Molecular biology of progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1)

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by age of onset at 6-15 years, stimulus-sensitive myoclonus, tonic-clonic epileptic seizures and a progressive course. Mutations in the cystatin B (CSTB) gene underlie EPM1. The most common mutation underlying EPM1 is a dodecamer repeat expansion in the promoter region of CSTB. In addition, nine other mutations have been identified. CSTB, a cysteine protease inhibitor, is a ubiquitously expressed inhibitor of cathepsins, but its physiological function is unknown. The purpose of this study was to investigate CSTB gene expression and CSTB protein function in normal and pathological conditions. The basal CSTB promoter was mapped and characterized using different promoter-luciferase gene constructs. The binding activity of transcription factors to one ARE half, five Sp1 and four AP1 sites in the CSTB promoter was demonstrated. The CSTB promoter activity was clearly decreased using a CSTB promoter with "premutation" repeat expansions and in individuals with alike expansions. The expression of CSTB mRNA and protein was markedly reduced in patient cells. The endogenous CSTB protein localized to the nucleus, cytoplasm and lysosomes, and in differentiated cells merely to the cytoplasm. This suggests that the subcellular distribution of CSTB is dependent on the differentation status of the cells. The proteins representing patient missense mutations failed to associate with lysosomes, implying the importance of the lysosomal association for the proper physiological function of CSTB. Several alternatively spliced CSTB isoforms were identified. Of these CSTB2 was widely expressed with very low levels whereas the other alternatively spliced forms seemed to have limited tissue expression. In patients CSTB2 expression was reduced similarly to that of CSTB. The physiological relevance of CSTB alternative splicing remains unknown. The mouse Cstb transcript was shown to be present in all embryonic stages and adult tissues examined. The expression was highest at embryonic day 7 and in thymus, as well as in postnatal brain in the cortex, caudate putamen, thalamus, hippocampus, and in the Purkinje cell layer of the cerebellum. Our data implies that CSTB expression is tightly temporally and spatially regulated. The data presented in my thesis lay the basis for further understanding of the role of CSTB in health and disease.

NMR Spectroscopy of Multi-domain Proteins : Immunoglobulin-like Domains of Human Filamin A

Proteins are complex biomacromolecules playing fundamental roles in the physiological processes of all living organisms. They function as structural units, enzymes, transporters, process regulators, and signal transducers. Defects in protein functions often derive from genetic mutations altering the protein structure, and impairment of essential protein functions manifests itself as pathological conditions. Proteins operate through interactions, and all protein functions depend on protein structure. In order to understand biological mechanisms at the molecular level, one has to know the structures of the proteins involved. This thesis covers structural and functional characterization of human filamins. Filamins are actin-binding and -bundling proteins that have numerous interaction partners. In addition to their actin-organizing functions, filamins are also known to have roles in cell adhesion and locomotion, and to participate in the logistics of cell membrane receptors, and in the coordination of intracellular signaling pathways. Filamin mutations in humans induce severe pathological conditions affecting the brain, bones, limbs, and the cardiovascular system. Filamins are large modular proteins composed of an N-terminal actin-binding domain and 24 consecutive immunoglobulin-like domains (IgFLNs). Nuclear magnetic resonance (NMR) spectroscopy is a versatile method of gaining insight into protein structure, dynamics and interactions. NMR spectroscopy was employed in this thesis to study the atomic structure and interaction mechanisms of C-terminal IgFLNs, which are known to house the majority of the filamin interaction sites. The structures of IgFLN single-domains 17 and 23 and IgFLN domain pairs 16-17 and 18-19 were determined using NMR spectroscopy. The structures of domain pairs 16 17 and 18 19 both revealed novel domain domain interaction modes of IgFLNs. NMR titrations were employed to characterize the interactions of filamins with glycoprotein Ibα, FilGAP, integrin β7 and dopamine receptors. Domain packing of IgFLN domain sextet 16 21 was further characterized using residual dipolar couplings and NMR relaxation analysis. This thesis demonstrates the versatility and potential of NMR spectroscopy in structural and functional studies of multi-domain proteins.

Neuronal K-Cl Cotransporter : Transcriptional Mechanisms of KCC2 Gene Regulation

K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.

