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Browsing by discipline "Genetik"

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  • Pennonen, Jana (2017)
    Puberty is a process of physiological changes, through which an immature individual becomes sexually mature. In humans, timing of puberty is highly variable within and between sexes and populations. Timing of puberty represents a complex trait, which is controlled both genetically and environmentally. Precocious pubertal timing is associated with development of metabolic diseases later in life, such as obesity and diabetes, and other disorders as ovarian and testicular cancer. Despite the estimated high heritability (50-80%) of pubertal timing, its genetic background is still poorly understood. Recently, the genome-wide association studies (GWASs) revealed many novel pubertal timing associated loci. Nevertheless, molecular mechanisms behind these associations remain elusive. This thesis focused on gene vestigial-like family member 3 (VGLL3), which is associated with pubertal timing in humans and maturation in Atlantic salmon (Salmo salar). Since the main physical structures, such as the hypothalamus and the pituitary gland, needed in reaching puberty are evolutionary conserved and start to develop in vertebrates during embryogenesis, the aim was to study the expression pat-terns and role of vgll3 in zebrafish (Danio rerio) during this period. In order to localize expression patterns of the vgll3 gene in zebrafish embryos, a whole-mount in situ RNA hybridization (ISH) was performed. mRNA overexpression and morpholino oligonucleotide (MO) knockdown techniques were used to alter the vgll3 gene expression levels in 0-5 dpf zebrafish. The combined injections of both mRNA and MO were performed to validate MO specificity. The ISH experiment showed the expression patterns in 0-1 dpf embryos. The expression was ubiquitous up to 6 hours post fertilization becoming more localized to specific regions in the head and trunk of the embryos during the later stages. Altering vgll3 expression with high concentrations of synthetic mRNA or MO lead to phenotypical abnormalities such as shortened and curved body axis, pericardial and yolk sack edemas, deformed heads and eyes. However, it remained unclear if these malformations appear only due to the alteration of vgll3 expression levels. The results suggest that vgll3 may play an important role in the embryonic development. However, the study does not show that vgll3 has impacts on the pubertal timing in vertebrates by affecting the development of the structures required for sexual maturation.
  • Kuusisto, Jukka (University of HelsinkiHelsingin yliopistoHelsingfors universitet, 1990)
    Ns. restriktioentsyymin keksimisen myötä on kehitetty uusi menetelmä tutkia yksilön perimää geenitasolla. Perimäns isältämä DNA pilkotaan lyhyemmiksi pätkiksi restriktioentsyymin tunnistamista paikoista. Nämä pätkät kokoerotellaan elektroforeesilla. Radioaktiivisella koettimella saadaan näkyviin ne perimän sisältämät pätkät, joissa on kyseisen koettimen tunnistama ns. minisatellittialue. Näin saatu juovasto on yksilöspesifinen, ja sitä nimitetään yksilön DNA-sormenjäljeksi (DNA-fingerprint). Koirilla menetelmää voidaan käyttää yksilön tunnistamiseen, isyystutkimuksiin ja rodun sisäsiittoisuuden arvioimiseen. Isyystutkimuksessa jälkeläisen jokaisen juovan on löydyttävä jommalta kummalta vanhemmalta. Rodun sisäsiittoisuutta tutkittaessa arvioidaan rodun eri yksilöiden juovastojen samankaltaisuutta. Tutkimuksessa asetettiin pohdittavaksi, voiko voimakas sisäsiivoisuus alentaa menetelmän tuloksellisuutta varsinkin isyysmäärityksiä tehtäessä. Aineistona käytettiin 3 bedlingtoninterrieriä ja verrokkiryhmänä 8 sekarotuista. DNA eristettiin 2 ml verinäytteestä (EDTA). Aineiston perusteella laskettiin todennäköisyydet, että isyystapaus jäisi ratkaisematta käytettäessä kahta koetinta. Bedlingtoneilla tämä prosentti oli 36.0% (eli 36 isyystapausta sadasta jäisi ratkaisematta) ja sekarotuisilla 6.4%. Saadut prosentit ovat muihin vastaaviin tutkimuksiin verrattuna melko suuria. Tulosten perusteella havaitaan sisäsiittoisuuden vähentävän menetelmän tuloksellisuutta merkittävästi. Menetelmän käyttö isyysmäärityksiin ei ole mielekästä rutiininomaisena menetelmänä johtuen menetelmän kalleudesta ja hitaudesta. Perinteisillä menetelmillä ratkeamattomissa kiistatapauksissa voidaan DNA-sormenjälkianalyysillä kuitenkin saada hyödyllistä lisätietoa. Myös eri koirarotujen sisäsiittoisuuden arvioimisessa voidaan menetelmää soveltaa tuloksellisesti.
