Browsing by Subject "bioteknik"

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  • Elf, Sonja (Helsingin yliopisto, 2019)
    Despite recent advances in understanding, diagnosis and treatment of cancer, this complex and versatile disease remains one of the leading causes of death worldwide. New and rapid diagnostic methods are needed to detect cancers at their early stages of development, thus enabling earlier prognosis, better risk assessment and more efficient treatment of the disease. There has been an increasing interest in specific molecular biomarkers as the hallmark for cancer research, and the detection of these markers from liquid biopsies using advanced molecular diagnostics methods provides major advantages over the conventional imaging methods currently used in oncology. The aims of this thesis were to examine the applicability of a novel molecular method, SIBA® (Strand Invasion Based Amplification), for the detection of cancer biomarkers, and to develop an assay targeting androgen receptor splice variant 7 (AR-V7) mRNA. The AR-V7 is proposed as a treatment-response biomarker in patients with castration-resistant metastatic prostate cancer (mCRPC). The expression of this variant can indicate resistance to hormonal therapies used for the treatment of advanced prostate cancer. Prostate cancer is the most common cancer after lung cancer in men worldwide and can gradually develop into a highly advanced lethal form, mCRPC, that is not responsive to androgen deprivation therapies. Positive AR-V7 status is suggested to represent the phenotype of this advanced stage of prostate cancer, and its detection can assist in treatment selection for the mCRPC patients. SIBA is a novel isothermal method for the amplification and detection of nucleic acids. The technology offers significant advantages over the more conventional molecular detection method, polymerase chain reaction (PCR), since the amplification reaction occurs at constant temperature and does not require sophisticated laboratory equipment for the thermal cycling. Reverse transcription SIBA (RT-SIBA) enables reverse transcription of RNA to cDNA as well as the simultaneous amplification and detection of the cDNA in one-step reaction under isothermal conditions. The method displays both high analytical sensitivity and specificity to the target nucleic acids. The RT-SIBA technology has not formerly been applied for the detection of human DNA or RNA. The main finding of this thesis was, that the RT-SIBA technology can be applied for rapid detection of specific molecular cancer biomarkers such as the AR-V7 mRNA. In this study, two RT-SIBA assays targeting the full-length androgen receptor (AR-FL) mRNA and the AR splice variant 7 mRNA were developed and optimized. Performance of the assays were evaluated by testing RNA isolates from AR-V7 positive and negative prostate cancer cell lines in the presence of human whole blood and plasma in the reaction. The developed RT-SIBA assays provided high analytical sensitivity and specificity: low copies of the target mRNA were amplified within 20 minutes without the production of non-intended amplicons. The results suggest that the RT-SIBA technology can be utilized for easy and rapid detection of AR-V7 and AR-FL mRNA directly from liquid sample material without a need for time-consuming sample treatment. Further performance evaluation using real AR-V7 positive clinical samples from mCRPC patients is necessary for the reliable validation of the developed assays.
  • Zaki, Urfa (Helsingin yliopisto, 2019)
    Cerebral dopamine neurotrophic factor (CDNF) belongs to the the family of neurotrophic factors that are evolutionary conserved, having a unique structure, with two domains: C-terminal domain and the N-terminal domain, and a cysteine bridge. It is known to be involved in the repair of the dopaminergic neurons when studied in the animal models of PD, which shows their different mode of action as compared to other neurotrophic factors, highlighting their therapeutic potential. Analysis of the crystal structure shows that CDNF and MANF consist of two domains: the saposin-like N-terminal domain with five α-helices stabilized by three disulphide bridges, and presumably unstructured C-terminal domain with a disulphide bridge. Characteristic feature of saposin-like proteins is their ability to interact with membranes or lipids. The lipid interaction may be crucial for the activity of CDNF and MANF proteins. In the first part of this project, the binding of CDNF was tested with several oxidized lipids, using two methods; Co-sedementation assay and lipid fluorescence assay;with two different types of probes. According to the results, CDNF seemed to show binding with POVPC. The second part of the project involved testing the binding and internalization of CDNF to mouse myoblast cells in the presence of oxidized lipid; POVPC. It was observed that CDNF seemed to show binding to the cell surface of the mouse myoblast cells (C2C12) and is also observed to be internalized to the cells as well. However, as these are the preliminary results, so we need to further test the binding between the protein and other lipids and devise more precise protocols for the testing the internalization to the cells.
