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  • Karkola, Sampo (Helsingin yliopisto, 2009)
    Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.
  • Isaksson, Camilla Kristina (Helsingin yliopisto, 2007)
    The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.
  • Rajala, Hanna (Helsingin yliopisto, 2014)
    Large granular lymphocytic (LGL) leukemia is a chronic incurable disorder characterized by expansion of cytotoxic T- or natural killer (NK)-lymphocytes infiltrating blood and bone marrow. The diagnostic criteria include persistent lymphocytosis in peripheral blood, and in the case of T-LGL leukemia, detection of a clonal rearrangement of the T cell receptor. Typical clinical and hematological characteristics, including anemia, neutropenia, and autoimmune disorders, further support the diagnosis. Current immunosuppressive treatment options, such as methotrexate, induce remission in only 50% of cases and no targeted therapies exist. The aim of this project was to characterize the molecular pathogenesis of LGL leukemia and find molecular markers that could be used for diagnosis and novel therapeutic approaches. In the first study, the molecular background of LGL leukemia was analyzed by exome sequencing of a T-LGL leukemia patient. The index patient carried a novel D661V mutation in the Signal transducer and activator of transcription 3 (STAT3) gene. STAT3 is a known oncoprotein and a transcription factor. In the subsequent screening, the prevalence of somatic STAT3 mutations in T-LGL cases was 40%. Based on functional studies, the mutations located in the SRC-like homology 2 (SH2) domain increased phosphorylation and transcriptional activity of STAT3. A larger LGL-leukemia patient cohort, including both T-LGL leukemia and chronic lymphoproliferative disorder of NK cells (CLPD-NK) patients, was analyzed in the second study. Activating STAT3 mutations were seen in 27% and 30% of cases, respectively. The analysis of clinical characteristics showed that patients with STAT3 mutations were more likely to have neutropenia and rheumatoid arthritis and also required more frequent treatment when compared with unmutated patients. The third study concentrated on the analysis of STAT3 mutation-negative patients by exome sequencing. Two T-LGL leukemia patients carried Y665F mutation in STAT5B gene. In the screening of over 200 T-LGL leukemia and CLPD-NK cases, two patients presented with additional N642H mutation in STAT5B. The mutations were situated in the SH2 domain and led to increased phosphorylation and transcriptional activity of STAT5B. The in vitro effects of N642H mutation were more prominent, and both patients harboring N642H mutation had an untypically aggressive clinical presentation. In the fourth study, the clonal hierarchy and STAT3 mutation spectrum during immunosuppressive treatment was analyzed by deep amplicon-sequencing of STAT3 and simultaneous deep sequencing analysis of a T cell receptor beta chain (TCRB) repertoire. A total of 22% of STAT3-mutated patients harbored multiple STAT3 mutations mostly in separate lymphocyte clones. Mutated clones did not share common T cell receptor beta chain (TCRB) sequences by deep sequencing method in three T-LGL leukemia cases analyzed. Complete remission after immunosuppressive therapy resulted in the eradication of the STAT3-mutated clone in most patients, whereas partial responses reflected only modest changes in the leukemic clone burden. In conclusion, the activating STAT3 and STAT5B mutations provide a basis for understanding the molecular pathogenesis of leukemic LGL expansion. LGL leukemia can result from chronic antigen exposure in combination with immunogenetic factors such as STAT mutations. The detection of STAT3 and STAT5B mutations can be included in the diagnostic criteria of LGL leukemia, and facilitate development of novel therapeutics.
  • Aula, Nina (Helsingin yliopisto, 2003)
  • Salovaara, Reijo (Helsingin yliopisto, 2004)
  • Sarparanta, Jaakko (Helsingin yliopisto, 2013)
    This study aimed at elucidating molecular pathways behind muscular dystrophies, inherited disorders causing progressive weakness and loss of skeletal muscle, with the perspectives of demonstrating the pathogenicity of newly identified mutations, understanding the biology of muscle diseases, and finding options for their treatment. Tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy type 2J (LGMD2J) are caused by mutations in the C-terminal (M-band) part of the sarcomeric protein titin, whereas LGMD2A results from mutations in the muscle-specific protease calpain 3 (CAPN3). In yeast two-hybrid studies aiming at identifying proteins secondarily affected in the diseases, the multifunctional TRIM-related protein myospryn (CMYA5) was identified as a novel binding partner for both M-band titin and CAPN3. The interactions were confirmed by coimmunoprecipitation, and localization of myospryn at the M-band level was supported by multiple methods. Coexpression studies identified myospryn as a proteolytic substrate for CAPN3, and suggested that myospryn may attenuate its autolytic activation. The biological role of the titin myospryn interaction remained unresolved, and the mouse model of TMD/LGMD2J showed normal myospryn localization. However, since the TMD/LGMD2J mutations disrupt the myospryn binding site in titin, they are likely to have a downstream functional effect on myospryn. LGMD1D is caused by dominant mutations in the ubiquitous co-chaperone DNAJB6. LGMD1D muscle showed a myofibrillar pathology, with cytoplasmic accumulations of DNAJB6, aggregated Z-disc-associated proteins, and autophagic rimmed vacuoles. Expression of DNAJB6 constructs in zebrafish embryos confirmed a toxic effect of the mutant cytoplasmic DNAJB6b isoform, and cell culture studies demonstrated a slower turnover and impaired anti-aggregation activity of mutant DNAJB6. Protein interaction studies indicated an association of DNAJB6 with the chaperone-assisted selective autophagy (CASA) pathway, and a modulatory effect of BAG3 on DNAJB6 pathogenicity in zebrafish suggested that CASA has active role in the pathogenesis of LGMD1D. Welander distal myopathy (WDM) results from a dominant mutation in the prion-related domain (PRD) of the RNA-binding protein TIA1, a regulator of splicing and translation, and a component of stress granules (SGs). RT-PCR analysis of selected TIA1 target genes did not show splicing changes in WDM muscle, suggesting that the pathogenesis does not involve extensive mis-splicing. IF microscopy revealed accumulation of TIA1 and other SG proteins in WDM muscle, while image analysis of transfected cells, and fluorescence recovery after photobleaching (FRAP) studies indicated a mild increase in the SG-forming propensity of mutant TIA1. These findings suggest that increased aggregation of the TIA1 PRD causes muscle pathology in WDM, either directly through inappropriate protein aggregation or indirectly by compromising cellular metabolism.
