Browsing by discipline "genetik"
Now showing items 21-36 of 36
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(2018)Recently, our group reported that mutations in KCNQ1, a potassium channel gene usually linked to long QT syndrome, cause growth hormone deficiency and maternally inherited gingival fibromatosis. Expression of the mutated KCNQ1 with KCNE2 subunit was shown to reduce pituitary hormone secretion in functional experiments in the original study. The aim of this thesis was to investigate if germline mutations in KCNQ1 and KCNE2, a gene encoding an auxiliary potassium channel subunit, could also play a role in the opposite phenomenon, growth hormone excess. Growth hormone (GH) excess causes acromegaly, a condition that is typically due to a GH secreting pituitary adenoma. I screened KCNQ1 and KCNE2 for germline mutations in 45 acromegaly patients by Sanger sequencing and predicted effects of the mutations on protein function by in silico tools. Only deep intronic and synonymous polymorphisms were detected in KCNQ1. These findings were likely insignificant based on the in silico predictions and the variants’ frequencies in the general population. In KCNE2, a heterozygous c.22A>G, p.(Thr8Ala) mutation with an unknown significance was found in two patients. It was present in the general population with a frequency of 0.0038. In conclusion, no evidence of KCNQ1 or KCNE2 mutations being associated with growth hormone excess was found. Mutation screenings of larger patient series and additional functional experiments are needed to shed more light on the roles of KCNQ1 related genes in growth hormone secretion.
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(2015)Charcot-Marie-Tooth disease is a clinically and genetically heterogeneous group of disorders of the peripheral nervous system. It affects both peripheral motor and sensory nerves that lead to progressive degeneration of muscles in the lower limbs. Prevalence of CMT is 1:2500. It can be divided into two groups: CMT1 is the demyelinating type and CMT2 is the axonal type. There are 20 known genes associated with CMT2 but not all of them have been found yet. Hereditary spastic paraplegia (HSP) is a group of inherited neurological disorders and it is also genetically and clinically heterogeneous. HSP is caused by degeneration of the upper motor neurons in the brain and spinal cord. HSP can be divided into pure and complex forms. In pure forms the main symptoms are progressive spasticity and weakness in the lower limbs. In complex forms there are additional symptoms such as epilepsy and mental retardation. The prevalence of HSP is 2-10:100 000. There are over 70 loci associated with HSP and 55 genes identified so far but there is probably more to be found. With both of these diseases it has been difficult to diagnose patients at the genetic level because of the great amount of disease genes. The aim of this study was to identify potential disease causing mutations by using targeted next-generation sequencing (NGS) and also to do functional testing with the discovered mutations. There were 48 patients, 28 diagnosed with CMT and 20 with HSP. For the targeted sequencing there was a gene panel with 167 genes, including genes associated with neuropathy, paraplegia, ALS and mitochondrial functions. Analyzing of the gene variants was largely based on their frequencies in the population and several exome databases were used. All possibly pathogenic mutations were confirmed with Sanger-sequencing and also sequenced from the patients’ relatives, if DNA samples were available. Possible genetic diagnosis was found for five CMT patients and six HSP patients. Only one of the mutations was previously known. One of the probably pathogenic mutations was a deletion, others were missense mutations. Large portion of the patients were left without a genetic diagnosis probably because there are many more genes associated with these diseases to be found. De novo heterozygous missense mutations in the KIF1A gene were found to cause a novel phenotype. Symptoms of these patients include classical HSP symptoms and additionally intellectual disability and other symptoms, such as cerebellar atrophy, in some patients. In this study we found a deletion in the gene HSPB1 that causes premature STOP codon that leads to disappearance of nearly the entire C-terminus. In this study I built a HSPB1-Western blot assay that was used in further research. It was found out that the truncated protein was stabile, it forms dimers with the normal protein and impairs the cells response against stress caused by misfolded proteins.