  • Niskanen, Julia (2016)
    Epidermolysis bullosa (EB) is a group of hereditary skin disorders caused by mutations in the genes that code for adhesion molecules in keratinocytes. The symptoms of the disease include blisters and erosions in the skin as well as abnormalities in the mucosal membranes, nails and tooth enamel. Depending on the causative mutation the severity of the disease ranges from mild to lethal. This master s thesis was carried out in professor Hannes Lohi s canine genetics research group. It is a case study aiming to identify the genetic cause of EB present in Central Asian shepherd dogs. Furthermore, the mode of inheritance, frequency of the mutation in the breed and the effect of the mutation on the tissue were also examined. Primary research material included blood and tissue samples from a family of Central Asian shepherd dogs. Additional samples were obtained from other Central Asian shepherd dogs as well as from dogs of closely related breeds. All dogs included in this study are owned by private persons, and participation in this study was voluntary. The research methods used in this study included both wet laboratory experiments and bioinformatic in silico procedures. The genome of one affected dog was sequenced in order to identify the gene causing EB, and data from the sequencing was filtered with multiple programs according to recessive model. The model was decided after analyzing the pedigree of the affected dogs. After finding the likely causative gene the mutation was validated in a larger cohort with Sanger sequencing. Protein expression in the tissue of affected dogs was also studied using immunofluorescence staining. As a result of this study, a new mutation causing recessive dystrophic EB was identified. The mutation is specific to Central Asian shepherd dogs. Affected dogs have a homozygous mutation in the COL7A1 gene, which codes for collagen VII α1 protein. The mutation causes a premature stop codon in the mRNA sequence, which results in abnormal protein production and separation of skin layers. The frequency of the mutation allele in the sample is approximately 18 % and more than a fourth of the dogs in the sample are carriers. Based on the pedigree analysis, the mutation is relatively new and it is only found in a small population. The disease can be prevented from becoming more common in the breed with the help of a gene test, and the test will be available in the commercial MyDogDNA gene test panel.
  • Väyrynen, Pia (2017)
    Follicle-stimulating hormone (FSH) regulates mammalian reproduction. The hormonal function of FSH is exerted through its receptor, FSHR. Hormone binding leads to signal transduction by the synthesis of the second messenger (intracellular cAMP) and the activation of other downstream signaling pathways. FSH receptors are mainly found in ovaries and testes where FSH stimulates e.g. follicular growth and spermatogenesis, respectively. It has been previously shown that FSHR mutations are linked to infertility through abnormalities in the receptor function. For example, inactivating FSHR mutation (Ala189Val) leads to arrest of follicular development in females and reduced sperm counts in males. FSH action seems to be especially critical for folliculogenesis and essential for female fertility. The development of gonads and dysfunctions affecting reproduction are the focus of our research group. Studies of recent years have demonstrated extragonadal FSHR expression, including endothelial cells of female reproductive tract and developing placenta. The physiological relevance and function of this extragonadal FSHR is still not well known, especially during embryogenesis. In addition, the expression or function of the mutated FSHR has not been studied in any cells endogenously expressing the receptor. As a consequence, the purpose of this thesis was to study FSHR expression and function in human pluripotent stem cells (hPSCs) as a model for early human development. The study was conducted in a human embryonic stem cell line (hESC H9, 46, XX) and two human induced pluripotent stem cell lines (hiPSC HEL127.6 and HEL128.5; 46, XX). iPSCs were obtained from female patients carrying the A189V mutation in the FSHR gene. Cells were differentiated for 8/12 days using two in vitro cell culturing protocols for distinct differentiation attempts. The first protocol (protocol D) was designed for embryonal cell differentiation, the other (trophoblast protocol) for extraembryonal differentiation. Cells at different stages of differentiation were studied with qRT-PCR and immunocytochemistry. At d8, cells were stimulated with FSH for qPCR studies and for cAMP assay. Control cells (H9) differentiated by protocol D endogenously expressed functional FSHR. These cells responded dose-dependently to FSH stimulation by substantially increasing cAMP production, downregulating FSHR mRNA expression and altering inhibin gene expression. Patient-derived iPSCs carrying the mutation expressed FSHR as well but the receptors were non-functional as expected. Both H9 and patient-derived cells that differentiated into trophoblast-like cells with the other protocol, also expressed FSHR at low level but did not respond to FSH stimulation. Preliminary results with protocol D indicate that the cells resemble extraembryonic cell types. The study has revealed novel information and insight about extragonadal FSHR expression and function during differentiation. However, the exact identity and the biological role of these cells is yet to be confirmed.