  • Banerjee, Rishi (Helsingin yliopisto, 2019)
    After birth, stem cells act as the source of reparative and regenerative potential in various tissues. Among different tissues and organs in human body, tooth is one of the organs which does not undergo continuous regeneration. Therefore, tooth regeneration must be studied in a different animal, which possesses continuously growing teeth. In mouse, the incisor undergoes continuous growth which is fueled by the interaction between epithelial and mesenchymal stem cell compartments located at its apical end. The inferior alveolar nerve, which supports mandibular dentition, and its surrounding blood vessels (combinedly known as neurovascular bundle or NVB) were previously shown to act as a source of the mesenchymal stem cells during incisor growth and regeneration. However, the regulation of the cells in the NVB is not well understood. The primary aim of my master’s thesis was to characterize the effect of the Hh pathway modification on cellular properties of the NVB and the MSCs within it. The Ptch2 KO mouse model used in this study demonstrated increase in the number of blood vessel in the NVB. Additionally, analysis of the structure of skin in the mouse model was the second aim of my project, which showed significant increase in the thickness of the dermis at the postnatal day 1. Collectively, the change in structure of skin and NVB showed that Ptch2 might regulates the cellular properties of tooth mesenchyme and dermis by modulating the structural components of the NVB of continuously growing mice incisor and skin, respectively.
  • Sultana, Nasrin (Helsingin yliopisto, 2020)
    Tiivistelmä – Referat – Abstract Plant lives and grows in variable environment and climate conditions. Everyday plants can be confronted with a variety of abiotic (temperature, light, salt, water availability) and biotic stress (pathogens, insects etc). These abiotic and biotic stress can halt plant growth and influence crop productivity. Plant has evolved signaling mechanism and different responses to adapt or respond with these unfavorable environmental conditions. Our group’s previous research identified a new mutant in the model plant Arabidopsis thaliana with a striking phenotype – when the plants ages it progressively becomes yellow and eventually the entire plant is white. The mutant was named “white” after its striking appearance. The phenotype is associated with increased accumulation of mRNA transcript for stress and senescence regulated genes. Mapping of the mutation identified a 4 bp deletion in a gene EGY1 that encodes a metalloprotease located in the chloroplast. To identify molecular mechanisms that regulate this unusual type of premature senescence, a suppressor mutants screen was performed in the white mutant, and three suppressors that restore normal appearance to the plant was identified. Mapping of one of these suppressors, identified a mutation in STAY GREEN1 (SGR1) as a likely candidate. SGR1 encodes the protein that catalyze the first step in chlorophyll breakdown, removal of Mg2+ from chlorophyll. The overall aim of my master thesis was to understand the molecular mechanisms behind the development of the age and chlorophyll related phenotypes in the white mutant and its two suppressors S1 and S2. Furthermore, with gene expression analysis, plant stress and senescence responses were studied in white, S1 and S2. By complementation method I proved that mutations in SGR1 gene caused the development of suppressor mutant phenotype and restoration of wild type allele of SGR1 gene restore white phenotype in suppressor mutant. Measurements of chlorophyll concentration provided further evidence that the mutation in SGR1 stabilizes the suppressor mutant phenotype, stops chlorophyll breakdown and keep the leaves green. Gene expression study using qPCR with marker genes provided insight of molecular changes within these phenotypes.