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(2020)Cancer-associated fibroblasts (CAF) form a heterogenous stromal cell population of a solid tumor. They are known to promote tumor growth and survival through metabolic reprogramming and inflammation. It is unclear though whether CAF are crucial component of tumor initiation and whether CAFs are dispensable altogether from the fully developed neoplasm. Tumor suppressor LKB1 regulates AMPK and AMPK-related kinases (ARK), and its function is compromised in familial disorder Peutz-Jeghers syndrome (PJS). Fibroblast specific haploinsufficiency of LKB1 alone is sufficient of initiating gastrointestinal polyposis but the mechanism through which LKB1 mediates this is only partially understood. We provide evidence that LKB1 is downregulated in multiple human malignancies including high grave serous ovarian cancer (HGSOC). Human ovarian cancer is the most lethal gynecological disease, characterized by metastasis of omentum. Loss-of LKB1 in ovarian fibroblasts was accompanied with metabolic changes associated with CAF-transformation. We screened down critical LKB1 substrates through transcriptomic and functional assays revealing AMPKa1 and MARK3 as potential downstream effectors of oxidative phosphorylation. AMPKa1, MARK1 and SIK-family were the glycolytic counterparts. We also took an initiative of cataloguing published human cancer stroma data in order to gain more comprehensive look of tumor heterogeneity. Metabolic rewiring was also observable in published cancer-stroma datasets. Human cancer-stroma divided into metabolically active and highly inflamed subtypes. These results highlights LKB1’s role as a conserved metabolic caretaker in fibroblasts. Our data also support mechanistic model in which LKB1 and ARKs regulate mitochondrial metabolism, essential for CAF transformation.
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(2019)Retrotransposons are genetic elements with the ability to make a copy of themselves and insert the copy into a new location in a genome. Most of the retrotransposons in the human genome are not transposition competent and the remaining copies are prevented from moving by epigenetics. However, some tumors experience abnormal retrotransposon activity resulting in many copies of retrotransposons inserted into new locations. Retrotransposons can be detected from sequenced genome data by bioinformatic tools. One of them is TraFiC, a tool designed to detect somatic retrotransposon insertions from tumor samples. In this Master’s thesis, I test TraFiC with 201 colorectal cancer tumors and one colorectal adenoma and develop tools to further analyze retrotransposon insertions. These tools are TraID, a pipeline to detect transductions, insertions with flanking sequence from source elements, and InSeqR, a pipeline to recreate the inserted sequence from known insertion sites. TraFiC detected 4744 somatic insertions and TraID detected 346 somatic transductions from the tumor samples. 80 % of the detected insertions were identified as true somatic insertions based on visual examination of a subset of the calls. 87 % of insertions detected by TraFiC and 82 % of the insertions detected by TraID had their insertion breakpoints and the sequence flanking them recreated by InSeqR. The detected insertions with their sequence form a reliable and comprehensive call set that can be used to create new knowledge of somatic retrotransposon insertions in colorectal cancer.