  • Gass, Natalia (Helsingin yliopisto, 2010)
    Background: Adenosine is a potent sleep-promoting substance, and one of its targets is the basal forebrain. Fairly little is known about its mechanism of action in the basal forebrain and about the receptor subtype mediating its regulating effects on sleep homeostasis. Homeostatic deficiency might be one of the causes of the profoundly disturbed sleep pattern in major depressive disorder, which could explain the reduced amounts of delta-activity-rich stages 3 and 4. Since major depression has a relatively high heritability, and on the other hand adenosine regulates sleep homeostasis and might also be involved in mood modulation, adenosine-related genes should be considered for their possible contribution to a predisposition for depression and disturbed sleep in humans. Depression is a complex disorder likely involving the abnormal functioning of several genes. Novel target genes which could serve as the possible common substrates for depression and comorbid disturbed sleep should be identified. In this way specific brain areas related to sleep regulation should be studied by using animal model of depression which represents more homogenous phenotype as compared to humans. It is also important to study these brain areas during the development of depressive-like features to understand how early changes could facilitate pathophysiological changes in depression. Aims and methods: We aimed to find out whether, in the basal forebrain, adenosine induces recovery non-rapid eye movement (NREM) sleep after prolonged waking through the A1 or/and A2A receptor subtype. A1 and A2A receptor antagonists were perfused into the rat basal forebrain during 3 h of sleep deprivation, and the amount of NREM sleep and delta power during recovery NREM sleep were analyzed. We then explored whether polymorphisms in genes related to the metabolism, transport and signaling of adenosine could predispose to depression accompanied by signs of disturbed sleep. DNA from 1423 individuals representative of the Finnish population and including controls and cases with depression, depression accompanied by early morning awakenings and depression accompanied by fatigue, was used in the study to investigate the possible association between polymorphisms from adenosine-related genes and cases. Finally to find common molecular substrates of depression and disturbed sleep, gene expression changes were investigated in specific brain areas in the rat clomipramine model of depression. We focused on the basal forebrain of 3-week old clomipramine-treated rats which develop depressive-like symptoms later in adulthood and on the hypothalamus of adult female clomipramine-treated rats. Results: Blocking of the A1 receptor during sleep deprivation resulted in a reduction of the recovery NREM sleep amount and delta power, whereas A2A receptor antagonism had no effect. Polymorphisms in adenosine-related genes SLC29A3 (equilibrative nucleoside transporter type 3) in women and SLC28A1 (concentrative nucleoside transporter type 1) in men associated with depression alone as well as when accompanied by early morning awakenings and fatigue. In Study III the basal forebrain of postnatal rats treated with clomipramine displayed disturbances in gamma-aminobutyric acid (GABA) receptor type A signaling, in synaptic transmission and possible epigenetic changes. CREB1 was identified as a common transcription denominator which also mediates epigenetic regulation. In the hypothalamus the major changes included the expression of genes in GABA-A receptor pathway, K+ channel-related, glutamatergic and mitochondrial genes, as well as an overexpression of genes related to RNA and mRNA processing. Conclusions: Adenosine plays an important role in sleep homeostasis by promoting recovery NREM sleep via the A1 receptor subtype in the basal forebrain. Also adenosine levels might contribute to the risk of depression with disturbed sleep, since the genes encoding nucleoside transporters showed the strongest associations with depression alone and when accompanied by signs of disturbed sleep in both women and men. Sleep and mood abnormalities in major depressive disorder could be a consequence of multiple changes at the transcriptional level, GABA-A receptor signaling and synaptic transmission in sleep-related basal forebrain and the hypothalamus.
