Browsing by discipline "Genetics"
Now showing items 41-48 of 48
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(2020)Tiivistelmä – Referat – Abstract Ikääntymiseen liittyvien biologisten ilmiöiden, kuten rappeutumisen ja sairauksien tutkiminen on ensiarvoisen tärkeää, koska näistä ilmiöistä on haittaa niin yksilölle kuin koko yhteiskunnalle. Erityisen kiinnostavaa on, jos sairaudet ja rappeutuminen pystytäisiin parantamaan tai niiden haitallisia vaikutuksia voitaisiin vähentää. Ikääntymiseen voidaan yhdistää monia vakavia sairauksia kuten hermostorappeumasairaudet, syöpä ja diabetes. Hermostorappeumasairauksille on tunnusomaista proteiiniaggregaattien muodostuminen. SBC003-yhdiste on luonnon yrtistä eristetty molekyyli, joka on potentiaalinen lääke hermostorappeumasairauksiin. Aikaisemmin tehdyt tutkimukset hermostorappeuma sairausmalleilla osoittavat yhdisteellä olevan voimakas konsentraatiosta riippuvainen hermosoluja suojaava vaikutus. Malliorganismit ovat tärkeitä, koska niillä pystytään testaamaan potentiaalisten molekyylien vaikutusta vahingoittamatta kuitenkaan ihmistä. Hiivasta on tullut mielenkiintoinen malliorganismi ikääntymisen ja ikääntymiseen liittyvien sairauksien tutkimuksessa. Monet biologiset prosessit ja aineenvaihduntareitit ovat konservoituneet hiivasta aina ihmiseen asti. Lisäksi geneettiset muutokset on helppo jäljittää. Ikääntymisen tutkimisessa leivinhiiva (Saccharomyces cerevisiae) on osoittautunut erinomaiseksi malliorganismiksi. Hiivan kronologinen ikääntyminen on osoittautunut myös hyväksi menetelmäksi tutkittaessa solujen post-mitoottista ikääntymistä. Hiivasoluissa pystytään havaitsemaan nopeasti, edullisesti ja helposti proteiiniaggregaattien aiheuttamat haitalliset vaikutukset. Käytettäessä hiivaa malliorganismina tutkimuksessa pystytään luomaan suora linkki geenien ja kemiallisten yhdisteiden välille. Hiivalla tehdyt solujen sairausmekanismien tutkimukset ovat vertailukelpoisia ihmisten sairauksien kanssa. Nämä seikat tekevät hiivasta kiinnostavan malliorganismin myös hermostorappeumasairauksien lääkeainetutkimuksessa. Työn tavoitteena oli selvittää, voidaanko hiivasairausmalleja käyttää SBC003-yhdisteen vaikutuksien arvioimiseen hermostorappeumasairauksissa, ja onko tutkittavalla yhdisteellä pelastava vaikutus haitallisia aggregaatteja muodostavia proteiineja vastaan hiivasairausmalleissa. Lisäksi tarkoitus oli tutkia, onko SBC003-yhdisteellä vaikutusta ikääntymisestä aiheutuvaan elinvoimaisuuden heikkenemiseen hiivasoluissa. Tutkimuksissa käytettiin seuraavia menetelmiä ja koejärjestelyjä 1. spottitestejä ja 2. elinkiertotestejä ravistelukasvatuksina, joiden elinvoimaisuutta mitattiin näytteillä, joita puolestaan analysoitiin edelleen Bioscreen-kasvatuksia apuna käyttäen. Spottitestien ja elinkiertotestien tuloksien mukaan SBC003-yhdisteellä saattaa olla pelastava vaikutus haitallisia FUS-aggregaatteja vastaan hiivan ihmisen sairausmallin soluissa. Tutkimustulosten mukaan SBC003-yhdisteellä on solun elinvoimaisuutta parantava vaikutus soluihin, jotka rappeutuvat ikääntymisen seurauksena. Saatujen tulosten varmentamiseksi pitäisi elinkierotestit ja spottitestit toistaa ja lisäksi tehdä sekä geneettisiä että epigeneettisiä tutkimuksia.
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(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.
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(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.