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(2019)Ihmistoiminnan vaikutukset ovat muuttaneet voimakkaasti ekosysteemien rakennetta ja toimintaa maailmanlaajuisesti viimeisten vuosikymmenten aikana. Ekologisen tilan heikentyminen aiheuttaa vakavan uhan virtavesien eliöyhteisöjen monimuotoisuudelle. Tulevaisuudessa tarve kustannustehokkaille, luotettaville ja nopeille keinoille arvioida vesistöjen ekologista tilaa kasvaa. Biomonitorointi, eli eliöiden käyttö ympäristömuutosten seurantaan ja mittaamiseen, pyrkii tunnistamaan, seuraamaan ja arvioimaan ekosysteemien toimintaa uhkaavia ympäristön stressitekijöitä. Tulevaisuudessa yksi mahdollinen biomonitoroinnin työkalu ja ekologisen tilan mittari yhdessä muiden biologisten (esim. piilevät, pohjaeläimet, vesikasvit ja kalat) ja fysikaalis-kemiallisten mittarien kanssa voi olla mikrobit. Uuden polven sekvensointimenetelmien kehitys ja sekvensointikustannusten aleneminen viime vuosina on mahdollistanut yhä useamman ympäristömikrobiaineiston analysoinnin uuden polven sekvensointimenetelmillä. Tämän pro gradu -tutkielman tarkoitus on tuoda lisätietoa siitä, mitkä eri ympäristötekijät vaikuttavat virtaavien vesien biofilmibakteerien yhteisörakenteeseen, yhteisö-ympäristösuhteen voimakkuuteen, yhteisöjen koostumusta selittävien muuttujien identiteettiin ja miten harvinaisten bakteerisukujen poisto aineistosta vaikuttaa erilaisiin ympäristömuuttujiin. Tutkimusaineistoa analysoidaan redundanssianalyysin (redundancy analysis, RDA) ja monimuuttujaregressiopuiden (Multivariate regression trees, MRT) avulla. Tutkielman tavoitteena on myös kuvata länsisuomalaisten virtavesien koskipaikkojen kivien pinnalla elävien biofilmibakteerien ominaispiirteitä pääjakso-, luokka-, lahko-, heimo- ja sukutasoille tehtävien BLAST-analyysien (basic local alignment search tool) avulla. Pro gradu tutkielmani bakteeriaineiston näytteet on otettu 21 suomalaiselta kolmannen jakovaiheen jokivaluma-alueelta, joilta eristettiin erillisten purojen koskipaikoista bakteerinäytteitä yhteensä 84 kappaletta. Samaan aikaan bakteerinäytteiden ottamisen kanssa tutkimuskohteilla toteutettiin vesinäytteenotto, ja kohteista määritettiin myös piilevä- ja pohjaeläinlajisto. Näytteet sekvensoitiin Ion Torrent Semiconductor -sekvensaattoria käyttäen ja valmis sekvenssiaineisto analysoitiin QIIME (Quantitative Insights Into Microbial Ecology) –ohjelmalla. Sekvenssit klusteroitiin toiminnallisiksi taksonomisiksi yksiköiksi (OTUiksi) 97 % samankaltaisuusastetta käyttäen. OTUille haettiin BLASTia (basic local alignment search tool) käyttäen paras mahdollinen pääjakso-, luokka-, lahko-, heimo- ja sukutason vastaavuus. Yleisimmät havaitut luokat olivat Alphaproteobacteria (28.9%), Betaproteobacteria (27.7%), Gammaproteobacteria (7.5%) Cytophagia (7.3%), Saprospirae (6.5%) ja Flavobacteriia (5.1%). Tuloksista havaitaan, että yhteisön ympäristösuhteisiin vaikuttavat erityisesti yleiset lajit. Harvinaisten lajien poistolla ei tässä tutkimuksessa siten havaita olevan suurta merkitystä, vaan merkitsevät muuttujat pysyvät lähes samoina huolimatta siitä, poistetaanko aineistosta ne lajit, jotka esiintyvät vain yhdellä paikalla vai kaikki ne lajit, jotka esiintyvät vain ≤ 40 paikalla. Myös selitysasteet jäävät alhaisiksi ja satunnaisten eli stokastisten tekijöiden vaikutus yhteisön ympäristöolosuhteisiin luultavasti korostuu. Jatkossa eri bakteeritaksonien roolista muuttuvissa ympäristöolosuhteissa tarvitaan tarkempaa tietoa ennen kuin virtavesien biofilmibakteereja voidaan laajamittaisesti käyttää biomonitoroinnin täysimittaisena työkaluna ja ympäristön tilan kuvaajana. Tämän tutkielman redundanssianalyysin, vaihtelun osituksen ja monimuuttujaregressiopuiden avulla saatujen tulosten perusteella voidaan todeta virtaavien vesien mikrobiyhteisöjen yhteisö-ympäristösuhteiden olevan heikkoja. Ympäristön tilan seurannassa tulisikin jatkossa tulosten perusteella käyttää mikrobien osalta muita vastemuuttujia kuin yhteisörakennetta.