  • Jurgens, German (Helsingin yliopisto, 2002)
    Suurin osa luonnossa havaitsemistamme mikrobeista on sellaisia, joita emme edelleenkään osaa kasvattaa laboratorio-oloissa, vaikka tietomme mikrobien monimuotoisuudesta paranevat koko ajan. Luonnontilaisen mikrobieliöstön kokoonpano eri ympäristöissä on paljolti epäselvä ja ymmärrämme vielä hyvin puutteellisesti mikrobien ekologiaa ja niiden rooleja eliöyhteisöissä. Nykyaikaiset molekulaariset tutkimusmenetelmät auttavat selvittämään mikrobien monimuotoisuutta kokonaisvaltaisesti ja nopeasti. Ympäristöstä kemiallisesti puhdistetut ribosomaalista RNA:ta koodaavat geenit edustavat periaatteessa kaikkia eliöyhteisön geneettisesti toisistaan poikkeavia eliöitä. Niistä voidaan valikoida halutut genomit jatkotutkimuksia varten. Uusien menetelmien käyttö on tuonut esiin sen merkittävän seikan, että "tavanomaisten" elinympäristöjen eliöyhteisöihin kuuluu suuri joukko entuudestaan tuntemattomia arkkieliöitä. Aiemmin kuviteltiin, että arkkieliöt asuttavat vain sellaisia "epätavallisia" tai "äärimmäisiä" elinympäristöjä, joita luonnehtii joku seuraavista ominaisuuksista: hyvin korkea lämpötila, korkea suolapitoisuus, korkea happamuus tai emäksisyys, hapettomuus. Tutkijat ovat viimeisen noin kymmenen vuoden aikana osoittaneet, että arkkieliöt asuttavat hyvin monenlaisia kylmän ja lauhkean vyöhykkeen ympäristöjä, yhtä hyvin maaperää kuin suolaisen ja makean veden pohjaa tai pintakerroksia. Nämä löydöt ovat avanneet uuden alun arkkieliöiden tutkimukselle, erityisesti sen selvittämiselle, mitkä ovat niiden fysiologiset ja ekologiset roolit monimuotoisissa mikrobiyhteisöissä. Tämä väitöskirja kuvaa entuudestaan tuntemattomien arkkieliöiden löytymistä havumetsävyöhykkeen metsämaasta. Arkkieliöitä löytyi myös lauhkean vyöhykkeen vuorovesialueelta, murtoveden huuhtelemasta pohjasta. Nämä löydöt ovat perustavalaatuisia vuorovesialueen eliöyhteisöjen ymmärtämiseksi. Suomalaisen metsäjärven vedestä määritettiin molempien arkkieliöiden pääryhmien - tieteellisiltä nimiltään Crenarchaeota ja Euryarchaeota - edustajia. Euryarchaeota-ryhmän edustajia voitiin havainnoida myös fluoresenssi-mikroskopoinnilla. Löydöt viittaavat siihen, että arkkieliöillä on oma biogeokemiallinen roolinsa makeanveden ravintoketjujen hiilen käytössä. Tässä työssä määritetyt uudet arkkieliöiden genomien nukleotidisekvenssit on toimitettu ARB-tietokantaan, jonka kasvava vertailuaineisto edelleen parantaa uusien arkkieliösekvenssien analyysiä ja auttaa hybridisaatiokoetinten ja polymeraasiketjureaktioalukkeiden suunnittelussa ja arvioinnissa. Tässä väitöskirjassa esitellyt tulokset yhdessä lukuisien vesi-, maaperä- ja muiden ympäristöjen arkkieliöitä käsittelevien julkaisujen kanssa osoittavat, että arkkieliöt asuttavat monia erilaisia elinympäristöjä ja että ne ovat ekologisesti paljon menestyneempiä, kuin tieteenalalla on kuviteltu. Voimme olettaa, että heti kun joitain näistä eliöistä onnistutaan kasvattamaan ja ylläpitämään laboratorio-oloissa, niiden joukosta löydetään aivan uusia, entuudestaan tuntemattomia fysiologisia fenotyyppejä, jotka avaavat mielenkiintoisia näkymiä aineenvaihdunnan ja perinnöllisten ominaisuuksien tutkimukselle.
