed that central metabolism genes that responsible for lyase activity, degradation activity, disaccharides and monosaccharides metabolism are warm induced genes. The genes play role in citrate metabolism pathways were significantly down-regulated during cold room, citrate degradation pathways are critical for buttery flavor products, therefore buttery flavor compounds are produced by LC705 during warm room. Finally, during cold room ripening, the genes of LC705 that produces ethanol and acetyl-CoA from pyruvate was up-regulated, so we may say that LC705 uses pyruvate to produce ethanol and acetyl-CoA instead of lactic acid.
  • Domènech Moreno, Eva (Helsingin yliopisto, 2017)
    In this Master’s project, I have studied a mammalian serine-threonine kinase NUAK2 implicated in human disease but whose molecular functions and interacting proteins are as of yet poorly characterized. The goal was to identify new interacting proteins to increase understanding of the molecular functions and potentially link to human physiology and disease. Recent work from the host lab shows NUAK2 loss in cultured primary cells mimics loss of the tumor suppressor LKB1 which also acts upstream of NUAK2, together suggesting NUAK2 could be involved in tumor suppression. Currently, only two protein-protein interacting proteins with NUAK2 have been identified: NUAK2 is targeted to actin stress fibers by the myosin phosphatase Rho-interacting protein (MRIP), and it is involved in regulating cell contractility by affecting indirectly the phosphorylation cycle of the myosin light chain through inactivation of the myosin phosphatase target subunit 1 (MYPT1). In this project, I utilized a novel protein-protein interaction screening method that utilizes proximity-dependent biotin labeling to identify new interacting proteins with NUAK2 in human embryonic kidney cells (HEK 293). This method is based on fusing an E.Coli promiscuous biotin ligase, BirA*(R118G), to the investigated protein. The BirA*(R118G) ligase biotinylates all the proteins in close proximity of the fusion protein creating a history of protein-protein associations over time. Afterwards, the biotinylated proteins can be isolated by affinity purification methods and identified by mass-spectrometry. The screening identified the previously known interaction partners of NUAK2 indicating it was technically successful. In addition, I also identified in total 108 novel potential protein interaction partners for NUAK2. One of the top hits was Cytospin-A, a cross-linking protein between microtubules and actin cytoskeleton, supporting a role of NUAK2 as regulator of cytoskeleton. Supporting the validity of our finding, Cytospin-A depletion in mammalian cells causes defective actin-cytoskeleton reorganization, a very similar phenotype seen with NUAK2 depletion. In future studies, I will continue to investigate the specific role of NUAK2 and Cytospin-A aiming for detailed information on the function of NUAK2 in regulation of microtubules and actin cytoskeleton. Validation of some of the other identified interactions is expected to provide novel insights to the biology and role of NUAK2 in LKB1 tumor suppressor functions.
  • Sylvänne, Tuulia (Helsingfors universitet, 2013)
    Lipoproteins play a central role in the disease mechanisms of cardiovascular diseases (CVD) and therefore they have been studied widely. They carry several classes of apolipoproteins where apo-A1 and apo-B are the major classes. The sucrose based sequential lipoprotein isolation method can retrieve the lipoprotein fractions suitable for lipidomics analyses. The main lipoprotein classes are very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high density lipoprotein (HDL) that can be isolated easily by their density from human blood plasma or serum. Lipidomics analyses can quantify lipids that lipoproteins carry in the circulation. Mainly they carry cholesterol and its esterified forms, glycerolipids, small amounts of sphingolipids and phospholipids form their monolayer membrane. The isolation method was set-up together with scaled-down sample volumes. The protein and lipid content of the main lipoprotein fractions were evaluated by electrophoresis analysis, various enzymatic assays and lipidomics analyses. The total protein and apolipoprotein content was found to be similar as in the literature. Apo-B was found to be the main apolipoprotein in the VLDL and the LDL fractions whereas apo-A1 was the main apolipoprotein in the HDL fractions. Triglycerides (TG) were measured by enzymatic analysis and TG was mainly found in LDL and VLDL. The lipidomics analyses demonstrated the lipid content of the lipoproteins were similar as in the literature with minor changes. The main lipid class found in all the lipoproteins was cholesteryl esters (CE) followed by phosphatidylcholines (PC) that are commonly found in cell membranes. Sphingolipids such as ceramides were also detected in lipid class level only in small quantities in the lipoprotein fractions. The low initial sample volume did not correlate linearly with higher sample volume and low sample volume is not recommended to use in this specific isolation method. Based on the results of the comprehensive screening of isolated lipoproteins the isolation method was successfully established.