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(2020)MikroRNA:t ovat tärkeitä, transkription jälkeen geenien ilmenemiseen vaikuttavia säätelijöitä. MikroRNA:iden tuotantoa on löydetty konservoituneena useista eri eliöryhmistä ja kudoksista, mikä osaltaan kertoo niiden toiminnan tärkeydestä. Rakenteeltaan miRNA:t ovat lyhyitä, noin 22 nukleotidin mittaisia yksijuosteisia RNA:ita, jotka tuotetaan pidemmistä esi-RNA:sta entsymaattisesti leikkaamalla. Geenien säätelyssä miRNA:iden teho perustuu niiden kykyyn tunnistaa kohdegeenit miRNA:n ja lähetti-RNA:n sekvenssien komplementaarisuuden perusteella. MikroRNA:iden kohteiden tunnistuksen kannalta tärkeätä, 6-8 nukleotidin jaksoa kutsutaan miRNA:n ydinalueeksi (seed-region). Yhdellä miRNA:lla voi potentiaalisesti olla satoja kohdegeenejä, joita se säätelee, minkä vuoksi näitä miRNA-mRNAinteraktioita on pyritty bioinformatiivisesti ennakoimaan useiden ohjelmien ja algoritmien avulla. miRNA:iden toiminnan tutkimista mutkistavat isomiR:it, jotka ovat yleisesti tunnettujen, kanonisten, miRNA:iden vaihtoehtoisia muotoja. IsomiR:ejä tuottavat poikkeamat miRNA:iden esiasteiden prosessoinnissa ja mikroRNA:ihin kohdistuva RNA-editointi. Tuloksena saadaan mikroRNA:ita joiden pituus tai emäskompositio eroavat kanonisista sekvensseistä. Myös miRNA:iden toiminta voi muun muassa alkaa kohdistamaan säätelyä eri geeneihin, kuin mitä kanonisen sekvenssin perusteella voisi olettaa. Tässä pro gradu -projektissa analysoin suurtehosekvensoinnilla tuotettua dataa kahdesta aivoalueesta kuudelta eri laboratoriohiirikannalta. Normalisoin datan ja todettuani sen laadun hyväksi jatkoin miRNA:iden ilmenemisen tarkastelua eri analyyseillä: Selvitin mitkä miRNA:t ilmentyivät tutkituilla aivoalueille jamiten niiden ekspressio erosi eri aivoalueiden ja hiirikantojen välillä. Tämän jälkeen tarkastelin miRNA:sta tuotettujen vaihtoehtoisten sekvenssien, isomiR:ien ilmentymistä. Vertailimme lisäksi aineistossa havaitsemiemme isomiR:ien ekspressiossa eroja aivoalueiden ja hiirikantojen välillä. Lopuksi pyrimme osoittamaan havaittujen isomiR:ejä tuottavien RNAeditointitapahtumien olevan funktionaalisesti merkittäviä. Tätä tarkoitusta varten transfektoimme soluviljelmiä lusiferaasi-reportterivektorilla ja editoiduilla ja editoimattomilla miRNA:ta muistuttavilla sekvensseillä. Analyysien tuloksena havaitsimme lukuisia miRNA:ita, jotka olivat eritavoin ilmentyneitä joko aivoaluiden, hiirikantojen tai molempien välillä. Lisäksi havaitsimme lukuisia isomiR-sekvenssejä: Noin 90%:sta havaittuja miRNA:ita löydettiin vähintään yksi vaihtoehtoinen, ei-kanoninen sekvenssi. IsomiR:ien suuresta määrästä huolimatta,miRNA:iden kokonaisekspressiosta suurin osaoli yleensä vain muutaman isomiR:in aikaansaannosta. Aineistoista löysimme myös runsaasti merkkejä RNA-editoinnista, ja erityisesti ADAR-entsyymin toiminnasta. Funktionaaliset kokeemme antoivat myös vahvoja viitteitä siitä, että miRNA:iden ydinalueisiin kohdistuvat editoinnit ovat funktionaalisesti merkittäviä tapahtumia. Muutosten seurauksena havaitsimme muuttuneiden ydinaluiden tunnistavan eri kohdegeenejä kuin kanoninen muuntelematon miRNA-sekvenssi. Johtopäätöksenä miRNA:iden ja erityisesti niiden isomiR:ien muunteluun on jatkossa syytä kiinnittää nykyistä enemmän huomiota. Parempi ymmärrys isomiR:eistä ja niitä synnyttävistä mekanismeista voivat jatkossa mahdollistaa tehokkaamman koesuunnittelun ja helpottaa saatujen tulosten tulkintaa.