  • Pitkäranta, Miia (Helsingin yliopisto, 2012)
    Epidemiological studies have shown an elevation in the incidence of asthma, allergic symptoms and respiratory infections among people living or working in buildings with moisture and mould problems. Microbial growth is suspected to have a key role, since the severity of microbial contamination and symptoms show a positive correlation, while the removal of contaminated materials relieves the symptoms. However, the cause-and-effect relationship has not been well established and knowledge of the causative agents is incomplete. The present consensus of indoor microbes relies on culture-based methods. Microbial cultivation and identification is known to provide qualitatively and quantitatively biased results, which is suspected to be one of the reasons behind the often inconsistent findings between objectively measured microbiological attributes and health. In the present study the indoor microbial communities were assessed using culture-independent, DNA based methods. Fungal and bacterial diversity was determined by amplifying and sequencing the nucITS- and16S-gene regions, correspondingly. In addition, the cell equivalent numbers of 69 mould species or groups were determined by quantitative PCR (qPCR). The results from molecular analyses were compared with results obtained using traditional plate cultivation for fungi. Using DNA-based tools, the indoor microbial diversity was found to be consistently higher and taxonomically wider than viable diversity. The dominant sequence types of fungi, and also of bacteria were mainly affiliated with well-known microbial species. However, in each building they were accompanied by various rare, uncultivable and unknown species. In both moisture-damaged and undamaged buildings the dominant fungal sequence phylotypes were affiliated with the classes Dothideomycetes (mould-like filamentous ascomycetes); Agaricomycetes (mushroom- and polypore-like filamentous basidiomycetes); Urediniomycetes (rust-like basidiomycetes); Tremellomycetes and the family Malasseziales (both yeast-like basidiomycetes). The most probable source for the majority of fungal types was the outdoor environment. In contrast, the dominant bacterial phylotypes in both damaged and undamaged buildings were affiliated with human-associated members within the phyla Actinobacteria and Firmicutes. Indications of elevated fungal diversity within potentially moisture-damage-associated fungal groups were recorded in two of the damaged buildings, while one of the buildings was characterized by an abundance of members of the Penicillium chrysogenum and P. commune species complexes. However, due to the small sample number and strong normal variation firm conclusions concerning the effect of moisture damage on the species diversity could not be made. The fungal communities in dust samples showed seasonal variation, which reflected the seasonal fluctuation of outdoor fungi. Seasonal variation of bacterial communities was less clear but to some extent attributable to the outdoor sources as well. The comparison of methods showed that clone library sequencing was a feasible method for describing the total microbial diversity, indicated a moderate quantitative correlation between sequencing and qPCR results and confirmed that culture based methods give both a qualitative and quantitative underestimate of microbial diversity in the indoor environment. However, certain important indoor fungi such as Penicillium spp. were clearly underrepresented in the sequence material, probably due to their physiological and genetic properties. Species specific qPCR was a more efficient and sensitive method for detecting and quantitating individual species than sequencing, but in order to exploit the full advantage of the method in building investigations more information is needed about the microbial species growing on damaged materials. In the present study, a new method was also developed for enhanced screening of the marker gene clone libraries. The suitability of the screening method to different kinds of microbial environments including biowaste compost material and indoor settled dusts was evaluated. The usability was found to be restricted to environments that support the growth and subsequent dominance of a small number microbial species, such as compost material.
  • Lindholm, Pamela (Helsingin yliopisto, 2009)
    Malignant mesothelioma (MM) is a rare, usually incurable, disease mainly caused by former exposure to asbestos. Even though MM has a strong etiological link, genetic factors may play a role, since not all cases can be linked to former asbestos exposure. This thesis focuses on lung diseases, mainly malignant mesothelioma (MM), and idiopathic pulmonary fibrosis (IPF), which resembles asbestosis. The specific asbestos-related pathways associated with malignant as well as non-malignant lung diseases, still need to be clarified. Since most patients diagnosed with MM or asbestosis/fibrosis have a dismal prognosis and few therapeutic options are available, early diagnosis and better understanding of the disease pathogenesis are of the utmost importance. The first objective of this thesis was to identify asbestos specific differentially expressed genes. This was approached by using high-resolution gene expression arrays, and three different human lung cell lines, as well as with three different bioinformatics approaches. Since the first study aimed to elucidate potential early changes, the second study was used to screen DNA copy number changes in MM tumour samples. This was performed using genome wide microarrays for identification of DNA copy number changes characterstic for MM. Study III focused on the role of gremlin in the regulation of bone morphogenetic protein (BMPs) in IPF. Further studies were conducted in asbestos-exposed cell cultures as well as in an asbestos-induced mouse model. Furthermore, GATA-6 was studied in MM and metastatic pleural adenocarcinoma. The GATA transcription factors are important during embryonic development, but their role in cancer is still unclear. GATA-6 is a co-factor/target of thyroid transcription factor 1 (TTF-1), which is used in differential diagnostics of pleural MM and adenocarcinoma. Bioinformatics probed the genes and biological processes ordered in terms of significance, clusters, and highly enriched chromosomal regions. The study revealed several already identified targets, produced new ideas about genes which are central for asbestos exposure, as well as provided supplementary data for researchers to check their own novel findings or ideas. The analysis revealed DNA copy number changes characteristic for MM tumors. The most common regions of loss were detected in 1p, 3p, 6q, 9p, 13, 14, and 22, and gains at 17q. The histological features in asbestosis and IPF are very similar, wherefore IPF can be studied in asbestos models. The BMP antagonist gremlin was up-regulated by asbestos exposure in human epithelial cell lines, which was also observed in Study I. The transforming growth factor (TGF) -β and BMP expression and signaling activities were measured from murine and human fibrotic lungs. BMP-7 signaling was down-regulated in response to up-regulation of gremlin, and restoration of BMP-7 signaling prevented progression of fibrosis in mice. Therefore, the study suggests that the restoration of BMP-7 signaling in fibrotic lung could potentially aid in the treatment of IPF patients. Study IV revealed that GATA-6 was strongly expressed in the majority of the MM cases, and correlated statistically significant with longer survival in subgroups of MM.