  • Rahikainen, Jenni (Helsingfors universitet, 2009)
    Environmental concerns and limited availability of fossil hydrocarbons have boosted the research of renewable feedstocks and their processing into fuels and chemicals. Currently, vast majority of transportation fuels and bulk chemicals are refined from crude oil, but renewable lignocellulosic plant biomass has long been recognised as potential feedstock for liquid fuel and chemical production. Several alternative processes exist for biomass refining, lignocellulose-to-ethanol process being among the most studied processes. First, lignocellulose is pretreated in order to deconstruct the recalcitrant structures of plant cell walls and expose cellulosic fibrils. Subsequently, biotechnical process utilises cellulolytic enzymes of fungal origin to depolymerise cellulose down to glucose monomers and oligomers. Monomeric sugars serve as a source for platform chemicals in further conversions. Lignocellulose consists mainly of cellulose, hemicellulose and lignin. It is generally accepted that lignin has an inhibitory effect during enzymatic hydrolysis of cellulose and part of this effect is caused by irreversible cellulase adsorption on lignin. Fungal cellulase system consists of several enzyme components that contribute to the effective degradation of insoluble cellulosic substrate. Cellulases are traditionally divided to three groups according to enzymatic activity: exoglucanases, endoglucanases and ?-glucosidases. Different enzyme components are shown to have different affinity to lignin which enables screening or engineering of weak lignin-binding enzymes. However, too little is still known about enzyme-lignin interactions and competitive nature of enzyme binding on lignin. In this study, lignin-rich residues were isolated from steam pretreated spruce (SPS) using three different methods: enzymatic hydrolysis, acid hydrolysis and alkali extraction. Lignin residues were characterized and used in adsorption studies with commercial cellulase preparations from Trichoderma reesei (Celluclast 1.5L) and Aspergillus niger (Novozym 188). Enzyme activity measurements and protein analytics were employed to reveal competitive adsorption of cellulases and catalytic activity of solid-bound enzymes. Results showed that T. reesei enzymes had high affinity on lignocellulosic SPS and all SPS-derived lignins, but enzyme activity measurements revealed considerably divergent competitive adsorption patterns. Among all the isolated lignins, lignin-rich residue obtained by enzymatic hydrolysis of SPS and subsequent protease purification was evaluated as most suited adsorption substrate for further adsorption studies and screening purposes. ?-glucosidases from T. reesei and A. niger were shown to have highly distinctive adsorption behaviour on the lignin-rich substrates: A. niger ?-glucosidase lacked affinity to lignin, whereas T. reesei ?-glucosidase adsorbed to all lignin-rich particles. Lignin-bound Trichoderma reesei endoglucanases and CBH I exoglucanase were shown to retained high activity towards soluble substrates used in activity measurements. On the contrary, same enzymes were unable to processively hydrolyze insoluble crystalline cellulose.
  • Elbadri, Khalil (Helsingin yliopisto, 2020)
    An increased attention has been drawn towards porous silicon (PSi) based materials for biomedical applications, due to their promising features demonstrated through several scientific studies. Here, we further investigated the biological responses of PSi nanoparticles (NPs) with different surface chemistries, including immunomodulatory effects, inflammation mitigation and biocompatibility. In this collaborative study, the PSi NPs were investigated both in vitro and in vivo, using different molecular biology and biochemistry techniques, e.g., qPCR, ELISA, cell sorting and cell viability assays. Our results showed the capabilities of these PSi NPs to relieve the inflammatory conditions, whereas significant decrease was recorded of pro-inflammatory cytokines: TNF-α, IL-1β and IL-6. Likewise, these PSi NPs revealed a considerable consumption aptitude of pro-inflammatory reactive oxygen species molecules. Administrating PSi NPs in an acute liver inflammation (ALI) model, showed no conspicuous influence on cellular viability. Thus, the outcome of this study demonstrates the potential biocompatibility of PSi nanomaterials, in addition to their outstanding features as potential candidates for further incorporating in ALI applications.
