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Browsing by discipline "Biokemi"

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  • Analysis of neuronal transcripts of PGC-1α transgenic mice 
    Döhla, Julia Maria (2013)
    Peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) is a transcriptional coactivator involved in mitochondrial biogenesis, oxidative stress response, and energy metabolism. PGC-1α is part of an energy sensing network that translates environmental influences into alterations in gene expression of mainly mitochondrial molecular pathways. A role in neuroprotection has been implicated for PGC-1α in the context of mitochondrial expression networks. Our research group has previously established a transgenic mouse line with stable overexpression of PGC-1α in brain neurons. Transgenic overexpression of PGC-1α is associated with an enhanced functional state of mitochondrial energy production. In the context of neurodegenerative processes, brain neurons of PGC-1α transgenic mice are protected against oxidative stressors in the MPTP mouse model of Parkinson's Disease. To further characterize the transcriptional activity of PGC-1α regulated gene networks in brains of transgenic mice, a quantitative real-time PCR based system was established. Gene expression was measured for a subset of genes found to be differentially regulated in a microarray based screening of RNA obtained from hippocampus and cortex of PGC-1α transgenic mice. Increased PGC-1α gene expression was found in hippocampus and cortex of PGC-1α transgenic mice, and their translation into protein was confirmed immunohistochemically. Expression analysis revealed significant changes in mRNA levels of PGC-1α controlled molecular pathways involved in mitochondrial energy production and antioxidant responses. Furthermore, alterations in the expression of some non-mitochondrial genes with established links to neurodegeneration were observed. Furthermore, a change in GABAA receptor subunit expression was detected. In accordance with previous studies on the PGC-1α transgenic mouse line, these findings suggest that differential gene expression associated with PGC-1α overexpression contributes to an enhanced functional state of neurons in hippocampus and cortex of PGC-1α transgenic mice. Increased knowledge about the transcriptional modulation of neuronal genes regulated by PGC-1α can lead to better insights into mechanisms governing neurodegeneration and neuroprotective pathways. Pharmacological modulation of PGC-1α activity may be a feasible approach for neuroprotective treatments in neurodegenerative diseases, such as Parkinson's Disease.
  • Circadian Timekeeping is Disturbed in Rheumatoid Arthritis at Molecular level 
    Olkkonen, Juri (2015)
    Introduction: Patients with rheumatoid arthritis (RA) have disturbances in the hypothalamic-pituitary-adrenal (HPA) axis. These are reflected in altered circadian rhythm of circulating serum cortisol, melatonin and IL-6 levels and in chronic fatigue. We hypothesized that the molecular machinery responsible for the circadian timekeeping is perturbed in RA. The aim of this study was to investigate the expression of circadian clock in RA. Methods: Gene expression of thirteen clock genes was analyzed in the synovial membrane of RA and control osteoarthritis (OA) patients. BMAL1 protein was detected using immunohistochemistry. Cell autonomous clock oscillation was started in RA and OA synovial fibroblasts using serum shock. The effect of pro-inflammatory stimulus on clock gene expression in synovial fibroblasts was studied using IL-6 and TNF-a. Results: Gene expression analysis disclosed disconcerted circadian timekeeping and immunohistochemistry revealed strong cytoplasmic localization of BMAL1 in RA patients. Perturbed circadian timekeeping is at least in part inflammation independent and cell autonomous, because RA synovial fibroblasts display altered circadian expression of several clock components, and perturbed circadian production of IL-6 and IL-1b after clock resetting. However, inflammatory stimulus disturbs the rhythm in cultured fibroblasts. Throughout the experiments ARNTL2 and NPAS2 appeared to be the most affected clock genes in human immune-inflammatory conditions. Conclusion: We conclude that the molecular machinery controlling the circadian rhythm is disturbed in RA patients.
  • Experimental approach for studying structural changes in axonal membrane upon nerve excitation 
    Ahokas, Lauri Juhani (2011)
    The Hodgkin and Huxley (HH) model of action potential has become a central paradigm of neuroscience. Despite its ability to predict action potentials with remarkable accuracy, it fails to explain several biophysical findings related to the initiation and propagation of the nerve impulse. The isentropic heat release and optical phenomena demonstrated by various experiments suggest that action potential is accompanied by a transient phase change in the axonal membrane. In this study a method was developed for preparing a giant axon from the crayfish abdominal cord for studying the molecular mechanisms of action potential simultaneously by electrophysiological and optical methods. Also an alternative setup using a single-cell culture of an Aplysia sensory neuron is presented. In addition to the description of the method, the preliminary results on the effect of phloretin, a dipole potential lowering compound, on the excitability of a crayfish giant axon are presented.