  • Deb, Debashish (Helsingin yliopisto, 2019)
    There is significant reduction in number of approved drugs for acute myeloid leukemia in recent years. Partially it may be due to the failure of discovery and validation approach to new drugs as well as the complexity of the disease. Ex vivo functional drug testing is a promising approach to identify novel treatment strategies for acute myeloid leukemia (AML). In ideal condition, an effective drug should eradicate the immature AML blasts, but spare non-malignant hematopoietic cells. However, current strategies like conventional cell viability assay fail to measure cell population-specific drug responses. Hence, development of more advanced approaches is needed. Using multiparameter, high-content flow cytometry (FC), we simultaneously evaluated the ex vivo sensitivity of different cell populations in multiple (10) primary AML samples to 7 FDA/EMA-approved drugs and 8 drug combinations. Amongst the 7 tested drugs, venetoclax, cytarabine and dasatinib were very cytotoxic with venetoclax had the highest blast-specific toxicity, and combining cytarabine with JAK inhibitor ruxolitinib effectively targeted all leukemic blasts but spared non-malignant hematopoietic cells. Taken together, we show that the ex vivo efficacy of targeted agents for specific AML cell population can be assessed with a cell phenotype, FC-based approach. Furthermore, we put an effort to analyze the potential of this assay and biomarkers to predict the clinical outcome of individual patients and future perspectives.
  • Auvinen, Pauliina (Helsingin yliopisto, 2018)
    Assisted reproductive technology (ART) refers to treatments used for infertile couples to achieve pregnancy in vitro. The main technology of ART is in vitro fertilization (IVF), which may also include intracytoplasmic sperm injection (ICSI) and/or embryo cryopreservation and frozen embryo transfer (FET). ART treatments are well-accepted in Western countries and there is an increasing number of children being conceived in that way. Even though, majority of ART derived newborns appear healthy, they have been associated with increased risks of adverse perinatal outcomes, especially, alterations in birth size as well as higher frequencies of imprinting disorders and alterations in epigenetic modifications, such as in DNA methylation, of imprinted genes. Epigenetically regulated imprinted genes have crucial roles in fetal and placental growth during development and they are known to be affected by environmental factors. Since ART takes place in the early embryo in vulnerable time-period of epigenetic reprogramming, ART has been suggested to impact on epigenetic profiles of the embryo, consequently, affecting the phenotype of newborns, and therefore potentially causing long-term health effects. This thesis aimed to study whether ART has effects on DNA methylation in the placenta and whether ART has effects on the phenotype of newborns. To study these effects, this thesis focused on the sixth binding sequence of CTCF (CTCF6) of H19 ICR1 of the growth-related imprinted IGF2/H19 gene locus. The aim was also to study whether the possible changes associate with the rs10732516 G/A polymorphism locating at CTCF6 of H19 ICR1. DNA methylation levels of placental tissue as well as white blood cells in umbilical cord blood of ART derived, and spontaneously conceived newborns were explored by mass spectrometry-based Sequenom MassARRAY® EpiTYPER® method and traditional bisulfite sequencing. To study the effects of ART on the phenotype of newborns, the birth weight, length and head circumference of ART and control newborns were explored using international growth standards. Moreover, placental weights were compared. The results of this thesis showed slightly, but consistently decreased DNA methylation levels at H19 ICR1 in the paternal allele of ART derived placentas in rs10732516 patA/matG genotype, but not in patG/matA genotype. Thus, the results suggest that the changes in DNA methylation at IGF2/H19 in the placenta are genotype-specific and associate with the rs10732516 polymorphism. Similar decreased methylation levels in the paternal allele of patA/matG genotype was not detected in white blood cells suggesting that the effects on DNA methylation levels are also cell type-specific. The effects of ART on the phenotype also associated with the rs10732516 polymorphism. Fresh embryo transfer derived newborns with A/A genotype were seen to have smaller birth weight than newborns with G/G genotype. Moreover, in A/A genotype, frozen embryo transfer derived newborns were demonstrated to be heavier and to have heavier placentas than fresh embryo transfer derived newborns. The findings of this thesis suggest that ART has effects on DNA methylation in the placenta and on the phenotype of newborns, and the effects associate with the rs10732516 G/A polymorphism. This underlines the significance of the polymorphism when studying the effects of ART. However, further investigations are needed to confirm these findings and to discern whether the changes are due to the ART procedures or underlying infertility.