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(2018)nxiety disorders are the most common psychiatric disorders. Iiris Hovatta’s group (Neurogenomics lab) uses inbred mice to study effects of psychosocial stress. Mice can be divided to susceptible and resilient phenotypes, based on their behavioral response to psychosocial stress. Differences between phenotypes can be examined for example on brain structural or transcriptional levels. These studies have shown changes in brain myelination and expression levels of myelin-related genes between phenotypes. Transcriptomic studies have also revealed differences in microRNA (miRNA) expression profiles. Especially miR-219a, a known regulator of myelination, appears to be differentially expressed. Bioinformatic studies also revealed that many predicted target genes of miR-219a are connected to Wnt/β-catenin signaling pathway, which is also known to regulate myelination. This led to a hypothesis that miR-219a regulates myelination through Wnt/β-catenin signaling pathway in adult mouse brain. Prediction of miRNA target genes is based on algorithms using varying criteria, and their biological significance is uncertain. Assumptions about the biological role of specific miRNA require experimental validation of the silencing interaction between the miRNA and the target gene mRNA. The purpose of this study was to validate five Wnt-signaling related mouse genes (Gsk3b, Esr1, Tcf7l2, Fkbp5 and Acvr1b) as targets for miR-219a-5p or miR-219a-1-3p. For validation a luciferase-reporter assay was used, in which the target sequence of the gene’s mRNA is cloned to a reporter vector, then cotransfected to cultured cells (human HEK293 cells) with a miRNA-mimic. The light emitting luciferase activity is measured to examine the possible silencing effect of the miRNA. Results of this study showed that miR-219a mimics did silence the expression of the target genes. Although some variation was observed in the efficiency of silencing between the genes and based on the mimic concentration. The most efficient silencing was observed between miR-219a-5p and Gsk3b construct. These results implicate that miR-219a regulates several genes related to Wnt/β-catenin pathway, and that stress-related differences in its expression may be associated to differential myelination observed between phenotypes that react differentially to psychosocial stress.
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(2019)Myopatiat ovat laaja joukko erilaisia luurankolihasten heikkoutena ja rappeumana ilmeneviä sairauksia. Ne ovat geneettisesti ja ilmiasuiltaan heterogeenisiä: pelkän kliinisen taudinkuvan perusteella taudin alatyypin tarkka määrittely on hankalaa. Aiemmin näitä tauteja on tutkittu ja diagnosoitu lähinnä melko työläällä Sanger-sekvensointimenetelmällä. Sekvensointimenetelmien kehittyminen on mullistanut myopatioiden tutkimuksen ja diagnostiikan. Uusien massiivisten rinnakkaissekvensointimenetelmien (Massively parallel sequencing, MPS) suoritusteho on valtava ja esimerkiksi koko ihmisen genomi on mahdollista sekvensoida yhdessä sekvensointiajossa. Tampereen yliopiston Lihastautien tutkimuskeskus hyödyntää myopatioiden tutkimuksessa ja diagnostiikassa kohdennettua MPS-menetelmää. Tutkimus on nimeltään MYOcap ja se on kohdennettu myopatioita tunnetusti tai ennustetusti aiheuttavien geenien eksoneihin. MYOcap-geenipaneelia on asteittain laajennettu ja nykyään se kattaa 345 geenin eksonit. Intronaalisia alueita, joilta on kuvattu patogeenisia muutoksia, on sisällytetty paneelin viimeisimpään versioon. Onnistuneessakin MYOcap-geenipaneelitutkimuksessa havaitaan kuitenkin matalasti katettuja alueita (alle 20X-lukupeitto). Nämä alueet voivat pahimmillaan aiheuttaa patogeenisen muutoksen havaitsematta jäämisen. Näitä huonosti katettuja alueita ei aiemmin ollut systemaattisesti selvitetty. Tämän tutkielman tarkoituksena oli kartoittaa MYOcap-geenipaneelitutkimuksen matalan lukupeiton alueita geenipaneelin eri versioissa ja tunnistaa toistuvasti huonosti katettuja alueita. Lisäksi tämän tutkielman tarkoituksena oli selvittää, onko tietyn geenin koettimien suhteellisen määrän lisäämisestä (tehostuksesta) seurannut lukupeittoa parantavaa vaikutusta sekä, onko toistuvasti matalasti katetuilta alueilta kuvattu patogeenisia