  • Rice, Ritva (Helsingin yliopisto, 2004)
  • Voutilainen, Maria (Helsingin yliopisto, 2015)
    Mammary gland development begins during embryogenesis with the formation of species-typical number of mammary placodes that emerge along the flanks of the embryo at conserved positions. By birth, the mammary primordium has undergone branching morphogenesis and displays a small ductal tree with several branches. The organ development and growth continues throughout postnatal life and the mammary gland matures to functional form only during pregnancy and following lactation. Ectodysplasin (Eda), a member of the tumour necrosis factor family, is one of the key regulators of epithelial appendage development in all vertebrates. In humans, mutations in the Eda gene, or in other components of the signalling pathway, cause hypohidrotic ectodermal dysplasia (HED), a disorder characterized by sparse hair, missing teeth, and defects in several exocrine glands including the breast. Previous studies have shown that transgenic overexpression of Eda (K14-Eda mice) in the developing ectoderm leads to formation of ectopic mammary placodes, which give rise to supernumerary glands in the adult mice. Otherwise, effects of Eda signalling in the mammary gland have been fairly unknown. Here I have analysed the role of Eda in prepubertal mammary gland development. Characterization of the mammary glands of Eda gain- (K14-Eda) and loss-of-function (Eda−/−) mice revealed that the branching morphogenesis of the organs correlated with Eda levels. Overexpression of Eda induced precocious and accelerated branching whereas lack of Eda reduced number of ductal tips. Furthermore, Eda induced supernumerary mammary placode formation not only on the flank but also in the neck region. Analysis of the mouse line with suppressed NF-kappaB signaling (IκBαΔN mice) revealed that the transcription factor is a major mediator of Eda in the mammary gland. NF-kappaB activity was shown to be necessary for the ability of Eda to induce supernumerary mammary primordia and to accelerate branching morphogenesis. With a candidate gene approach and genome wide-profiling several potent Eda target genes were identified in the mammary gland. Among them were members of the Wnt/beta-cat pathway. The obtained results suggest that Eda promotes mammary cell fate by enhancing canonical Wnt pathway activity and other effects of Eda are cooperatively mediated by certain Wnt family members in addition to other factors. To study mammary placode formation and branching morphogenesis and to assess roles of individual downstream factors or pathways, ex vivo culture systems were developed and utilized in this thesis work.
  • Hakanen, Janne (Helsingin yliopisto, 2014)
    The development of the forebrain is dependent on controlled regulation of neural stem/progenitor cells, since the vast majority of all cells in the forebrain are generated by them. The cellular processes influencing forebrain development include cell proliferation, migration, differentiation, and apoptosis, which are regulated in a spatiotemporal manner by genetic programs and interactive protein networks in a given environment. In this thesis, the influence of Netrin1 (NTN1), Human papilloma virus (HPV) E6/E7, and actin-bundling protein with BAIAP2 homology (ABBA)proteins on neural stem/progenitor cells cellular processes was studied and evaluated. In vitro methods were used to uncover the cellular processes regulated and affected by these proteins. In addition, in vivo methods were used to assess their possible impact on forebrain development in mice. NTN1 belongs to a conserved family of laminin-related molecules and it is present in the developing and adult mouse brain. NTN1 has been thought to act as a diffusible long or short-range guidance cue, which influences growing axons and migrating cells in either a chemoattractive or repulsive manner, and NTN1 deficiency in the brain causes defects in axon guidance and cell migration. The main issue of this thesis was to determine the developmental impact of NTN1 in the formation of two forebrain structures, the olfactory bulb and the corpus callosum (CC). Olfactory bulb processes arriving odour information from the nasal cavity and transmit this information forward to various brain regions. During development, the different cell types of olfactory bulb are generated in distinct germinal regions of the brain. Projection neurons are mainly generated by the olfactory bulb stem/progenitor cells whereas majority of the interneurons are produced by the progenitors, which migrate from the forebrain germinal zones into the olfactory bulb via rostral migratory stream (RMS). The origin of non-neural cells, astrocytes and oligodendrocytes, is not as well understood as neurons. We observed that NTN1 has a significant impact on the beginning of stem/progenitor cells migration from the forebrain germinal zones into the olfactory bulb. In more detail, the migration of Ntn1 expressing stem/progenitor cells was delayed, which led to an accumulation of these cells in RMS, and to a substantial reduction of GABAergic interneurons and oligodendrocytes in the olfactory bulb. Thus, the results suggest that NTN1 acts mainly as a detachment/release factor for the Ntn1 expressing stem/progenitor cells in the beginning of their migration and this function is mediated in either cell-autonomous or paracrine manner. CC is the largest axon tract in the forebrain and it integrates motor, sensory and cognitive performances between cerebral hemispheres. In mice, the cerebral hemispheres have to fuse in the midline before the commissural axons can cross the midline and form the CC. This interhemispheric fusion normally includes the removal of leptomeningeal cells found between the hemispheres and disruption of pial basal lamina, which is produced and maintained by the leptomeningeal cells. In Ntn1 deficient mice the leptomeningeal cells were not removed and the pial basal lamina remained intact in the hemispheric midline. Thus, the results suggest that NTN1 is required for the interhemispheric fusion, which precedes midline crossing of commissural axons and is a prerequisite for the formation of the CC. ABBA belongs to the Bin amphiphysin Rvs167 (BAR) protein superfamily, which regulates plasma membrane morphology by directly influencing plasma membrane assembly or through rearrangement of the actin cytoskeleton. The functional and expression studies revealed that ABBA participates in the regulation of cell protrusions by enhancing actin dynamics and connecting plasma membrane deformation to actin cytoskeleton. ABBA was present in radial glia-like cells and radial glial cell extensions near meningeal pial basal lamina during CNS development. During early development of brain, interaction between radial glial end-feet and pial basal lamina is required to sustain radial migration and integrity of pial basal lamina. Thus, ABBA may have an important role in formation of radial glial extensions and their connections in CNS. Exquisite balance between proliferation, self-renew and differentiation of stem/progenitor cells is necessary to maintain appropriate tissue homeostasis. HPV16 E6/E7 oncoprotein mediated degradation of p53 and pRb family of proteins seemed to facilitate a defective coupling of proliferation, self-renewal, and differentiation processes inside neural stem/progenitor cells. In addition, these processes also seemed to be susceptible to the environmental signals. Hence, a defective coupling of proliferation, self-renewal, and differentiation processes in neural stem/progenitor cells may lead to an imbalance in normal tissue homeostasis and abnormal growth of tissue in the brain. In summary, even though NTN1, HPV E6/E7, and ABBA proteins affected different cellular processes of neural stem/progenitor cells, all these cellular processes participate in the development of forebrain conformation and, therefore, the future functionality of it. In addition, defects in regulation of neural stem/progenitor cells cellular processes may lead to congenital neural disorders in mice and humans. Thus, these results also increase our overall comprehension of the developmental cellular processes behind the different neural congenital disorders and diseases.