  • Ketola, Katri (2015)
    Darwinian selection can be measured and investigated from gene sequences. A certain gene form favored by positive selection will become more common in the population. Detecting strong positive selection is rare, but it has been found to affect genes involved in immune defense and perception of odorants. Genes under positive selection have a possible role in speciation or adaptation. This is why chemical communication, being based on the sense of smell, is an interesting topic for measuring natural selection and positive selection in particular. Social insects, such as ants, are model organisms for chemical communication. They use chemical communication not only for finding nutrition and detecting intruders, but also in coordinating the activities of several thousands of colony members. Insects perceive odorant signal molecules with their antennae. Odorant binding proteins (OBP) and chemosensory proteins (CSP) bind and transport odorant molecules through the sensillar lymph. OBPs and CSPs are also suspected to have a role in selecting odorants. This work focuses on two OBP genes and two CSP genes to study natural selection. All of these genes are conserved among all ant species. Three of these genes, OBP1, CSP1 and CSP7, are strongly expressed in the antennae suggesting that they function in chemical communication. CSP7 also has a known function in nest mate recognition in ants and OBP1 is known to bind a queen pheromone in the honeybee. The data includes gene sequences from 7 Formica ant species (270 sequences in total). The main goals of the research were to find out 1) the extent of variation between and within closely related ant species, 2) which evolutionary forces, natural selection or random drift, are behind the variation and 3) are there systematic differences between the two social forms of ants suggesting that these genes would affect the social structure of an ant colony. The variation in the sequence data was visualized by phylogenetic, principal coordinate and fixed differences analyses. Differences between populations were studied by FST values. The evolutionary forces shaping chemical communication genes within the species and populations, were studied by McDonald-Kreitman test, Tajima s D, Fu and Li test and MFDM test. The data shows that two of the species, F. cinerea and F. exsecta, significantly differ from the other five ant species, the F. rufa group species, and that the F. rufa group species don t significantly differ from each other based on these genes and this data. This could be due to their recent speciation or crossing between the species leading to hybrids in the data. The results of the evolutionary analyses are inconsistent. However, CSP7 has the strongest indication of selection based on all of the tests. Possible selection and sequence variation was detected at a predicted transcription factor binding site in F. cinerea. This indicates that selection might affect the regulation of CSP7. In the future it would be interesting to check the true transcription factor binding sites experimentally.