muutoksia. Tämän tutkielman käytössä oli koko Lihastautien tutkimuskeskuksen MYOcap-sekvensointiaineisto. Aineisto sisälsi 1898 näytteen sekvensointiaineistot. Sekvensointiaineistoista koottiin tietokanta, josta voitiin tunnistaa eri MYOcap-geenipaneeliversioilla toistuvasti matalasti katetut kohdealueet. Näillä kohdealueilla sijaitsevat tunnetut patogeeniset muutokset kerättiin Human Gene Mutation Database 2018.1 -tietokannasta. Sekvensointiaineistojen analysointi tehtiin R-ohjelmointikielellä. Keskeiset käytetyt paketit olivat tidyversen dplyr, tidyr, readr ja ggplot2. Kaikissa MYOcap-versioissa voitiin havaita toistuvasti matalasti katettuja kohdealueita. Näiden huonosti sekvensoituvien kohdealueiden määrä kuitenkin väheni merkittävästi uusien geenipaneeliversioiden myötä. Huonoa sekvensoituvuutta voitiin selittää kohdealueen korkealla GC-pitoisuudella ja kohdealueen sekvenssin epäuniikkiudella. Osa ongelmakohdealueista jäi selittämättömästä syystä toistuvasti matalasti katetuiksi. Tunnettuja patogeenisia muutoksia sijoittui vain osaan toistuvasti matalasti katetuista alueista. Lukupeiton parantamistoimenpiteet voisi keskittää näille alueille, mikäli tutkimuksesta halutaan ensisijaisesti tunnettujen muutosten osalta hyvin kattava. Toisaalta geenipaneeli sisältää laajasti myös ennustettuja tautigeenejä ja siten tutkimuksen ideologiaan sopisi kaikkien toistuvasti matalasti katettujen kohdealueiden lukupeiton parantaminen myös uusien, toistaiseksi tuntemattomien muutosten havaitsemiseksi. Tehostuksen vaikutusta kohdealueiden lukupeittoon oli vaikea arvioida menetelmän epäsystemaattisen käytön takia. Tämän tutkielman perusteella voitiinkin todeta, että tehostuksen kohdistaminen ilman järjestelmällistä lukupeiton kattavuuden arviointia on vaikeaa. Tulosten perusteella käytössä olevaa tehostusta voidaan haluttaessa purkaa ja puolestaan lisätä tehostusta osalle tutkielman perusteella ongelmallisille kohdealueille.
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(2019)Nucleosides are one of the fundamental building blocks of the cell and are precursors of DNA and RNA and serve as universal signalling molecules for cell surface receptors. These nucleosides play vital roles in myriad of physiological processes, such as cardioprotection, platelet aggregation and coronary vasodilation. In clinical settings, synthetic analogs of nucleosides are used as therapeutic against cancer and other viral diseases. In human, uptake and regulation of nucleosides and their analog drugs is facilitated by the two families solute carrier membrane transporter proteins (SLC); SLC28 and SLC29. SLC29 consists of four human equilibrative nucleoside transporter members (hENT1-4) with different sub-cellular localization, tissue distribution and substrate selectivities. Human equilibrative nucleoside transporter 1 (hENT1) is the major plasma membrane nucleoside transporter and has shown to play an important role in adenosine signalling and anticancer therapies. Despite the significant physiological and pathophysiological role, little is known about the structure and molecular function of ENT homologs. Structural studies of hENT1 and other nucleoside transporter is hampered by their low expression and hydrophobic nature. The aim of this thesis work is to find suitable nanobodies (Nbs) that may serve as crystallization chaperone to facilitate the structural studies of hENT1 transporter. Camelid heavy chain only antibodies (Nanobodies) raised against recombinant hENT1 were screened for their suitability for future structural investigation of hENT1. Selected nanobodies were expressed and purified from E.coli cell in milligram quantities. Affinity of nanobodies with hENT1 was assessed using co-elution on size exclusion chromatography. High affinity binders were further screened for their ability to conformationally stabilize hENT1. In future, high affinity nanobodies will be explored for x-ray crystallization and single particle cryo-electron microscopy of hENT1. For cryo-EM it's important to convert the selected Nbs into megabody which constitute large scaffold proteins. 3D structure determination of hENT1 will be significant in understanding its molecular function and to accelerate rational drug designing against cancer, HIV and other viral infections.