  • Morales Suárez, Luis Orlando (Helsingin yliopisto, 2014)
    Plant responses to solar ultraviolet radiation (UV, 280-400 nm) were assessed at different molecular levels using Betula pendula Roth (silver birch) and Arabidopsis thaliana (Arabidopsis) as model species in outdoor experiments to assess the possibly interacting roles of the UV-B and UV-A wavebands in acclimation to sunlight. Solar UV-B (280-315 nm) and UV-A (315-400 nm) irradiance was attenuated with plastic films. Both solar UV-B and UV-A promoted the acclimation of silver birch and Arabidopsis to UV in sunlight by regulating the expression of genes with functions in UV protection and also by inducing the accumulation of phenolic compounds in the leaves. Solar UV also regulated transcript accumulation of genes involved in the signaling and biosynthesis of auxin, brassinosteroids and jasmonic acid (JA) in Arabidopsis. A new role of Arabidopsis UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) in the regulation of some responses to solar UV-A radiation was observed in addition to its previously described role in UV-B perception. High UV-A irradiance as present in sunlight, had a large effect on plant metabolism and modulated some of the previously characterized UV-B responses most probably through interaction between UVR8 and CRY pathways. In contrast to UVR8, under UV-B irradiation conditions not inducing stress, RADICAL-INDUCED CELL DEATH1 (RCD1) played no active role in UV signaling and acclimation, but rather modulated UV responses under sunlight. We demonstrated that solar UV-A makes an important contribution to acclimation of plants to sunlight, independently and interacting with UV-B.
  • Järvinen, Päivi (Helsingin yliopisto, 2011)
    Various intrinsic and external factors are constantly attacking the cells causing damage to DNA and to other cellular structures. Cells in turn have evolved with different kinds of mechanisms to protect against the attacks and to repair the damage. Ultraviolet radiation (UVR) is one of the major environmental genotoxic carcinogens that causes inflammation, mutations, immunosuppression, accelerated aging of the skin and skin cancers. Epidermis is the outermost layer of the skin consisting mostly of keratinocytes, whose primary function is to protect the skin against e.g. UV radiation. LIM domain proteins are a group of proteins involved in regulation of cell growth, damage signalling, cell fate determination and signal transduction. Despite their two zinc fingers, LIM domains do not bind to DNA, but rather mediate protein-protein interactions and function as modular protein binding interfaces. We initially identified CSRP1 as UVR-regulated transcript by using expression profiling. Here we have further studied the regulation and function of CRP1, a representative of cysteine rich protein- family consisting of two LIM domains. We find that CRP1 is increased by UVR in primary human keratinocytes and in normal human skin fibroblasts. Ectopic expression of CRP1 protected the cells against UVR and provided a survival advantage, whereas silencing of CRP1 rendered the cells more photosensitive. Actinic keratosis is a premalignant lesion of skin caused by excess exposure to sunlight and sunburn, which may lead to formation of squamous cell carcinoma. The expression of CRP1 was increased in basal keratinocytes of Actinic keratosis patient specimens suggesting that CRP1 may be increased by constant exposure to UVR and may provide survival advantage for the cells also in vivo. In squamous cell carcinoma, CRP1 was only expressed in the fibroblasts surrounding the tumour. Moreover, we found that ectopic expression of CRP1 suppresses cell proliferation. Transforming growth factor beta (TGFbeta) is a multifunctional cytokine that regulates several functions in cell including growth, apoptosis and differentiation, and plays important roles in pathological disorders like cancer and fibrosis. We found that TGFbeta-signalling pathway regulates CRP1 at protein, but not at transcriptional level. The increase was mediated both through Smad and non-Smad signalling pathways involving MAPK/p38. Furthermore, we found that TGFbeta-mediated increase in CRP1 was associated with myofibroblast differentiation, and that CRP1 was significantly more expressed in idiopathic pulmonary fibrosis as compared to normal lung specimens. Since cell contractility is a distinct feature of myofibroblasts, and CRP1 is associated with actin cytoskeleton, we studied the role of CRP1 in cell contractility. CRP1 was found to localize to stress fibres that mediate contractility and to mediate myofibroblast contraction. These studies identify CRP1 as a stress responsive and cytokine regulated cytoskeletal protein that participates in pathological processes involved in fibrotic diseases and cancer.