  • Neittaanmäki, Henriikka (University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2017)
    Hammasresorptio on kissojen yleisin hammassairaus. Sairauden aiheuttavat odontoklastit eli hampaan luunsyöjäsolut, joiden aikaansaama hammaskudoksen tuhoutuminen johtaa lopulta hampaan menetykseen. Hammasresorptio voi alkaa mistä tahansa hampaan pinnalta, joten hampaiden röntgenkuvaus on olennainen osa diagnostiikkaa. Pitkälle edetessään muutokset ovat kivuliaita ja niiden ainoa hoito on tapauksesta riippuen hampaan poisto tai kruunuamputaatio. Hammasresorptioiden esiintyvyys on korkea, aikaisemmissa tutkimuksissa 29-67 % välillä. Sairauden etiologia on pitkälti epäselvä, vaikka lukuisia teorioita on esitetty. Kirjallisuudessa hammasresorptioiden on epäilty liittyvän mm. korkeaan ikään, rotuun, muihin suu- ja hammassairauksiin, virusinfektioihin sekä kissan ruokavalioon ja elinympäristöön liittyviin tekijöihin. Tämän tutkimuksen kirjallisuuskatsauksessa kartoitettiin hammasresorptioihin liitettyjä erilaisia etiologisia teorioita ja erilaisille populaatioille tehtyjä esiintyvyystutkimuksia. Sairauden patogeneesiä, diagnosointia ja hoitoa käsiteltiin lyhyesti. Tutkielma sisältää alkuperäistutkimuksen, joka on poikittaistutkimusaineistosta tehty tapaus-verrokkianalyysi. Tutkimuksessa käytettiin internetkyselynä toteutettua kissojen terveyskyselyaineistoa. Tähän analyysiin otettiin mukaan vastaukset 8115 kissasta. Tutkimuksen ensimmäisenä tavoitteena oli selvittää hammasresorption esiintyvyyttä suomalaisilla maatiais- ja rotukissoilla. Hypoteesina oli, että hammasresorption esiintyvyys olisi korkeampi puhdasrotuisilla kissoilla ja tietyissä roduissa. Toisena tavoitteena oli arvioida hammasresorption riskitekijöitä tarkastelemalla sairautta eri ikäryhmissä sekä suhteessa sukupuoleen, ruokavalioon, elinympäristöön, muihin suu- ja hammassairauksiin ja kissojen muihin sairauksiin. Hypoteesina oli, että hammasresorption riskitekijät olisivat kirjallisuudessa aikaisemmin kuvattujen kaltaisia. Monimuuttujaisen logistisen regressiomallin avulla laskettiin ennusteita hammasresorption esiintymisen todennäköisyydelle erilaisilla kissoilla. Kissojen hammasresorption esiintyvyys suomalaisille kissanomistajille suunnatussa internetkyselyssä oli 3,9 %. Esiintyvyys oli tässä tutkimuksessa alhaisempi kuin sellaisissa, joissa kissojen hampaita on tutkittu kliinisesti. Esiintyvyys oli tietyissä roduissa huomattavasti muita rotuja korkeampi. Hammasresorptioon tilastollisesti merkitsevästi assosioituvia ominaisuuksia olivat rotu, ruoan saatavuus, stomatiitti, parodontiitti ja interaktioina toistensa kanssa ikä, ientulehdus ja hammaskivi. Hammasresorptioon assosioituvat tekijät olivat pääasiassa aikaisemmin kirjallisuudessa kuvattujen kaltaisia. Tietyillä roduilla on lisääntynyt alttius, mikä voi viitata geneettiseen komponenttiin hammasresorption etiologiassa. Rodun yhteys hammasresorptioon vaatisi kuitenkin tarkempaa selvitystä kliinisillä jatkotutkimuksilla.