Clinical Studies on Epidural and Spinal Postoperative Analgesia : With Special Reference to Continuous Techniques of Administration and Adjuvant Drugs

Continuous epidural analgesia (CEA) and continuous spinal postoperative analgesia (CSPA) provided by a mixture of local anaesthetic and opioid are widely used for postoperative pain relief. E.g., with the introduction of so-called microcatheters, CSPA found its way particularly in orthopaedic surgery. These techniques, however, may be associated with dose-dependent side-effects as hypotension, weakness in the legs, and nausea and vomiting. At times, they may fail to offer sufficient analgesia, e.g., because of a misplaced catheter. The correct position of an epidural catheter might be confirmed by the supposedly easy and reliable epidural stimulation test (EST). The aims of this thesis were to determine a) whether the efficacy, tolerability, and reliability of CEA might be improved by adding the α2-adrenergic agonists adrenaline and clonidine to CEA, and by the repeated use of EST during CEA; and, b) the feasibility of CSPA given through a microcatheter after vascular surgery. Studies I IV were double-blinded, randomized, and controlled trials; Study V was of a diagnostic, prospective nature. Patients underwent arterial bypass surgery of the legs (I, n=50; IV, n=46), total knee arthroplasty (II, n=70; III, n=72), and abdominal surgery or thoracotomy (V, n=30). Postoperative lumbar CEA consisted of regular mixtures of ropivacaine and fentanyl either without or with adrenaline (2 µg/ml (I) and 4 µg/ml (II)) and clonidine (2 µg/ml (III)). CSPA (IV) was given through a microcatheter (28G) and contained either ropivacaine (max. 2 mg/h) or a mixture of ropivacaine (max. 1 mg/h) and morphine (max. 8 µg/h). Epidural catheter tip position (V) was evaluated both by EST at the moment of catheter placement and several times during CEA, and by epidurography as reference diagnostic test. CEA and CSPA were administered for 24 or 48 h. Study parameters included pain scores assessed with a visual analogue scale, requirements of rescue pain medication, vital signs, and side-effects. Adrenaline (I and II) had no beneficial influence as regards the efficacy or tolerability of CEA. The total amounts of epidurally-infused drugs were even increased in the adrenaline group in Study II (p=0.02, RM ANOVA). Clonidine (III) augmented pain relief with lowered amounts of epidurally infused drugs (p=0.01, RM ANOVA) and reduced need for rescue oxycodone given i.m. (p=0.027, MW-U; median difference 3 mg (95% CI 0 7 mg)). Clonidine did not contribute to sedation and its influence on haemodynamics was minimal. CSPA (IV) provided satisfactory pain relief with only limited blockade of the legs (no inter-group differences). EST (V) was often related to technical problems and difficulties of interpretation, e.g., it failed to identify the four patients whose catheters were outside the spinal canal already at the time of catheter placement. As adjuvants to lumbar CEA, clonidine only slightly improved pain relief, while adrenaline did not provide any benefit. The role of EST applied at the time of epidural catheter placement or repeatedly during CEA remains open. The microcatheter CSPA technique appeared effective and reliable, but needs to be compared to routine CEA after peripheral arterial bypass surgery.

Content of zinc, iron and their absorption inhibitors in Nicaraguan common beans (Phaseolus vulgaris L.).

Protein-energy malnutrition and mineral deficiencies are two of the three forms of nutritional deficiencies that affect most developing countries due to inadequate access to food and diets based on a sole crop. Common bean (Phaseolus vulgaris L.) is the staple crop of Nicaragua and it has the potential to improve the nutritional status of the poorest group of the nation. Its high content of both protein and nonhaem iron provides many nutrients, but inhibitors also may prevent absorption of iron and zinc by the human consumer. A proper production chain must be followed to ensure the best grain quality for the consumer. To achieve food security, both production and high nutritional content must be maintained. Four nationally important accessions of common bean, with different harvesting dates, were selected to be submitted to two treatments: to evaluate the impact of storage conditions on the end quality of the grain. The duration of the study was six months with sampling every six weeks, and the two treatments were controlled one stored at 40°C and 75 RH %, and the other was stored in in-situ conditions. Proximate and mineral composition was evaluated as well as tannin, phytate and bioavailability. Significant differences among different accessions were found, being the most significant in protein, Fe and Zn content, tannins and phytate. Protein values ranged from 21-23%. Iron content was 61-81 mg/kg but only 3-4% was bioavailable. Zinc content was 21-25 mg/kg and 10-12% was bioavailable. The concentration of phytate ranged from 8.6-9.6 mg/g while tannin values ranged within 37.7-43.8 mg/g. Storage at high temperatures was demonstrated to have an impact on certain nutritional compounds and proved detrimental to final grain quality. Soluble sugar content and tannin content decreased after six months in both storage conditions, IDF decreased in the in-situ and SDF in the stress. The iron content and bioavailability in INTA Biofortificado were not as outstanding as expected, so experiments should be conducted to compare its iron uptake and delivery with other cultivars.

