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  • Lagus, Markus (Helsingin yliopisto, 2013)
    BACKGROUND Sleep disturbances and mood alterations are highly interrelated. The majority of patients suffering from depression report a reduced sleep quality. Inversely, people with sleep complaints are at elevated risk to develop depression. The complex regulation of these phenomena involves several brain areas and mechanisms. The susceptibility to change in this system is influenced by several factors, such as age and stressful lifestyle that are considered in this study. HYPOTHESIS The hypothesis of this study was that sleep and mood share common genetic/molecular regulatory networks and that both are also regulated by epigenetic mechanisms and neural plasticity. METHODS The studies were conducted both on humans and using an animal model for depression. In the animal model we measured the genome wide expression of genes in different brain areas of clomipramine-treated pups and adults. Using these data we conducted both individual area and inter-area network analyses of basal forebrain, frontal cortex, hypothalamus and hippocampus. We also measured the amount of BDNF, one of the plasticity-related factors, in sleep restriction and under aging. In the human study we conducted epigenetic analysis of the serotonin transporter gene and related the epigenetic changes to stress in a stressful working environment. RESULTS In the models investigated changes were observed on the system, protein, transcript and transcriptional regulatory levels. Inter-tissue pathways related to synaptic transmission, regulation of translation and ubiquitinylation were disrupted. The involved pathways are within the cellular components of the axons, growth cones, melanosomes and pigment granules. The disturbed networks are centred around serotonin, Mn(II) and Rhoa. In the basal forebrain the imbalance in gene expression is widely controlled by CREB1. Some of the changes seem to be epigenetically induced by sleep deprivation and stress. Individuals working in a high stress environment have significantly less methylation in the promoter area of serotonin transporter gene SLC6A4, as compared to individuals working in a low stress environment. We also found that the expression of cortical BDNF correlated with the recovery non-REM (NREM) slow wave activity (SWA) response, and that both the cortical BDNF and the SWA response to sleep deprivation were decreased in the aged animals, as were the changes in sleep latency. CONCLUSIONS The disturbances in the models investigated, arise, largely, but not solely, due to disruption in neurological systems previously related to the regulation of sleep and mood. Novelty value could be ascribed to findings that suggest involvement of inter-tissue networks, and more precisely, imbalance of melanosome related gene expression and gene networks connected to Mn(II). The stress induced demethylation of the SLC6A4 promoter suggests a mechanism for the body to cope with prolonged excessive stress. The downside of this coping mechanism is the possibility that this reprogramming increases the long-term risk for mood disorders. The findings in the sleep deprived aging rats support the hypothesis that the age related decrease in homeostatic NREM SWA is related to a reduced sleep need.
  • Wasik, Anita Agnieszka (Helsingin yliopisto, 2014)
    Diabetic nephropathy (DN) is a major microvascular complication of diabetes and a common cause of end-stage renal disease. Mechanisms leading to the development of DN are not fully understood, but podocyte injury is involved. Interestingly, in respect to glucose uptake podocytes are uniquely insulin sensitive cells. Insulin rapidly induces remodeling of the actin cytoskeleton and leads to glucose uptake via glucose transporters GLUT1 and GLUT4. Defects in the trafficking of the glucose transporters may affect the insulin sensitivity of podocytes. Thus, regulators of glucose transporter trafficking may provide suitable targets to enhance insulin sensitivity of podocytes and prevent the development and progression of DN. However, the precise mechanisms regulating glucose transporter trafficking and glucose uptake into podocytes are largely uncharacterized. To identify changes in the expression of glomerular proteins at an early stage of DN we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Ezrin was found to be downregulated in diabetic glomeruli. In cultured podocytes depletion of ezrin increased glucose uptake by increasing translocation of GLUT1 to the plasma membrane. Loss of ezrin also induced actin remodelling, which involved cofilin-1. Phosphorylated cofilin-1 was upregulated in diabetic glomeruli suggesting altered actin dynamics. Furthermore, reduced expression of ezrin was found in the podocytes of human patients with diabetes. We found that the filament-forming septin 7 forms a complex with CD2AP and nephrin, both of which are essential for glomerular ultrafiltration. We showed that septin 7 negatively regulates GLUT4 storage vesicle (GSV) trafficking by forming a physical barrier between the vesicles and the plasma membrane. The novel interaction partner of septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA), was found to positively regulate insulin-stimulated glucose uptake into podocytes. Loss of NMHC-IIA reduced formation of the SNARE complex involved in GSV exocytosis. Furthermore, we presented that insulin regulates the association of septin 7 and phosphorylated RLC (pp-RLC), a part of myosin hexameric complex, with a plasma membrane SNARE, SNAP23. pp-RLC is upregulated in diabetic glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with Type 1 diabetes. Our findings indicate that ezrin, septin 7 and NMHC-IIA regulate glucose uptake into podocytes and may play a role in the development of the renal complication in diabetes by regulating glucose transport and organization of the actin cytoskeleton in podocytes.