  • Kaya, Meryem Ecem (Helsingin yliopisto, 2019)
    Synthetic biology is an emerging interdisciplinary field of biology that aims to system-atically design artificial biological systems. As synthetic biologists seek increasingly complex control over cellular processes to achieve robust and predictable systems. A new frontier in synthetic biology is engineering synthetic microbial consortia. This ap-proach employs the concept of division of labor, instead of introducing large genetic cir-cuitry to homogenous cell populations. In this approach, different cell types are assigned to execute a portion of the overall circuit. Each cell type communicates with their co-worker subpopulations to complete the circuit. The main advantage of this strategy is the reduced metabolic burden on each cell type. Thus, leading to more reliable and stable overall performance. In this work, to simplify cellular communication between the mem-bers of the consortium, we used the simple architecture of quorum sensing machinery. We constructed a toolbox that contains promoter, receptor and quorum sensing signal synthase genes along with fluorescent reporters. Using this toolbox, we constructed dif-ferent cell types that can be used in synthetic consortia forming various communication topologies. We characterized the constructed cell types individually and in co-cultures.
  • Partti, Edvard (Helsingin yliopisto, 2018)
    Kaurapohjaiset elintarvikkeet ovat terveellisiä. Monet niiden terveyshyödyt johtuvat kauran liukoisen ravintokuidun suuresta β-glukaanipitoisuudesta. β-glukaanin terveysvaikutukset ovat riippuvaisia sen molekyylipainosta ja viskositeetista. Viskositeetilla on myös muuta merkitystä kaurapohjaisissa elintarvikkeissa kuten kaurajugurteissa ja kauramaidoissa. Aiemmassa Folafibre-tutkimusprojektissa oli tutkittu kaurakuidun folaattipitoisuuden (B9 vitamiini) kasvattamista fermentoimalla sitä eri mikrobeilla. Hyvin folaattia tuottaneet mikrobit myös alensivat kaurakuituvalmisteen viskositeettia, ja erittivät glykosyylihydrolaaseja ja/tai proteaasia. Oli kuitenkin jäänyt epäselväksi, olivatko entsyymit ainut syy viskositeetin alenemiseen, ja kuinka paljon kullakin niistä oli vaikutusta viskositeettiin. Lisäksi tämän alan kirjallisuudessa on pidetty epäselvänä, onko esim. tärkkelyksen ja β-glukaanin välillä jotain interaktioita jotka mm. nostavat niiden viskositeettia yli yksittäisten polymeerien viskositeetin summan. Näitä voitaisiin selvittää puhdistamalla viskositeettia alentaneista mikrobikannoista kyseiset entsyymit ja tutkimalla niiden vaikutusta kaurakuidun viskositeettiin. Yhdeksi mikrobiksi valittiin Exiguobacterium sp. RB3 kanta, koska em. syiden lisäksi tässä bakteerisuvussa esiintyy psykrofiilejä ja mikrobeja jotka voivat kasvaa korkeassa pH:ssa, jolloin sen erittämät entsyymit saattaisivat olla aktiivisia matalissa lämpötiloissa ja korkeassa pH:ssa, ja olla siten teollisesti kiinnostavia. Toiseksi mikrobiksi valittiin Bacillus sp. ABM5119, koska sitä oli käytetty monissa Folafibre-projektin tutkimuksissa. Työn tavoitteena oli puhdistaa Bacillus sp. ABM5119:n endo-β-1,4-glukanaasi, ja Exiguobacterium sp. RB3:n α-amylaasi ainakin siinä määrin, että ne ovat muista endoaktiivisista glykosyylihydrolaaseista ja proteaaseista puhtaita, ja sitten mitata puhdistettuja entsyymejä ja proteaasia käyttäen niiden vaikutus yhdessä ja erikseen keitetyn kaurakuituvalmisteen viskositeettiin. Lisäksi tavoitteena oli karakterisoida Exiguobacterium sp. RB3 amylaasin olennaisimmat biokemialliset ominaisuudet, sekvensoida sen geenin amylaasia koodaava alue, ja selittää sen ominaisuuksia myös sekvenssistä johdettavissa olevan tiedon perusteella. Keitetyn kaurakuitupreparaatin viskositeettia alentavat eniten α-amylaasi ja endo-β-1,4-glukanaasi. Proteaasi ei vaikuta kaurakuitupreparaatin viskositeettiin, kun kuitupreparaatti on keitetty. Endo-β-1,4-glukanaasi alentaa viskositeettia yhtä hyvin kuin β-1,3-1,4-glukanaasi. Synergiaa