  • Madhav, Hema (Helsingin yliopisto, 2019)
    Abstract Antibiotics are used to prevent microbial diseases in both animals and humans. Because of the overuse of antibiotics, the microorganism now gained the ability to resist the drugs through genetic changes. Integrons are widely known for their role in the dissemination of antibiotic resistance. The class1 integrons are mostly studied in Gram-negative bacteria of clinical strain as they are reported mostly in the human and animals. The integrons having antibiotic resistance genes are linked with mobile genetic elements which help them to disseminate by the lateral gene transfer method. Previous research has proved that the class 1 integrons have sulfonamide and tetracycline resistance gene by using Long-Range PCR, Inverse PCR, and metagenomics. However, it is not clear what other possible combination of antibiotic resistance genes the class1 Integrons may carry. My thesis focuses on the class 1 integron from wastewater (both inflow and outflow water) by Long-range PCR, which can amplify fragments more than 15kb and PacBio RS long-read sequencing. Its a novel method of combining Long-range PCR and may illuminate what other possible resistance genes the class 1 integrons carry. The antibiotics resistance genes such as CatB8, -aadA2, blaOxA-1 0, IMP-38 were amplified using our designed primers from IntI1 to QacEdelta1, thus the designed primers and the optimization of Long-Range were successful. The combination of inverse PCR and Pac-Bio sequencing was successful to amplify the antibiotic resistance genes from Class 1 integrons. The Long-Range PCR saves time and gives DNA amplified products longer than 1500kb. The purified samples from long range PCR can be studied by direct sequencing using the Pac-Bio sequencer. Thus, the future implementations of the above combination of two techniques can be very useful to study the antibiotic resistance genes in the soil and polluted water. More in-depth information about antibiotic resistance genes in class 1 integrons will help to understand their dissemination.
  • March, Alexander (Helsingin yliopisto, 2016)
    This body of research focuses on establishing a drug screening pipeline for discovering drugs which increase the differentiation of pluripotent stem cells into cardiac myocytes, known as cardiogenic molecules. Cardiomyocytes can be utilized in regenerative medicine by offering a platform for testing molecules or drugs which may increase cardiomyocyte proliferation and for using cardiomyocytes produced outside of the body for clinical transplant, in order to heal the damage caused by heart attacks. Building on known models and developmental pathways three assays were designed and implemented for in vitro cardiogenic molecule screening. A pipeline comprised of three primary screening systems; an embryoid body (EB) model, a cardiomyocyte directed differentiation model, and a magnetic activated cell sort (MACS) model. The MACS model uses the cell surface receptors Fetal Liver Kinase 1 (FLK1) and/or Platelet Derived Growth Factor Receptor alpha (PDGFRα) as the most practical platform for screening drugs against an enriched mesodermal population of cells. The MACS system was confirmed with flow cytometry to ensure the enrichment of Myl2-eGFP+ (ventricular cardiomyocytes) cells in the FLK1+ cells. Furthermore unique known molecules help elucidate the molecular mechanisms governing cardiomyocyte differentiation, measured by cardiomyocyte purity in in vitro models. Also demonstrated are assay controls which decrease purity and acts as negative controls for the MACS assay such as a late stage GSK-3 Inhibitor treatment used to constitutively activate the canonical Wnt/β-catenin pathway and effectively reduce the cardiomyocyte proliferation. Additionally, an early stage Wnt Inhibitor compound IWP-4 was used as a potential positive control effectively blocking late stage activation of canonical Wnt/β-catenin pathway and increase the in vitro purity of cardiomyocytes. These controls provide two important reference points for the many molecules screened over the course of these experiments for the 3i Regeneration project. Additional molecular inhibitors are used to elucidate the mechanism of action within the MACS cells; including a Sonic Hedgehog inhibitor (cyclopamine), an NKX2.5 activator (ISX-9) and a novel small molecule (C1). These models act as an effective pipeline bringing a potential drug through first an EB model, followed by a cardiomyocyte enriched model, to finally a MACS model targeting FLK1. This pipeline tests the molecules against conditions of increasing resemblance to the native microenvironment of a cardiomyocyte.