  • Identifying human epithelial architecture genes with tumour suppressor functions 
    Myllynen, Mikko (2013)
    Epithelial tissue is characterized by close cell-cell and cell-extracellular matrix (ECM) contacts as well as by apico-basal polarization. Integrity of these two features is important for functionality of epithelium. Additionally, proteins regulating polarity and cell junctions have been linked to cell cycle and apoptosis control. Consequently, defects in many of the polarity proteins have been linked to oncogenic events and loss of polarity is a hallmark of advanced cancers but whether it is causal to tumorigenesis is yet unknown. However, large body of knowledge on apico-basal polarity regulation and its connection on homeostasis control is derived from studies in Drosophila. This is mainly due to fact that efficient high throughput organotypic three dimensional (3D) culture methods enabling apico-basal polarization have not been available until the last decade. Large screens for epithelial polarity regulators have not been carried out in mammalian cells. Moreover, as cancer is the leading cause of death in developed countries and most of the cancers originate from epithelial tissues, knowledge of polarity regulation can be medically relevant. Oncogene MYC is overexpressed or amplified in variety of human cancers. The tumorigenic function of MYC is mainly due to its ability to drive cell cycle. We have previously shown that intact epithelial architecture is protective from cell cycle deregulating activities of MYC in 3D MCF10A mammary epithelial cell model and in vivo. This resistance can be overcome by inactivating LKB1 which is the human homologue of the polarity protein PAR4 implying a tumour suppressive role for epithelial architecture in mammalian cells. To identify regulators of epithelial architecture in mammalian cells, we have established lentiviral shRNA library (human epithelial architecture library, hEAL) encompassing 219 constructs targeting 77 genes associated with polarity regulation in Drosophila. We have previously screened the shRNA constructs for downregulation and quantified their effects on acinar morphology in the MCF10A 3D model. In this Master's Thesis I have validated the downregulation and phenotypes observed in a subset of the shRNAs during primary screening of the constructs of the library. Additionally, the possible co-operation with downregulation of the polarity regulators and conditional activation of MYC was determined. Most dramatically, downregulation of Wnt pathway gene DVL3 was shown to cause formation of enlarged multiacinar structures, which have increased proliferation. Additionally, downregulation of another Wnt pathway gene, GSK3β, resulted in acini with increased size and filled lumens. Thus these results propose a role for these genes in epithelial architecture regulation and tumour suppression in the used model even though apico-basal polarization of the acini was intact and no synergy with MYC was observed. Interestingly, no role in epithelial architecture regulation for Hippo pathway related genes FAT4 and MOBKL1A was found. Importantly, this study was able to validate primary screen showing relevance of the pipeline. Lastly, the study characterized the synthetic lethality phenotype found in the primary screen caused by downregulation of GTPase RHOA and chronic MYC activation. The shRHOA acini exhibited perturbed α6-integrin localization. When combined with MYC activation, the percentage of apoptotic acini was significantly increased. Importantly, the results suggest the observed synthetic lethality to be specific for the 3D context and to be associated with MEK/ERK and ROCK pathways. Taken together, in this study I have validated the role of novel epithelial architecture regulators and candidate tumour suppressors in MCF10A cells which may have medical relevance by helping to characterize tumorigenic processes. Furthermore, I characterized a novel 3D specific RHOA-MYC synthetic lethal interaction, which may prove to have therapeutic significance in MYC-driven cancers in future.