POP-inhibiittorin KYP-2047 vaikutukset angiogeneesiin in vitro

Angiogeneesi on tärkeä ilmiö elimistön fysiologiassa, mutta myös lukuisissa patologisissa tiloissa. Angiogeneesi on monivaiheinen prosessi, joka sisältää angiogeneesiä indusoivia ja sitä inhiboivia tekijöitä tasapainossa keskenään. Useat tutkimukset puoltavat sitä, että tymosiini ȕ4 (Tȕ4) ja tetrapeptidi Ac-SDKP (N-asetyyliseryyli- aspartyyli-lysyyli-proliini) indusoivat angiogeneesiä in vitro ja in vivo. Tutkimukset viittaavat myös siihen, että prolyylioligopeptidaasi (POP) hydrolysoi peptidifragmentin Ac- SDKP Tȕ4:n (43 ah) proliinin jälkeen. POP on laajalti esiintyvä seriiniproteaasi, joka pystyy pilkkomaan vain alle 30 aminohapon oligopeptidejä. Tȕ4:n tulee siksi pilkkoutua ensin jonkin, vielä tuntemattoman peptidaasin johdosta. POP:ia on löydetty eniten aivoista, minkä vuoksi sitä on tutkittu varsinkin muistin ja oppimisen häiriötiloissa sekä neurodegeneratiivisten sairausten yhteydessä. POP:in todellinen fysiologinen merkitys on kuitenkin vielä selvittämättä. Tämän pro gradun kirjallisuusosiossa selvitetään angiogeneesiin liittyvien tekijöiden yhteyksiä sekä kuvataan angiogeenisten Tȕ4:n, Ac-SDKP:n ja POP:in ominaisuuksia, esiintymistä ja toimintaa. Kokeellisen osion tarkoituksena oli osoittaa, osallistuvatko POP ja Tȕ4 tetrapeptidin Ac-SDKP muodostumiseen ja kapillaarimuodostumiseen ja edelleen, voidaanko POPaktiivisuutta, tetrapeptidi- ja kapillaarimuodostumista estää spesifisellä POP-inhibiittorilla, KYP-2047:llä. Kokeellinen osa oli kaksiosainen. Ensimmäisessä osassa tutkittiin POPaktiivisuutta ja suoritettiin Ac-SDKP –pitoisuusmittauksia ajanjaksolla 0-180 min Wistarkannan rotista tehdyillä homogenaateilla. Tutkimusryhminä olivat 0,1 ja 0,5 μM KYP-2047 (+2 μM Tȕ4), 1:20 (0,625 μM) humaaniperäinen rekombinantti-POP (+ 2 μM Tȕ4), 2 μM Tȕ4 (pos. kontrolli) ja raakahomogenaatti (neg. kontrolli). Toisessa osassa tutkittiin kapillaarimuodostumista ajanjaksolla 0-180 min humaaniperäisillä napanuoralaskimon primaariendoteelisoluilla MatrigelTM Matrix -päällystetyllä 48- kuoppalevyllä, jolle oli siirrostettu 50 000 solua/kuoppa. Naudan seerumilla ja antibiooteilla käsitellyt tutkimusryhmät olivat 5 ja 10 μM KYP-2047 (+4 μM Tȕ4), 1:20 (0,625 μM) humaaniperäinen rekombinantti-POP (+4 μM Tȕ4), 4 μM Tȕ4 (pos. kontrolli) ja DMEM (neg. kontrolli). Kuoppia inkuboitiin ja kapillaarimuodostuminen kuvattiin valomikroskoopilla digitaalikameralla. Kutakin tutkimusryhmää pipetoitiin kolmeen rinnakkaiseen kuoppaan ja kokeet toistettiin neljästi. Sulkeutuneiden kapillaarien lukumäärä laskettiin manuaalisesti ja tuloksista tehtiin tilastollinen analyysi. 7ȕ4:n ja POP:in havaittiin molempien osallistuvan tetrapeptidin AC-SDKP muodostumiseen munuaishomogenaateissa. Primaariendoteelisolut muodostivat selkeitä kapillaareja Matrigelilla, erityisesti POP- ja Tȕ4–ryhmissä. KYP-2047 inhiboi tehokkaasti POP:ia kaikissa kokeissa osoittautuen hyväksi antiangiogeeniseksi yhdisteeksi. Angiogeneesin mekanismien ja POP:in, Tȕ4:n ja Ac-SDKP:n yhteyksien selvittäminen vaatii luonnollisesti vielä lisätutkimuksia.