  • Salo, Annukka (University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2015)
    Tässä tutkimuksessa selvitettiin sairauksien ja ongelmien esiintyvyyttä norjalaisilla metsäkissoilla Suomessa. Ensisijaisena tavoitteena oli kerätä tietoa rodulla esiintyvistä sairauksista, mutta samalla selvitettiin myös kissojen taustatietoja (kissan ja vanhempien rekisteröintitiedot, sukupuoli, ikä, paino, synnyinmaa, vieroitus- ja omistajalle saapumisikä, elinympäristö, ravinto, veriryhmä), perinnöllisten sairauksien (glykogeenin kertymäsairaus tyyppi IV ja hypertrofinen kardiomyopatia) varalta tehtyjä tutkimuksia ja niiden tuloksia, kuolinsyitä ja jalostukseen liittyviä tekijöitä (kasvattajien osuus, pentujen ja pentueiden kissakohtainen määrä, kissan käyttö jalostukseen tulevaisuudessa). Rotukissoilla, kuten norjalaisilla metsäkissoilla, ei ole aiemmin Suomessa tehty terveystietojen kartoitusta tieteellisesti tai yhtä kattavasti kuin tässä tutkimuksessa. Tutkimusta tehdessä ei myöskään ollut tiedossa vastaavanlaisia tutkimuksia ulkomailta. Nollahypoteesina oli, että norjalaisilta metsäkissoilta ei löytyisi mitään sairautta epätavallisen paljon. Tutkimus toteutettiin sähköisen kyselylomakkeen avulla. Tavoitteena oli kerätä 400 kissan terveystiedot. Tietoja kerättiin kolmen kuukauden ajan vuonna 2012 ja niitä saatiin 604 kissalta. Tutkimustulosten perusteella todettiin, että yleisimmät eläinlääkärissä todetut sairaudet ja ongelmat kuuluivat suun ja hampaiden (11,4 %), munuaisten ja virtsateiden (9,6 %) ja sukupuolielinten (6,8 %) sairauksiin ja ongelmiin. Omistajien itse toteamat ongelmat liittyivät useimmin käytöshäiriöihin (35,3 %). Käytöshäiriöiden osuus oli odotettua korkeampi ja joitain sairauksia, kuten korvasairauksia, esiintyi odotettua vähemmän. Saavutettu otoskoko on tilastollisesti riittävä kuvaamaan norjalaisilla metsäkissoilla esiintyvien sairauksien ja ongelmien esiintyvyyttä Suomessa. Useimpien taustatietojen osalta saatiin myös riittävän kattavasti tietoa, jotta tuloksia voidaan yleistää koskemaan koko populaatiota. Kunkin elin- tai ongelmaryhmän osalta on kuvattu yksittäisten sairauksien ja ongelmien jakauma ryhmän sisäisesti, jotta todennäköisimmät ja harvinaisimmat ryhmänsisäiset sairaudet saataisiin selville. Nämä luvut kuvaavat luotettavasti niiden jakaumaa vain tutkimuksen kissoilla. Sama pätee kuolinsyihin. Tämän tutkimuksen tuloksista on apua etenkin norjalaisten metsäkissojen kasvattajille. Tiedostamalla rodun keskeisimmät ongelmat, voidaan niiden ehkäisemiseen kiinnittää huomiota jalostuksessa ja valistaa kissojen omistajia todennäköisimmistä terveysongelmista. Tutkimuksen tuloksia voidaan hyödyntää rodulle rakennettavassa terveystietorekisterissä ja jalostuksen tavoiteohjelmassa. Eläinlääkäreille tulokset antavat tärkeää tietoa mihin ongelmiin rodulla pitäisi erityisesti kiinnittää huomiota. Lisäksi tutkimus edesauttaa ja kannustaa kaikkien suomalaisten kissojen terveystietojen keräystä. Tutkimus on myös ollut osa laajempaa käynnissä olevaa kissojen geenitutkimushanketta Suomessa (Kissojen geenitutkimus 2015).
  • Aho, Kukka (2012)
    In multicellular organisms, complex signalling mechanisms have evolved to guide the behaviour of individual cells. Growth factors are secreted proteins that can stimulate the proliferation and/or differentiation of cells. Vascular endothelial growth factor D (VEGF-D) is a ligand for VEGF receptor 2 (VEGFR-2) and for VEGFR-3, which are predominantly expressed on blood vascular endothelial cells and on lymphatic endothelial cells, respectively. Thus VEGF-D can contribute to growth of both blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). Although there have been many reports showing the angiogenic and lymphangiogenic effects of VEGF-D, its physiological role is still largely unknown. Most of these reports are severely hampered by incomplete characterization of the specific form of VEGF-D that was used. During or after secretion, VEGF-D undergoes complicated proteolytic processing. Alternative Nterminal cleavage results in two different fully processed forms, VEGF-D major and VEGF-D minor. Processing significantly increases the activity of VEGF-D towards its receptors. Surprisingly, it is still unknown whether the differential N-terminal cleavage of VEGF-D has any effect on receptor binding activity or on receptor activation. The goal of this study was to produce and purify high quality biologically active VEGF-D which is needed for studying the physiological role of this growth factor. Several different forms of recombinant human VEGF-D were produced using the Drosophila Schneider 2 insect cell system. A bioassay utilizing the Ba/F3 cells expressing chimeric VEGFR/EpoR receptors was used to determine the receptor binding activities of recombinant VEGF-Ds. Two constructs producing biologically active VEGF-Ds were chosen for chromatographic purification (untagged major and his-tagged major forms). During purification, the activity of both VEGF-D forms towards their receptors decreased significantly. In case of the untagged form, this was presumably due to some residual proteolytic activity during purifications. The results might indicate that only the major form is responsible for the activation of VEGFR-3. The fact that no activity of the minor forms was detected when screening the cell supernatants with Ba/F3-VEGFR-3-EpoR-bioassay, supports this explanation. If this explanation can be verified, the role of the alternative N-terminal cleavage becomes obvious: By proteolysis the activity of VEGF-D can be redirected from the lymphatics towards the blood vessels.