  • Färkkilä, Anniina (Helsingin yliopisto, 2012)
    Granulosa cell tumor (GCT) is the second most common subtype of ovarian cancer, representing 5% of all ovarian malignancies. GCTs are characterized by an indolent course of disease, with a 5-year survival rate of over 90%. However, recurrences occur in 20-30% of patients, also those with early-stage disease, leading to increased mortality. The pathogenesis and factors affecting prognosis of GCTs are largely unknown. Further, the treatment of advanced or recurrent GCT is difficult underscoring the need for biologically targeted treatments for aggressive GCTs. The molecular mechanisms leading to GCT formation are likely to include regulators of granulosa cell proliferation and apoptosis. We studied granulosa cell regulators and tumor angiogenesis in GCTs utilizing tumor tissue and serum samples, and cell culture assays. The objectives of this study were to find new molecular prognostic factors and to identify targets for future biological treatments for GCT. GCTs express Anti-Müllerian Hormone (AMH) and knockout mouse models suggest that AMH acts as a growth inhibitor in GCT pathogenesis. We found that GCTs expressed AMH receptors, with the AMH type II receptor (AMHRII) being characteristic of GCTs. AMH expression was decreased in large GCTs, and recombinant AMH inhibited growth of GCT cells in vitro. The results support the premise that AMH acts as a growth inhibitor in GCTs, and AMH and AMHRII emerge as targets for treatment of GCT. Vascular Endothelial Growth Factor-A (VEGF) is a key factor in tumor angiogenesis that has been successfully targeted in cancer treatment. We found that VEGF and its functional receptor VEGFR-2 are highly expressed in GCTs; VEGFR-2 was also expressed in the active, phosphorylated form. GCTs produced significant amounts of VEGF that could also be detected in the serum of GCT patients. In cell culture assays, the inhibition of VEGF by soluble anti-VEGF antibody (bevacizumab) inhibited growth and induced apoptosis of GCT cells. These results indicate a pro-tumorigenic role of VEGF in GCTs and encourage targeting VEGF and VEGFR-2 in the treatment of GCTs. Transcription factor GATA4 associates with GCT pathogenesis and prognosis. We found that high tumor GATA4 expression was an independent prognostic factor for GCT recurrence. GATA4 was also prognostic of shorter disease-specific survival along with higher tumor stage (II-III) and nuclear atypia. These results suggest that GATA4 can be used as a new prognostic marker for GCT. Human Epidermal Growth Factor Receptor 2 (HER2) is a known oncogene and a target for treatment in breast and gastric cancer. We found that high expression of HER2 delineated an aggressive subset of GCTs and HER2 is thus a potential target for treatment also in this disease.
  • Jokela-Määttä, Mirka (Helsingin yliopisto, 2009)
    Visual pigments of different animal species must have evolved at some stage to match the prevailing light environments, since all visual functions depend on their ability to absorb available photons and transduce the event into a reliable neural signal. There is a large literature on correlation between the light environment and spectral sensitivity between different fish species. However, little work has been done on evolutionary adaptation between separated populations within species. More generally, little is known about the rate of evolutionary adaptation to changing spectral environments. The objective of this thesis is to illuminate the constraints under which the evolutionary tuning of visual pigments works as evident in: scope, tempo, available molecular routes, and signal/noise trade-offs. Aquatic environments offer Nature s own laboratories for research on visual pigment properties, as naturally occurring light environments offer an enormous range of variation in both spectral composition and intensity. The present thesis focuses on the visual pigments that serve dim-light vision in two groups of model species, teleost fishes and mysid crustaceans. The geographical emphasis is in the brackish Baltic Sea area with its well-known postglacial isolation history and its aquatic fauna of both marine and fresh-water origin. The absorbance spectrum of the (single) dim-light visual pigment were recorded by microspectrophotometry (MSP) in single rods of 26 fish species and single rhabdoms of 8 opossum shrimp populations of the genus Mysis inhabiting marine, brackish or freshwater environments. Additionally, spectral sensitivity was determined from six Mysis populations by electroretinogram (ERG) recording. The rod opsin gene was sequenced in individuals of four allopatric populations of the sand goby (Pomatoschistus minutus). Rod opsins of two other goby species were investigated as outgroups for comparison. Rod absorbance spectra of the Baltic subspecies or populations of the primarily marine species herring (Clupea harengus membras), sand goby (P. minutus), and flounder (Platichthys flesus) were long-wavelength-shifted compared to their marine populations. The spectral shifts are consistent with adaptation for improved quantum catch (QC) as well as improved signal-to-noise ratio (SNR) of vision in the Baltic light environment. Since the chromophore of the pigment was pure A1 in all cases, this has apparently been achieved by evolutionary tuning of the opsin visual pigment. By contrast, no opsin-based differences were evident between lake and sea populations of species of fresh-water origin, which can tune their pigment by varying chromophore ratios. A more detailed analysis of differences in absorbance spectra and opsin sequence between and within populations was conducted using the sand goby as model species. Four allopatric populations from the Baltic Sea (B), Swedish west coast (S), English Channel (E), and Adriatic Sea (A) were examined. Rod absorbance