  • Ilander, Mette (Helsingfors universitet, 2011)
    Chronic myeloid leukemia (CML) is one of the most studied human malignancies. It is caused by an autonomously active tyrosine kinase BCR-ABL, which is a result from a translocation between chromosomes 9 and 22 in the hematopoietic stem cell. As an outcome, a Philadelphia (Ph) chromosome is formed. BCR-ABL causes disturbed cell proliferation among other things. Although targeted tyrosine kinase inhibitor therapy has been developed in the beginning of the millenium and the survival rate has increased significantly, it is still not known why some patients benefit more from the treatment than others. Furthermore, the therapy is not considered to be curative. Before the era of tyrosine kinase inhibitors, the first-line treatment for CML was interferon-? (IFN-?). However, only a small proportion of patients benefitted from the treatment. Of these patients, a few were able to discontinue the treatment without renewal of the disease. The mechanism of IFN-? is not completely understood, but it is believed that differences in the immune system can be one of the reasons why some patients have better therapy response. Kreutzman, Rohon et al. have recently discovered that patients who have been able to stop IFN-? treatment have an increased number of NK- and T-cells. They also have a unique clonal T-cell population and more cytotoxic CD8+ T-cells and less CD4+ T-cells. The aim of this master’s thesis was to study the function of T- and NK-cells in IFN-? treated patients. Although it was shown earlier that IFN-? treated patients have increased NK-cell count, the function of these cells was unknown. Therefore, we have now investigated the killing potential of patients’ NK-cells, their activation status and cell surface antigen expression. In addition, we have also studied the activation status of patients’ T-cells and their cytotoxic properties. We observed that NK-cells from patients treated with IFN-? are unable to kill leukemic cells (K562) than NK-cells from healthy controls. In addition, patients on IFN-? treatment have more active T-cells and their NK-cells have an undifferentiated immunoregulatory phenotype. Patients that have been able to stop the treatment have anergic T-and NK-cells. As a conclusion our results suggest that IFN-? therapy induces increased NK-cell count, NK-cell immunoregulatory functions and more active T-cells. After stopping IFN-? therapy, NK- and T-cells from CML patients restore anergy typical for CML.
  • Järvinen, Maija (Helsingfors universitet, 2010)
    The growing interest for sequencing with higher throughput in the last decade has led to the development of new sequencing applications. This thesis concentrates on optimizing DNA library preparation for Illumina Genome Analyzer II sequencer. The library preparation steps that were optimized include fragmentation, PCR purification and quantification. DNA fragmentation was performed with focused sonication in different concentrations and durations. Two column based PCR purification method, gel matrix method and magnetic bead based method were compared. Quantitative PCR and gel electrophoresis in a chip were compared for DNA quantification. The magnetic bead purification was found to be the most efficient and flexible purification method. The fragmentation protocol was changed to produce longer fragments to be compatible with longer sequencing reads. Quantitative PCR correlates better with the cluster number and should thus be considered to be the default quantification method for sequencing. As a result of this study more data have been acquired from sequencing with lower costs and troubleshooting has become easier as qualification steps have been added to the protocol. New sequencing instruments and applications will create a demand for further optimizations in future.
  • Shah, Saundarya (Helsingin yliopisto, 2019)
    Tiivistelmä – Referat – Abstract OCRL1 is a phosphatase that cleaves phosphatidylinositol 4,5-bisphosphate (PI4,5P) to phosphatidylinositol-4-phosphate (PI4P). ORP4 is a lipid binding/transport protein implicated in G-protein coupled signaling, cellular calcium homeostasis, proliferation and viability. OCRL1 and ORP4 are found in the endoplasmic reticulum and membrane contact sites throughout the endosomal system and the Golgi complex. OCRL1 is also present in the plasma membrane and vesicular structures. ORP4 has high affinity for binding sterols through the OSBP related domain (ORD). ORP4 also interacts with vimentin intermediate filaments via the ORD and influences the localization and organization of vimentin. The membrane lipid phosphatidylinositol (PI) can be phosphorylated either singly or in combination at three different positions on its inositol ring (D-3, D-4, and D-5)—yielding 8 possible phosphoinositides; the interconversion between the species is regulated by kinases and phosphatases that either add or remove phosphate groups from the various positions on the ring. Phosphoinositide metabolism is heavily involved in signal transduction pathways and cytoskeletal organization. Interestingly, it has also been found to be spatially regulated with distinct phosphoinositides being enriched in particular membrane compartments. BiFC assays can provide an important tool for visualizing protein-protein interactions. Not only is BiFC able to determine protein-protein proximity but it is also able to localize the interaction with regard to compartment membranes and structures. This study examined the interaction of ORP4 with OCRL1 by using BiFC analysis. We were able to determine that the protein pair seems to be in close proximity in the endoplasmic reticulum near the Golgi. Interaction only took place when the OCRL1 was tagged with VB at the C-terminal.