  • Korsisaari, Nina Kristiina (1998)
    Cells in tissues have only three serious options in life; they can grow and divide, remain static, or die by apoptosis. Upon growth factor stimulation a cell enters the so called cell cycle which will eventually lead to the division of the cell. Cell cycle can be divided into four phases; G1, S, G2 and M. The current model of the cell cycle control holds that the transitions between different cell cycle states are regulated by cyclin dependent kinases (CDK) with their activator subunits, the cyclins. CDK regulation can be separated into four distinct mechanisms, one of which being phosphorylation on the so called T-loop leading to complete activation. This phosphorylating activity is mediated by apparently a single enzymatic activity termed the CDK activating kinase, CAK. CAK activity was originally isolated as a biochemical purification extract and the enzyme was surprisingly noticed to be structurally related to CDKs. Since a novel cyclin was identified to be associated to it, the enzyme exercising CAK activity was named CDK7 and the cyclin was designated cyclin H. An entirely new perspective on CDK7 function was opened when CDK7 was identified as a subunit of transcription factor IIH (TFIIH) and shown to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). CDK7 has also been suggested to be involved in irradiation sensitivity pathways and nucleotide excision repair functions. To elucidate the intriguing in vivo role of CDK7, proteins interacting with CDK7 were screened for using the yeast two-hybrid method as part of previous studies of the laboratory. The results showed that 15 out of 144 (10,4%) positive clones were identified to encode a peptide sequence of a protein previously known as the inhibitor/interactor of protein kinase C (PKCI). These yeast colonies had an unexpected phenotype; contradictory to a dark blue color of the colonies, indicating strong interaction, the size of the PKCI colonies was small compared to others, indicating a possible growth inhibition effect. Several DNA open reading frames (ORF) coding for proteins related to human PKCI have been identified in a broad range of species representing mammalian, plant, fungal and bacterial kingdoms, all these forming a HIT (conserved triad of histidines) protein family. Another human member, part of this now super family, named FHIT (fragile triad of histidines) was identified with a dinucleoside 5’,5’’’-P1,P3-triphosphate hydrolase activity. These molecules; substrates of FHIT and related enzymes have been proposed to have various intracellular functions, including signalling stress responses. The aim of this study was to extend the investigation of the interaction between CDK7 and PKCI observed in yeast two-hybrid by means of several genetic and biochemical approaches to determine if this observed interaction and growth phenotype has any physiological significance. Investigations included performing yeast two-hybrid screening for PKCI, developing yeast three-hybrid system and carrying out growth rate assays for yeast liquid cultures. These studies also included performing biochemical purifications of over-expressed proteins, immunoprecipitations, western blot analysis and kinase activity assays. Protein extracts originated from transformed yeast cells, transfected mammalian cells or from in vitro transcription and translation reactions. On basis of growth rate assays it can be concluded that PKCI has an inhibitory growth effect in yeast. The preliminary finding of a specific PKCI-CDK7 interaction in yeast two-hybrid, however could not be conclusively verified by the other methods that were used in this study. Studies of PKCI characterisation also included examination of the subcellular localisation of PKCI in mammalian cells by immunofluorescence labelling of HA-PKCI. Results showed PKCI to localize both in the nucleus and in the cytoplasm. Also, studies to elucidate the function of PKCI were performed; whether it possesses enzymatic activity related to that of FHIT. By NMR spectroscopy using bacterially produced GST-PKCI, hydrolase activity towards ADP was indeed observed. Future studies will include elucidation of possible links between growth inhibition and hydrolase activity, in the form of stress signalling functions. The main focus of our future studies will be the generation of mice with targeted PKCI alleles offering powerful means to reveal the function of PKCI through observing phenotypes and through detailed analysis of these mice harbouring wild type, hypomorphic or null alleles.