  • Nuotio, Krista (Helsingin yliopisto, 2007)
    Carotid artery disease is the most prevalent etiologic precursor of ischemic stroke, which is a major health hazard and the second most common cause of death in the world. If a patient presents with a symptomatic high-grade (>70%) stenosis in the internal carotid artery, the treatment of choice is carotid endarterectomy. However, the natural course of radiologically equivalent carotid lesions may be clinically quite diverse, and the reason for that is unknown. It would be of utmost importance to develop molecular markers that predict the symptomatic phenotype of an atherosclerotic carotid plaque (CP) and help to differentiate vulnerable lesions from stable ones. The aim of this study was to investigate the morphologic and molecular factors that associate with stroke-prone CPs. In addition to immunohistochemistry, DNA microarrays were utilized to identify molecular markers that would differentiate between symptomatic and asymptomatic CPs. Endothelial adhesion molecule expression (ICAM-1, VCAM-1, P-selectin, and E-selectin) did not differ between symptomatic and asymptomatic patients. Denudation of endothelial cells was associated with symptom-generating carotid lesions, but in studies on the mechanism of decay of endothelial cells, markers of apoptosis (TUNEL, activated caspase 3) were found to be decreased in the endothelium of symptomatic lesions. Furthermore, markers of endothelial apoptosis were directly associated with those of cell proliferation (Ki-67) in all plaques. FasL expression was significantly increased on the endothelium of symptomatic CPs. DNA microarray analysis revealed prominent induction of specific genes in symptomatic CPs, including those subserving iron and heme metabolism, namely HO-1, and hemoglobin scavenger receptor CD163. HO-1 and CD163 proteins were also increased in symptomatic CPs and associated with intraplaque iron deposits, which, however, did not correlate with symptom status itself. ADRP, the gene for adipophilin, was also overexpressed in symptomatic CPs. Adipophilin expression was markedly increased in ulcerated CPs and colocalized with extravasated red blood cells and cholesterol crystals. Taken together, the phenotypic characteristics and the numerous possible molecular mediators of the destabilization of carotid plaques provide potential platforms for future research. The denudation of the endothelial lining observed in symptomatic CPs may lead to direct thromboembolism and maintain harmful oxidative and inflammatory processes, predispose to plaque microhemorrhages, and contribute to lipid accumulation into the plaque, thereby making it vulnerable to rupture.
  • Vainio, Anni (Helsingin yliopisto, 2012)
    Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae are major health problems worldwide, both found in symptomless carriage but also causing even life-threatening infections. The aim of this thesis was to characterise MRSA and S. pneumoniae in detail by using several molecular typing methods for various epidemiological purposes: clonality analysis, epidemiological surveillance, outbreak investigation, and virulence factor analysis. The characteristics of MRSA isolates from the strain collection of the Finnish National Infectious Disease Register (NIDR) and pneumococcal isolates collected from military recruits and children with acute otitis media (AOM) were analysed using various typing techniques. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and the detection of Panton-Valentine leukocidin (PVL) genes were performed for MRSA isolates. Pneumococcal isolates were analysed using antimicrobial susceptibility testing, serotyping, MLST, and by detecting pilus islet 1 (PI-1) and 2 (PI-2) genes. Several international community- and hospital-associated MRSA clones were recognised in Finland. The genetic diversity among MRSA FIN-4 isolates and among FIN-16 isolates was low. Overall, MRSA blood isolates from 1997 to 2006 were genetically diverse. spa typing was found to be a highly discriminatory, rapid and accurate typing method and it also qualifies as the primary typing method in countries with a long history of PFGE-based MRSA strain nomenclature. However, additional typing by another method, e.g. PFGE, is needed in certain situations to be able to provide adequate discrimination for epidemiological surveillance and outbreak investigation. An outbreak of pneumonia was associated with one pneumococcal strain among military recruits, previously healthy young men living in a crowded setting. The pneumococcal carriage rate after the outbreak was found to be exceptionally high. PI-1 genes were detected at a rather low prevalence among pneumococcal isolates from children with AOM. However, the study demonstrated that PI-1 has existed among pneumococcal isolates prior to pneumococcal conjugate vaccine and the increased antimicrobial resistance era. Moreover, PI-1 was found to associate with the serotype rather than the genotype. This study adds to our understanding of the molecular epidemiology of MRSA strains in Finland and the importance of an appropriate genotyping method to be able to perform high-level laboratory-based surveillance of MRSA. Epidemiological and molecular analyses of S. pneumoniae add to our knowledge of the characteristics of pneumococcal strains in Finland.
  • 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.