spectra, characterized by the wavelength of maximum absorbance (λmax), differed between populations and correlated with differences in the spectral light transmission of the respective water bodies. The greatest λmax shift as well as the greatest opsin sequence difference was between the Baltic and the Adriatic populations. The significant within-population variation of the Baltic λmax values (506-511 nm) was analyzed on the level of individuals and was shown to correlate well with opsin sequence substitutions. The sequences of individuals with λmax at shorter wavelengths were identical to that of the Swedish population, whereas those with λmax at longer wavelengths additionally had substitution F261F/Y in the sixth transmembrane helix of the protein. This substitution (Y261) was also present in the Baltic common gobies and is known to redshift spectra. The tuning mechanism of the long-wavelength type Baltic sand gobies is assumed to be the co-expression of F261 and Y261 in all rods to produce ≈ 5 nm redshift. The polymorphism of the Baltic sand goby population possibly indicates ambiguous selection pressures in the Baltic Sea. The visual pigments of all lake populations of the opossum shrimp (Mysis relicta) were red-shifted by 25 nm compared with all Baltic Sea populations. This is calculated to confer a significant advantage in both QC and SNR in many humus-rich lakes with reddish water. Since only A2 chromophore was present, the differences obviously reflect evolutionary tuning of the visual protein, the opsin. The changes have occurred within the ca. 9000 years that the lakes have been isolated from the Sea after the most recent glaciation. At present, it seems that the mechanism explaining the spectral differences between lake and sea populations is not an amino acid substitution at any other conventional tuning site, but the mechanism is yet to be found.
  • de Haan, Astrid (Helsingin yliopisto, 2012)
    Campylobacter is the main bacterial genus of gastroenteritis in Finland and C. jejuni accounts for more than 90% of the Campylobacter infections. The majority of infections occur during the summertime (June-August). Risk factors associated with the disease are the handling and consumption of undercooked chicken meat, drinking of raw milk or contaminated water and foreign travel. Nevertheless, the source of Campylobacter often remains obscure, due to the sporadic nature of the infection. Multilocus sequence typing (MLST) has been proven to be useful in molecular epidemiology and source attribution studies of this zoonotic food- and water-borne pathogen. Using MLST, we typed 454 C. jejuni isolates from domestically acquired human infections and 208 isolates from chicken meat and caecal samples collected between 1996 and 2006. In this study period, clonal complexes (CCs) ST-45, ST-21 and ST-677 covered 73.9% of all isolates. The annual overlap between STs from human and chicken isolates decreased from 76% in 1999 to 58% in 2006. CC ST-677 occurred with an increasing trend among chicken isolates, but with a decreasing trend among human isolates. A high ST diversity was revealed among 102 Finnish bovine C. jejuni isolates. CC ST-21 alone accounted for 56% of the bovine C. jejuni isolates and encompassed 23 different STs. CC ST-61 was the second largest CC and included 20% of the isolates. Source attribution was determined by the Bayesian Analysis of Population Structure (BAPS) program, which showed that 44.3% of the human isolates were found in the bovine-associated BAPS cluster; 45.4% were found in the chicken-associated BAPS cluster and 10.3% remained unassigned. A high diversity of STs including novel and unassigned STs were found among the 113 C. jejuni isolates sampled from natural water bodies, wild birds and zoo animals. However, the most common ST found among human and poultry isolates, ST-45, was also the most common ST typed among the natural water body isolates. In the BAPS analysis 65.4% of the human isolates grouped into the poultry-associated cluster. A new cluster associated with natural water bodies emerged that largely consisting of uncommon and unassigned STs. Population differentiation revealed that the human and poultry populations and the human and natural water populations were the most similar to each other. The distributions of fspA1 and fspA2 among isolates taken from different sources and STs were studied. The human and poultry isolates, in addition to CC ST-45 and ST-283 were associated with fspA1, whereas the bovine isolates, CC ST-21 and ST-61 were associated with fspA2. FspA1 was highly conserved among the strains in contrast to fspA2, which was profoundly heterogeneous. Sequence analysis of the fspA2 allele in different STs showed that several predicted protein sequences contained premature stop codons. The presence of certain unique metabolic traits was found in some but not all C. jejuni strains. Gamma-glutamyl transpeptidase (GGT) and the gene for periplasmic L-asparaginase (ansBs) were often mutually exclusive with fucose permease (fucP). This particular combination, and also the presence of fucP only was highly ST dependent. The exception to this phenomenon was ST-45, which was very heterogeneous. Total absence of all studied metabolic traits occurred in 22% of the strains. The research conducted here conclusively showed a great diversity of C. jejuni STs among the human, bovine and environmental isolates. The overlap between Finnish human and chicken C. jejuni isolates declined between 1999 and 2006. However, natural waters could be an underestimated source of infection. Pronounced fspA2 diversity was found. The distribution of the secondary metabolic traits GGT, ansBs and fucP was promiscuous among the isolates obtained from different sources which suggests that animal colonization and human infection are not necessarily dependent on these traits.