  • Osteoclast Activators Are Elevated in Intervertebral Discs with Modic Changes Among Patients Operated for Herniated Nucleus Pulposus 
    Torkki, Matias (2014)
    Modic-muutokset (MC) liittyvät alaselkäkipuun (LBP). Tulehdusta on pidetty erityisesti 'tyypin I' -MC:n avaintekijänä, mutta tähän mennessä potentiaalisista tulehduksellisista välittäjäaineista vain TNFα on yhdistetty MC:n. Tutkimuksen tavoitteena oli analysoida tiettyjä tulehduksen välittäjäaineita ihmisen kirurgisesti poistetuista välilevyistä, ja määrittää niiden yhteys MC:n kanssa viereisissä selkänikamissa. Tutkimuspopulaatio koostui 55 välilevynäytteestä; 20 'Ei MC' -välileyä, 20 'tyypin I' -välilevyä ja 15 'tyypin II' -välilevyä. 47 välittäjäaineen mRNA -ekspressio määritettiin eristetystä RNA:sta. Märitetyistä välittäjäaineista tilastollisesti merkittävät assosiaatiot löydettiin RANKL:sta (p=0.020), M-CSF1:stä (p=0.019), NFATc1:stä (p=0.021) ja RUNX1:stä (p=0.023), jotka olivat lisääntyneet 'tyypin II' MC -ryhmässä, kun taas OSCAR (p=0.023) oli lisääntynyt 'tyypin I' –ryhmässä verrattuna 'ei MC' -ryhmään. Kun nämä välittäjäaineet ovat yhteydessä osteoklastien erilaistumiseen ja lisääntymiseen, hypoteesimme mukaan välilevyjen erittämien aineiden aiheuttama nikamien osteoklastien stimulaatio on osallisena MC:n patofysiologiassa.
  • TOMM40-APOE-geenialueen vaikutus Alzheimerin tautiin 
    Isoviita, Veli-Matti (2014)
    Alzheimerin tauti (AT) on yleisin dementian syy ja perintötekijöiden vaikutus tautiriskiin on suuri. APOE-geenialue on vahvimmin AT:iin liittyvä geenialue. APOE:sta on kolme yleistä alleelia, joiden koodamassa proteiinissa on 1-2 aminohapon eroja: E2, E3 ja E4, joista APOE4 on voimakas AT-riskitekijä. Suomalaistutkimuksissa on osoitettu, että eräs yleinen APOE3-haplotyyppi lisää ja toinen APOE3-haplotyyppi pienentää AT-riskiä. Etiologinen variantti APOE3-haplotyypissä on tuntematon. Tässä tutkimuksessa selvitettiin kolmen pistepolymorfismin avulla APOE:n viereisen TOMM40-geenialueen vaikutusta tautiriskiin, erityisesti APOE3-haplotyypin osana. Aikaisemmin kerätystä vantaalaisesta ikäkohortista monistettiin tutkittavat alueet polymeraasiketjureaktiolla (PCR), käsiteltiin PCR-tuotteet restriktioenstyymeillä ja tutkittiin restriktiotuotteet geelielektroforeesilla. Saatuja tuloksia vertailtiin aikaisemmin aineistosta kerättyyn kliiniseen, neuropatologiseen ja geneettiseen tutkimustietoon. Tulokset osoittivat TOMM40-geenialueen olevan kytkentäepätasapainossa APOEpromoottorialueeseen, mutta ei APOE-proteiinia määrittävään eksoni-4:ään. TOMM40-polymorfismien avulla voitiin erottaa myös APOE-tyypistä riippumaton heikko AT-riskivaikutus. TOMM40- ja APOE-polymorfismien analyysi viittasi siihen, että primaari geneettinen riskitekijä APOE3-haplotyypeissä on APOE-geenin ei-koodavalla alueella, mahdollisesti promoottori/enhancer-alueella. Jatkotutkimuksissa selvitetään, vaikuttaako tämä riskitekjä APOE:n, TOMM40:n tai muiden lähigeenien ekspressioon.
  • Uusi DNA:n puhdistusmenetelmä 
    Laitinen, Jens (2014)
    Nykyiset DNA:n eristämiseen ja puhdistamiseen käytetyt myrkyttömät proteiinien ulossuolausmenetelmät (protein-salting-out) ovat vielä melko aikaavieviä ja eräissä tilanteissa jopa epäluotettavia. Tämän työn tarkoituksena on kehittää luotettava ja entisiä menetelmiä nopeampi proteiinien ulossuolausmenetelmä kromosomaalisen DNA:n eristämiseksi viljellyistä ihmisen soluista (Jurkat, K562 ja U937). Tämän uuden proteiinien ulossuolausmenetelmän avulla voidaan 60 minuutissa eristää puhdasta ja suurimolekyylimassaista DNA:ta erilaisiin vaativiin sovelluksiin, kuten genomisten DNA kirjastojen valmistukseen ja niiden analyysiin. Tässä työssä kehitetty DNA:n eristysmenetelmä validoitiin tekemällä puhdistetusta DNA:sta geenikirjasto, josta vektoretti-PCR:ään perustuvalla kromosomikävelyllä eristettiin ihmisen kasvurajoitegeenin, tp53-geenin, promoottorialue, joka analysoitiin restriktioentsyymikartoituksella ja sekvennoinilla.

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