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(2018)The insulin/insulin-like growth factor signaling pathway plays an important role in the regulation of growth and development, metabolism, lifespan, reproduction and stress response of an organism. As the pathway is evolutionarily conserved, Drosophila melanogaster serves as a powerful model for characterizing the underlying molecular mechanisms and genetic components, which may further appear as potential therapeutic targets for metabolic disorders. Drosophila has eight genes encoding insulin-like peptides (DILPs), which display stage- and tissue-specific expression patterns. Furthermore, the expression of DILPs is regulated by nutritional status. To date, only few transcription factors have been shown to regulate the expression of the dilp genes: for instance Dachshund and Eyeless are known transcriptional activators of dilp5 and dFOXO is a known transcriptional activator of dilp6. Since transcription factors directly regulating the expression of dilps are not yet well known, there is a need to study them further. The aim of this master’s thesis was to screen for novel transcriptional regulators of the dilp genes and study their phenotypic effects in vivo. For this purpose Drosophila S2 cells were transfected with vectors containing the promoter regions of all the dilp genes and with a library of 822 transcription factors. Promoter activity of the dilp genes was further measured by using the Dual-Luciferase® Reporter Assay System. Some of the revealed potential regulators were further studied in vivo to confirm their regulatory functions by using RNA interference to silence these factors or overexpressing them and by performing quantitative real-time PCR to measure dilp expression levels. Subsequently, organismal growth and development were assessed and effects on larval metabolic profiles were monitored. The screen revealed a few potential transcriptional regulators, among which Spalt-related (Salr) was selected for further study. It was shown to act as an activator of dilp6 expression. dilp6 is an interesting target among the dilp network due to its central role as a communicator between two of the important organs involved in insulin signaling: the brain and the fat body. Subsequently, both Salr and DILP6 were shown to have a role in controlling growth and development of larvae. Based on the experiments in this thesis Spalt-related seems to have a broader role as a metabolic regulator, which is not limited to its function as a regulator of dilp6. Thus, it might act as an important link between the insulin signaling pathway and other pathways regulating growth and metabolism in Drosophila.
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(2019)Epithelial cells form a barrier between the tissue and the external environment. Epithelial morphogenesis refers to the shaping of epithelial layers and is a key step in the development of organisms. The actin cytoskeleton provides the cell its form and during epithelial morphogenesis, produces force to shape the cells. To achieve this, the actin cytoskeleton is organized into protrusive and contractile networks. In a living cell, these actin networks are dynamic, as the filaments are constantly undergoing assembly and disassembly. Actin-binding proteins regulate the turnover of actin filaments, but in epithelial morphogenesis, the regulatory role of most of these proteins is still relatively unknown. In all multicellular organisms, actin disassembly is controlled by ADF/cofilin. ADF/cofilin activity is furthermore enhanced by other actin-binding proteins, one of which is cyclase-associated protein (CAP). CAP promotes actin turnover by accelerating ADF/cofilin mediated actin disassembly and in recycling actin monomers to sites of actin polymerization. Unlike ADF/cofilin that regulates actin disassembly throughout the whole cell, CAP could be subject to more specific spatial regulation, as loss of CAP leads to F-actin accumulation on the apical side of epithelial cells. However, the role of CAP in morphogenetic cell rearrangements remains poorly known. In addition, the in vivo role of the biochemical functions of CAP has not been elucidated. The aim of this master’s thesis is to describe the role of CAP in regulating the actin cytoskeleton in the follicular epithelium of the fruit fly Drosophila melanogaster. For this purpose, chimeric mutant flies with homozygous CAP loss of function mutation were generated. Subsequently, the effect of the CAP loss of function was observed in follicle cell populations undergoing morphogenetic changes. In addition, CAP loss of function was rescued with different transgenes producing mutant CAP proteins to identify the protein domains of CAP with in vivo significance. In addition, a Drosophila CAP specific antibody was purified to be used in immunostaining. The ovaries were imaged using confocal microscopy. In this thesis, it is shown that CAP loss of function caused accumulation of filamentous actin in all observed follicular cell populations. Surprisingly, the actin turnover was rescued by all of the used CAP rescue transgenes, but the mutant transgenes exhibited phenotypes resembling the CAP loss of function in other epithelial tissues. Moreover, CAP loss of function caused defects in the follicle cell movement and cell spreading. The loss of function also caused expression changes in other actin-binding proteins. The findings of these thesis support the current knowledge of CAP importance for functional actin turnover in the follicle cells, even though the protein domain necessary for in vivo function could not be deciphered. Moreover, this project provides indication that CAP has an indispensable role in dynamic morphogenetic processes in the epithelium. Together with other actin-binding proteins, CAP could regulate epithelial actin turnover in spatially directed manner, providing force for epithelial cell adhesions or protrusions.
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(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.