  • D'amato, Dalia; Bartkowski, Bartosz; Droste, Nils (2020)
    The bioeconomy is currently being globally promoted as a sustainability avenue involving several societal actors. While the bioeconomy is broadly about the substitution of fossil resources with bio-based ones, three main (competing or complementary) bioeconomy visions are emerging in scientific literature: resource, biotechnology, and agroecology. The implementation of one or more of these visions into strategies implies changes to land use and thus ecosystem services delivery, with notable trade-offs. This review aims to explore the interdisciplinary space at the interface of these two concepts. We reviewed scientific publications explicitly referring to bioeconomy and ecosystem services in their title, abstract, or keywords, with 45 documents identified as relevant. The literature appeared to be emerging and fragmented but eight themes were discernible (in order of decreasing occurrence frequency in the literature): a. technical and economic feasibility of biomass extraction and use; b. potential and challenges of the bioeconomy; c. frameworks and tools; d. sustainability of bio-based processes, products, and services; e. environmental sustainability of the bioeconomy; f. governance of the bioeconomy; g. biosecurity; h. bioremediation. Approximately half of the documents aligned to a resource vision of the bioeconomy, with emphasis on biomass production. Agroecology and biotechnology visions were less frequently found, but multiple visions generally tended to occur in each document. The discussion highlights gaps in the current research on the topic and argues for communication between the ecosystem services and bioeconomy communities to forward both research areas in the context of sustainability science.
  • Kovac, Bianca (Helsingfors universitet, 2010)
    Actin stress fibers are dynamic structures in the cytoskeleton, which respond to mechanical stimuli and affect cell motility, adhesion and invasion of cancer cells. In nonmuscle cells, stress fibers have been subcategorized to three distinct stress fiber types: dorsal and ventral stress fibers and transverse arcs. These stress fibers are dissimilar in their subcellular localization, connection to substratum as well as in their dynamics and assembly mechanisms. Still uncharacterized is how they differ in their function and molecular composition. Here, I have studied involvement of nonmuscle alpha-actinin-1 and -4 in regulating distinct stress fibers as well as their localization and function in human U2OS osteosarcoma cells. Except for the correlation of upregulation of alpha-actinin-4 in invasive cancer types very little is known about whether these two actinins are redundant or have specific roles. The availability of highly specific alpha-actinin-1 antibody generated in the lab, revealed localization of alpha-actinin-1 along all three categories of stress fibers while alphaactinin-4 was detected at cell edge, distal ends of stress fibers as well as perinuclear regions. Strikingly, by utilizing RNAi-mediated gene silencing of alpha-actinin-1 resulted in specific loss of dorsal stress fibers and relocalization of alpha-actinin-4 to remaining transverse arcs and ventral stress fibers. Unexpectedly, aberrant migration was not detected in cells lacking alpha-actinin-1 even though focal adhesions were significantly smaller and fewer. Whereas, silencing of alpha-actinin-4 noticeably affected overall cell migration. In summary, as part of my master thesis study I have been able to demonstrate distinct localization and functional patterns for both alpha-actinin-1 and -4. I have identified alpha-actinin-1 to be a selective dorsal stress fiber crosslinking protein as well as to be required for focal adhesion maturation, while alpha-actinin-4 was demonstrated to be fundamental for cell migration.
  • Coles, Eric Anthony (Helsingin yliopisto, 2019)
    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.
  • Warren, Andrew (Helsingin yliopisto, 2019)
    A novel method of somatic cell reprogramming employing CRISPR/Cas mediated gene activation (CRISPRa) may lead to improvements in the quality and efficiency of induced pluripotent stem cell (iPSC) generation by directly activating the endogenous factors of the cell. However, this method is yet to be optimized and is inefficient in its current form. Thorough characterization of the molecular events that occur during CRISPRa-mediated reprogramming could permit the fine-tuning of this method to improve iPSC production. B-lymphoblastoid cell lines (LCLs) adhere to culture plates during reprogramming, offering a form of selection for reprogramming cell populations. This thesis aimed to establish a system using LCLs for the characterisation of CRISPRa-mediated pluripotent reprogramming at the single-cell transcriptomic level. In this thesis LCL reprogramming conditions were characterized using alkaline phosphatase staining, immunocytochemistry, embryoid body formation, and live cell imaging. CRISPRa-mediated reprogramming efficiency was greatly increased by the targeting of the miR-302/367 cluster, a group of microRNAs known to improve the efficiency of transgenic reprogramming. Samples were collected for single-cell RNA sequencing (scRNA-seq) at multiple stages of reprogramming, the pluripotency of the iPSC samples was assessed, and a subset of the samples was sequenced. Clustering analysis of the sequencing data showed that the samples clustered apart from one another distinctly based on gene expression. The expression of notable genetic markers of LCLs, pluripotency, and developmental stages was consistent with the loss of somatic cell identity and rise of subpopulations characteristic of reprogramming. These results show that this is a functional system for scRNA-seq sample preparation that can be used to investigate reprogramming kinetics, and the samples collected will be part of a larger study of CRISPRa reprogramming.