viskositeetin alentamisessa α-amylaasilla ja endo-β-1,4-glukanaasilla ei havaittu, mutta havaittiin että β-glukaani saattaa estää tärkkelyksen retrogradaatiota. Hyvin pienetkin entsyymiaktiivisuudet vaikuttavat viskositeettiin. Jos halutaan β-glukaanin viskositeetin ja siten terveysvaikutusten säilyvän, täytyy esim. kauramaidon valmistuksessa käytettävien entsyymivalmisteiden olla β-glukanaaseista hyvin puhtaita. Mikrobifermentaatioissa tapahtuvat viskositeetin alenemat johtuvat lähinnä mikrobien erittämistä glykosyylihydrolaaseista, ei niinkään esim. niiden metabolian sivutuotteista kuten happiradikaaleista. Exiguobacterium sp. RB3 α-amylaasi on rakenteeltaan Bacillus licheniformis α-amylaasin kaltainen glykosyylihydrolaasiryhmän 13 entsyymi. Se sitoo rakenteeseensa kolme kalsiumiatomia, ja kalsiumpitoisuus vaikuttaa sen aktiivisuuteen. Se on aktiivisimmillan pH alueella 5,0 – 7,5. Se sietää detergenttejä, toisin kuin eräs aiemmin karakterisoitu Exiguobacterium-α-amylaasi. RB3 α-amylaasin turnover number oli korkea, 29000 1/s. Exiguobacterium-suvun psykrofiilisessä haarassa esiintyy kahta eri α-amylaasia, joista yksi on tässä karakterisoitu, ja toinen on selvästi erilainen rakenteeltaan ja biokemiallisilta ominaisuuksiltaan.
  • Martins, Beatriz (Helsingin yliopisto, 2020)
    According to the latest estimations, cancer is the second leading cause of death worldwide. Despite the significant advances in the range of drugs and treatment modalities to treat cancer, the number of deaths is estimated to continue rising, posing serious challenges for the patients, their families, and the healthcare systems. Conventional treatments tend to be associated with severe adverse side effects and treatment resistance. Consequently, safer and more efficient therapy options are urgently needed, especially for the treatment of metastatic tumors refractory to conventional treatments. A new and revolutionizing field in oncology is immunotherapy, in which oncolytic viruses are included. Oncolytic viruses have an inherent or acquired selectivity to replicate exclusively in tumor cells, ultimately destroying them. Simultaneously, they also activate the dormant host’s immune system to fight against the tumor. Adenoviruses, particularly, have shown to be safe, inducing only mild adverse side effects in clinical trials, making them a great candidate for further clinical development. Adenoviruses can be genetically modified to increase their infectivity or improve the anti-cancer immune responses induced by the virus, e.g., through the expression of immunostimulatory molecules. The focus of this thesis was to develop and characterize several genetically modified oncolytic adenoviruses expressing either OX40L alone or OX40L and CD40L, two co-stimulatory molecules capable of engaging both the innate and adaptive arms of the immune system to fight the tumor. The insertion of the transgenes into the E3B-14.7k region of the Ad5/3-∆24 adenovector plasmid was performed using Gibson Assembly® cloning approach. After successful cloning, the recombinant viral genomes were transfected into A549 cells for viral amplification, followed by CsCl purification to produce a high titer viral preparation. The expression of the transgenes was studied in vitro by ELISA and functional assays, showing promising expression levels of functional OX40L and CD40L. However, when the infectivity and virus killing potency were analyzed, in vitro by immunocytochemistry and MTS assay; and in vivo using an immunodeficient mouse model, the data showed that the cloned viruses performed sub-optimally when compared to the control unarmed virus (Ad5/3-∆24). These findings suggest that the insertion of the two transgenes in place of the E3-14.7k gene was detrimental to the fitness of the virus.
  • 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.
  • 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.
  • 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.