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(2020)Since the establishment of pathologic and cytogenetic laboratories, left-over material in the form of G-banded slides and cytogenetic fixed cells, as well as formalin-fixed, paraffin-embedded (FFPE) material, tissue samples, blood and bone marrow have been stored in archives for possible later reference. This material, which potentially contains rare and special cases, has been a welcome source for retrospective studies or e.g. for trying out new methods of analysis. Molecular genetic and molecular cytogenetic techniques such as Southern hybridization, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) have been successfully applied on different kinds of archival specimens. With this study we wanted to explore, both through a literature review and through a practical experiment, the history, present day and future use of such archival material in the light of molecular cytogenetics, including the challenges of DNA extraction, sample degradation, data analysis and interpretation as well as ethical issues. The experimental part had two main objectives, (1) to investigate the use of archived cytogenetic material in the form of G-banded slides and cytogenetic fixed cells for array-based CGH (aCGH), and (2) to explore abnormalities on chromosome 1q in hematologic malignancies. Extra material on the long arm of chromosome 1 is a common recurrent chromosomal abnormality that is present in many classes of hematologic cancers as either primary or secondary aberration. It is the most common structural aberration in multiple myeloma (MM), myeloproliferative disease (MPD) and myelodysplastic syndrome (MDS) and is also a frequent aberration in pediatric acute lymphoblastic leukemia (ALL). It has been associated with increased cell proliferation, disease progression and poor outcome, the mechanisms of which are not fully understood yet. To combine these two aims, we screened the patient database for relevant cases and searched the archive for corresponding samples. The idea was to find cases of hematologic malignancies with extra material on chromosome 1q that were available as cytogenetic slides, fixed cells and frozen bone marrow, find an ideal method of DNA extraction from slides (for other material ready protocols were available), analyze the samples with aCGH and compare the results. We wanted to prove the eligibility of archived cytogenetic material for aCGH analysis and at the same time study rearrangements of 1q in our samples. Starting with 38 patient cases, DNA extraction was performed with 2 different protocols, the latter of which, using a modified version of the Puregene® DNA Purification Kit Protocol for Blood Smears, turned out to be more successful. After having obtained sufficient DNA from several slide samples, we assessed DNA quality with agarose gel electrophoresis. Because slide DNA was too fragmented to be used for aCGH and whole genome amplification (WGA) was not a choice, the experiment was continued with archived fixed cells, bone marrow and archived DNA only. Using a high resolution 60-mer oligonucleotide 44K human CGH microarray platform, we analyzed 15 patient cases that were available both as fixed cell and DNA samples (2 cases), both as fixed cell and bone marrow samples (1 case), both as fixed cell and CPT™ cell samples (1 case), frozen bone marrow (7 cases) and DNA samples (4 cases). The malignancies were pediatric ALL (6 cases), adult ALL (1 case), chronic myeloid leukemia (CML, 2 cases), non-Hodgkin lymphoma (NHL, 1 case), Burkitt lymphoma (BL, 3 cases), Hodgkin's disease (HD, 1 case) and one undefined malignancy. After analysis with CGH Analytics software, we saw that aCGH results of fixed cells compared to results from bone marrow or DNA were almost identical, which confirmed that cytogenetic fixed cells were a reliable source for aCGH analysis. Challenges of working with archived material were witnessed in the form of CG-waves and centralization errors and deviations of the hybridization ratio diagram caused by partially degraded DNA. Concerning aberrations calls, 12 of the 15 cases had detectable aberrations on 1q, which included amplification of the whole 1q arm (1 pediatric ALL, 3 BL cases) and duplications or amplifications of parts of 1q (5 pediatric ALL, 2 CML and 1 undefined malignancy cases) in addition to other aberrations. Common break points were 1q21.2 (2 CML cases), 1q23.2 (1 pediatric ALL and 1 CML case) and 1q32.3 (2 pediatric ALL cases), and we detected 2 large common overlapping areas, at 1q21.2q23.2 and 1q25.3q32.2. The areas were, however, too vast for disease gene screening, spanning several Mb each. We could thus prove and confirm the value of cytogenetic archives for scientific studies. Array CGH with fixed cells was confirmed to work well, also frozen bone marrow and archived DNA were valuable sources. We would suggest further aCGH experiments with cytogenetic slides by applying WGA but would also suggest slide and fixed cell material to be used for Next Generation Sequencing, which has not been reported yet. Regarding aberrations on 1q, further studies to more accurately delimit relevant break points and common overlapping areas are recommended.
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(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.
Now showing items 41-48 of 48