  • Moureen, Faiza (Helsingin yliopisto, 2019)
    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.
  • Ojha, Ravi Kant (Helsingin yliopisto, 2019)
    One of the first cellular response to a variety of stress inducers is the inhibition of cap-dependent protein synthesis. This also occurs as a first defense mechanism against viruses, which must evolve counter-defense strategies to overcome the attempt of the cell to block viral protein production. On the other hand, viruses have developed strategies to overcome the host translational shutoff and are resistant to cellular stress. Some viruses use specific proteins that interfere with PKR activation and prevents eukaryotic initiation factor 2 alpha (eIF2a inactivation, while others utilize the internal ribosome entry site (IRES) to achieve translation independently of eIF2a. Following the lead of a previously performed genome-wide screen, I found that ouabain, a potent cardiotonic steroid that specifically inhibits the cellular Na+ K+ ATPase pump leading to decreased intracellular levels of K+ and inhibition of cellular bulk protein synthesis, inhibits viruses at post entry step of their life cycle. Three viruses were used: Respiratory syncytial virus (RSV, Paramyxoviridae), Semliki forest virus (SFV, Togaviridae), and Vesicular stomatitis virus (VSV, Rabdoviridae). Of the three viruses, VSV was the less inhibited by ouabain. The effect of the drug was specific for the Na+K+ATPase because point mutations that disrupted the ouabain binding site in the pump abrogated the antiviral effect of ouabain. In addition, the drug-induced inhibition of virus infection was reversed by exogenous addition of K+ ions, indicating the effect is dependent of the activity of the Na+K+ATPase. Follow up experiments using the small molecule regulator of proteostasis ISRIB, an inhibitor of the integrated stress response, indicated that the antiviral effect of ouabain does not involve the inactivation of the eIF2a, a central regulator of protein synthesis and stress responses. The finding that VSV can replicate in the presence of concentrations of ouabain that strongly inhibit other viruses suggest a mechanism of resistance that could be further investigated to shed light into the regulation of viral protein synthesis and mRNA regulation and potential use of cardiotonic steroids as antivirals or in oncolytic therapy.
  • Avila Pulido, Alan (Helsingin yliopisto, 2020)
    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.
  • Wang, Hao (Helsingfors universitet, 2008)
    Anabaena is a common member of the phytoplankton in lakes, reservoirs and ponds throughout the world. This is a filamentous, nitrogen-fixing cyanobacterial genus and is frequently present in the lakes of Finland. Anabaena sp. strain 90 was isolated from Lake Vesijärvi and produces microcystins, anabaenopeptilides and anabaenopeptins. A whole genome shotgun sequencing project was undertaken to obtain the complete genome of this organism in order to better understand the physiology and environmental impact of toxic cyanobacteria. This work describes the genome assembly and finishing, the genome structure, and the results of intensive computational analysis of the Anabaena sp. strain 90 genome. Altogether 119,316 sequence reads were generated from 3 genomic libraries with 2, 6 and 40 kb inserts from high throughput Sanger sequencing. The software package Phred/Phrap/Consed was used for whole genome assembly and finishing. A combinatorial PCR method was used to establish relationships between remaining contigs after thorough scaffolding and gap-filling. The final assembly results show that there is a single 4.3 Mb circular chromosome and 4 circular plasmids with sizes of 820, 80, 56 and 20 kb respectively. Together, these 4 plasmids comprise nearly one-fifth of the total genome. Genomic variations in the form of 79 single nucleotide polymorphisms and 3 sequence indels were identified from the assembly results. Sequence analysis revealed that 7.5 percent of the Anabaena sp. strain 90 genome consists of repetitive DNA elements. The genome sequence of Anabaena sp. strain 90 provides a more solid basis for further studies of bioactive compound production, photosynthesis, nitrogen fixation and akinete formation in cyanobacteria.