  • Karppinen, Kristiina (University of HelsinkiHelsingin yliopistoHelsingfors universitet, 1992)
    Tutkimuksessa selvitettiin suomenpystykorvien (n=255) plasmaproteiinien polymorfismia elektroforeettisin menetelmin. Yksisuuntaisen ja kaksisuuntaisen elektroforeesin, sekä isoelektronisen fokusoinnin avulla saatiin määritettyä kahdeksan polymorfista proteiinia, joiden geenifrekvenssit laskettiin. Tutkimuksen yhteydessä löydettiin kaksi ennen julkaisematonta alleelia. Aineisto jaettiin populaatio- ja perhemateriaaliryhmiin, joiden frekvenssejä vertailtiin. Frekvenssit erosivat tilastollisesti merkitsevästi ainoastaan yhden alleelin kohdalla. Geenifrekvenssien avulla laskettiin todennäköisyyksiä saada selville virheelliset vanhemmat. Käytettäessä kahdeksaa proteiinia on todennäköisyys saada erotettua väärä isä tai emä 96 %. Tämä on maksimaalinen todennäköisyys, joka saavutetaan, mikäli pentuja on viisi kappaletta. Jos pentuja on vain yksi, on todennäköisyys 73 %. Populaatio- ja perhemateriaaleilla ei ole eroa. Pennunvaihtotapaus (molemmat vanhemmat väärät) saadaan selvitettyä 89 % todennäköisyydellä. Käytännössä plasmaproteiineja käytetään koiralla eniten isyysmäärityksiin. Niitä voidaan käyttää myös koiran identifioimiseen ja lajin/rodun historian tutkimiseen.
  • Ahvenainen, Terhi (2015)
    Huntington's disease (HD) is a progressive neurodegenerative disorder that causes involuntary muscle movements, deteriorates muscle coordination and cognitive decline. Typical onset age of the disease is in mid age, although a juvenile form of HD is also known. The disease is inherited in an autosomal dominant manner via a mutation in the huntingtin gene (HTT). The characteristic mutation in HTT is an expansion of the glutamine stretch at the 5 end of the gene. Excessive amounts of glutamine residues alters the conformation and chemical features of the huntingtin protein (HTT) leading to accumulation of cellular aggregates. Although to date there are several known alterations in the cell that contribute to the disease, the pathogenesis of HD is not fully understood. Ubiquitin proteasome system (UPS) dismantles proteolytically unneeded or damaged proteins, which are targeted to proteolysis when ubiquitin tags are added to them. Deubiquitinating enzymes (DUB) recycle ubiquitin molecules by releasing them from proteasome substrates. Recycling of ubiquitin is critical to a cell as it maintains the free pool of the targeting molecule. Ubiquitin-specific protease 14 (USP14) is one of the DUB family enzymes and its distinctive function is to remove ubiquitin molecules from the tip of the ubiquitin chain and thus antagonize protein degradation. Although the specific function of the protein is unclear, it seems that USP14 operates as a fine regulator of protein turnover rate and in ER stress both in catalytic and non catalytic manner. The role of USP14 is especially emphasized in the nervous system, as it regulates synaptic transmission and neuronal development. Although it is suggested that dysfunction of UPS is involved in the pathogenesis of HD, the role of USP14 in the disease remains to be unknown. IU1 is a novel inhibitor of the catalytic domain of USP14. Studies with IU1 indicate that inhibition of USP14 enhances the clearance of aggregate prone proteins. The approach of this thesis was aimed to elucidate the routes of HD pathogenesis from diverse approaches. The general aim of the thesis was to investigate the role of USP14 in the wild-type PC6.3 cell model, and in the pathogenesis of HD by expressing HTT proteins with different lengths of glutamine stretches in PC6.3 cells. The specific aim of the study was to examine by western blot and microscopy analysis the pathogenic routes of HD that involve ER stress, oxidative stress, autophagy and mutant HTT aggregate dynamics. The function of USP14 was studied with overexpression of USP14, or by inhibiting its catalytic activity by IU1. The findings of this thesis show that overexpression of USP14 enhances the clearance of mutant HTT aggregates, and this effect is obtained in catalytic activity dependent manner. I show that upregulated USP14 is connected to improved clearance of mutant HTT and inhibition of autophagy, suggesting that the degradation is mediated via UPS. The catalytic activity of USP14 might also be important in ER stress regulation, as the results indicate that IU1 activates phosphorylation of both JNK and eIF2α. I was also able to establish a connection between USP14 and GADD34, as I show that GADD34 upregulates USP14. Finally, I show that catalytic inhibition of USP14 decreases the expression of antioxidant SOD2. The data in this thesis is lacking statistical significance, and it can be considered solely as a guideline. However, together these results indicate that the deubiquitinating activity of USP14 increases survival in PC6.3 cells in both a healthy and a HD model.