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(2020)Mammalian dentitions exhibit extraordinary diversity in morphology and function. Yet,the mechanisms governing dental development are considered highly conserved across Mammalia. The inhibitory cascade (IC) model is a developmental model explaining variation in molar size proportions observed in mammals. The IC model predicts a range of dental phenotypes based on a dynamic of cumulative inhibition and activation in the sequential development of molars. Whereas most mammals fit these predictions well, bears(Ursidae) are a known exception.Here I employ dental topographic analysis to examine the developmental basis of ursine molar dentitions defying the IC model.I quantified two aspects of tooth shape:molar complexity using orientation patch count (OPC), and tooth size as the area of occlusal surface. As the complexity and size of a tooth mainly emerge based on two different developmental processes –patterning and growth, respectively –these measurements were used to decompose the two phases of tooth development producing the final phenotype. To this end, also an estimate of feature density was calculated.As in previous studies, the molar size proportions of bears were highly incongruent with the IC model. However, complexity along the molar row followed a trend more closely matching the model. Feature density was highest in the third molar. Altogether, these observations suggest an early arrest in the growth of the third molar as the principal cause for bears falling outside the predictions of the IC model –consequently supporting the idea of the inhibitory cascade as a plesiomorphy of Mammalia.As an auxiliary part of this project, I assessed the functionality of Morphoviewer, a new piece of software for measuring complexity. Morphoviewer was successfully applied to infer diet from tooth complexity in a limited sample of carnivorans; and was thus used for all further OPC analyses.
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(2020)Endocytosis is the process responsible for internalising membrane components and as such plays a key role in the biology of this structure. Mammalian cells have evolved various endocytic strategies, but Clathrin-Mediated Endocytosis (CME) is the most common type. Since the discovery of CME, around 50 years ago, the field has built a remarkable wealth of knowledge on the core CME components. In stark contrast, our understanding on the relationship between CME and the actin cytoskeleton, which is present throughout the process, is still in its infancy. In this thesis, I show the production and characterisation of recombinant, SpyCatcher tagged transferrin (TF), a canonical CME ligand. TF was expressed in E. coli and using an optimised protocol, successfully solubilised and refolded from inclusion bodies. The protein was then labelled with a fluorophore and purified to a high level of purity. Tests in mammalian cells showed that home-made TF has the same endocytic behaviour as TF purified from human plasma. Moreover, I could show that the SpyCatcher moiety attached to our home-made TF is capable to mediate its covalent linkage to its counterpart SpyTag. The successful production, refolding and functional characterization of recombinant TF in this study is an important first step to examine the participation of the actin cytoskeleton during CME.
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(2020)Tiivistelmä – Referat – Abstract Celiac disease (CD) is a serious lifelong condition, in which the immune system attacks an individual’s own tissue when eating gluten. This leads to inflammation and damage to the small intestine. Celiac disease often goes undiagnosed because many of its symptoms are nonspecific. The prevalence of combined undiagnosed and diagnosed CD is estimated to affect 1 in 100 people throughout Europe and USA. CD is a polygenic disease, it is known that the human leukocyte antigen (HLA) system plays a crucial role. HLA-DQ2/DQ8 risk allele genotyping screening test from a whole blood sample (B -HLAKeli) is routinely used to estimate the genetic risk of a patient having CD. HLA genotyping test result is routinely used to rule out celiac disease rather than confirming it; if an individual does not have celiac disease related risk alleles, it is very unlikely that he or she has celiac disease. The Celiac disease diagnosis decision making process is based on the classic triple combination of serological antibody tests, the HLA-DQ2/DQ8 genotyping test and duodenal biopsies. The aim of this master’s thesis was to study evaluate how the two different risk classification praxis for HLA-DQx.5 allele used for celiac disease diagnostics in SYNLAB Finland and Estonia central laboratory and in SYNLAB Suomi central laboratory might influence the clinical process and final diagnosis. In SYNLAB Suomi central laboratory HLA-DQx.5 is classified and interpreted as a risk allele predisposing to celiac disease. In SYNLAB Finland and Estonia central laboratory this allele is classified as CD-non-risk-allele based on recommendations in international guideline. In addition, the aim was to get a general understanding of celiac disease prevalence and risk allele distributions among the study population. From the study population of 196 celiac disease suspect patients, 9% had a celiac disease positive laboratory result and the HLA risk genotype distribution among positive cases was well aligned with the expected values described in the literature. Study results indicated that there’s no additional clinical value if HLA-DQx.5 is classified as a celiac disease predisposing risk allele; the study data implies that it is very unlikely to find celiac disease positive cases from laboratory test perspective among HLA-DQx.5 carriers. Based on the study, approximately 7% of the celiac disease suspects carry the allele HLA-DQx.5 and therefore probably go through additional celiac disease related laboratory testing if this allele is interpreted as a risk allele. According to the study findings and general recommendations based on international guideline of celiac disease diagnosis, it seems that there is no clear clinical benefit if HLA-DQx.5 is classified as a CD risk allele.
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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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(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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(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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