  • Syrjänen, Emmi (University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2014)
    Epilepsia on yksi yleisimpiä kohtauksia aiheuttavista kroonisista neurologisista sairauksista, johon eläinlääkärit törmäävät työssään. Epilepsian perinnöllisyyttä on tutkittu usealla eri koirarodulla, ja tähän mennessä on löydetty kymmenen rodun epilepsian aiheuttajageenit. Whippetien idiopaattisesta epilepsiasta ei löydy tieteellisiä julkaisuja, mutta rodun yksilöitä on ollut mukana yksittäisissä epilepsiatutkimuksissa. Kirjallisuuskatsauksessa käsitellään koirien epilepsiaa yleisesti, epilepsian perinnöllistä taustaa koirilla ja ihmisillä, perinnöllisten sairauksien geenitutkimusta sekä whippetiä rotuna ja lyhyesti myös rodun perinnöllisiä sairauksia. Kokeellinen osuus koostuu kolmesta erilaisesta osa-alueesta: kyselytutkimus, sukupuun arviointi sekä ehdokasgeenitutkimus. Kyselytutkimuksen avulla määritettiin 31 whippetin idiopaattisen epilepsian kohtauskuva. 77 % whippetien kohtauksista määriteltiin yleistyneiksi ja 23 % paikallisiksi epilepsiakohtauksiksi. Epilepsiaa sairastavissa whippeteissä esiintyi huomattavasti enemmän uroksia (65 %) kuin narttuja (25 %). Kohtausten keskimääräinen alkamisikä oli 3,2 vuotta, ja 32 % whippeteistä oli epilepsian pitkäaikaislääkityksellä. Yhdeksi mahdolliseksi whippetien idiopaattisen epilepsian periytymismalliksi arvioitiin sukupuun perusteella X-kromosomaalinen väistyvä periytyminen. Ehdokasgeenitutkimuksessa tutkittiin ihmisillä tunnetun X-kromosomaalisen epilepsiageenin SLC6A8:n assosiaatiota whippetien epilepsiaan. Tutkimukseen valittiin 24 epilepsiaa sairastavaa whippetiä ja näille 24 tervettä verrokkia. Assosiaatiota tutkittiin yhden emäsparin vaihteluiden (SNP) avulla. Assosiaatiota whippetien epilepsialla ja tutkituilla yhden emäsparin vaihteluilla ei havaittu. SLC6A8-geenin assosiaation tutkimiseksi seuraava mahdollinen tutkimus olisi koko SLC6A8-geenin sekvensointi. Whippetien epilepsian periytyminen voi mahdollisesti olla myös monitekijäistä, kuten monella muulla rodulla on todettu. Koska whippetien kohtaukset jakautuivat selkeästi kahteen eri kategoriaan, on sekin mahdollista, että epilepsian taustalla olisi kaksi geneettisesti täysin erilaista tautia. Whippetien idiopaattista epilepsiaa aiheuttavan geenin tai geenien löytämiseksi vaaditaan vielä lisätutkimuksia, esimerkiksi koko perimänlaajuista kartoitusta.