Environmental and climatic dependences of stable isotope ratios in tree rings on different temporal scales

This work examines stable isotope ratios of carbon, oxygen and hydrogen in annual growth rings of trees. Isotopic composition in wood cellulose is used as a tool to study past climate. The method benefits from the accurate and precise dating provided by dendrochronology. In this study the origin, nature and the strength of climatic correlations are studied on different temporal scales and at different sites in Finland. The origin of carbon isotopic signal is in photosynthetic fractionation. The basic physical and chemical fractionations involved are reasonably well understood. This was confirmed by measuring instantaneous photosynthetic discrimination on Scots pine (Pinus sylvestris L.). The internal conductance of CO2 was recognized to have a significant impact on the observed fractionation, and further investigations are suggested to quantify its role in controlling the isotopic signal of photosynthates. Isotopic composition of the produced biomass can potentially be affected by variety of external factors that induce physiological changes in trees. Response of carbon isotopic signal in tree ring cellulose to changes in resource availability was assessed in a manipulation experiment. It showed that the signal was relatively stable despite of changes in water and nitrogen availability to the tree. Palaeoclimatic reconstructions are typically based on functions describing empirical relationship between isotopic and climatic parameters. These empirical relationships may change depending on the site conditions, species and timeframe studied. Annual variation in Scots pine tree ring carbon and oxygen isotopic composition was studied in northern and in central eastern Finland and annual variation in tree ring latewood carbon, oxygen and hydrogen isotopic ratio in Oak (Quercus robur L.) was studied in southern Finland. In all of the studied sites at least one of the studied isotope ratios was shown to record climate strongly enough to be used in climatic reconstructions. Using the observed relationships, four-century-long climate reconstructions from living Scots pine were created for northern and central eastern Finland. Also temporal stability of the relationships between three proxy indicators, tree ring growth and carbon and oxygen isotopic composition was studied during the four-hundred-year period. Isotope ratios measured from tree rings in Finland were shown to be sensitive indicators of climate. Increasing understanding of environmental controls and physiological mechanisms affecting tree ring isotopic composition will make possible more accurate interpretation of isotope data. This study also demonstrated that by measuring multiple isotopes and physical proxies from the same tree rings, additional information on tree physiology can be obtained. Thus isotopic ratios measured from tree ring cellulose provide means to improve the reliability of climate reconstructions.

Studies of the Human Transcriptome

Gene expression is one of the most critical factors influencing the phenotype of a cell. As a result of several technological advances, measuring gene expression levels has become one of the most common molecular biological measurements to study the behaviour of cells. The scientific community has produced enormous and constantly increasing collection of gene expression data from various human cells both from healthy and pathological conditions. However, while each of these studies is informative and enlighting in its own context and research setup, diverging methods and terminologies make it very challenging to integrate existing gene expression data to a more comprehensive view of human transcriptome function. On the other hand, bioinformatic science advances only through data integration and synthesis. The aim of this study was to develop biological and mathematical methods to overcome these challenges and to construct an integrated database of human transcriptome as well as to demonstrate its usage. Methods developed in this study can be divided in two distinct parts. First, the biological and medical annotation of the existing gene expression measurements needed to be encoded by systematic vocabularies. There was no single existing biomedical ontology or vocabulary suitable for this purpose. Thus, new annotation terminology was developed as a part of this work. Second part was to develop mathematical methods correcting the noise and systematic differences/errors in the data caused by various array generations. Additionally, there was a need to develop suitable computational methods for sample collection and archiving, unique sample identification, database structures, data retrieval and visualization. Bioinformatic methods were developed to analyze gene expression levels and putative functional associations of human genes by using the integrated gene expression data. Also a method to interpret individual gene expression profiles across all the healthy and pathological tissues of the reference database was developed. As a result of this work 9783 human gene expression samples measured by Affymetrix microarrays were integrated to form a unique human transcriptome resource GeneSapiens. This makes it possible to analyse expression levels of 17330 genes across 175 types of healthy and pathological human tissues. Application of this resource to interpret individual gene expression measurements allowed identification of tissue of origin with 92.0% accuracy among 44 healthy tissue types. Systematic analysis of transcriptional activity levels of 459 kinase genes was performed across 44 healthy and 55 pathological tissue types and a genome wide analysis of kinase gene co-expression networks was done. This analysis revealed biologically and medically interesting data on putative kinase gene functions in health and disease. Finally, we developed a method for alignment of gene expression profiles (AGEP) to perform analysis for individual patient samples to pinpoint gene- and pathway-specific changes in the test sample in relation to the reference transcriptome database. We also showed how large-scale gene expression data resources can be used to quantitatively characterize changes in the transcriptomic program of differentiating stem cells. Taken together, these studies indicate the power of systematic bioinformatic analyses to infer biological and medical insights from existing published datasets as well as to facilitate the interpretation of new molecular profiling data from individual patients.

T-type calcium channel:from physiological function to therapeutical targeting – Exploring neuronal development

Tämän tutkimuksen tarkoitus oli tutkia T-tyypin kalsiumkanavan toimintaa ja sen mahdollista roolia neuronaalisten kantasolujen migraatiossa. T-tyypin kalsiumkanavan tehtävän kehittyneissä aivoissa tiedetään olevan elektroenkefalografisten oskillaatioiden tuottaminen. Nämä taas ovat eräiden fysiologisten ja patofysiologisten tapahtumien säätelyssä avainasemassa. Tällaisia tapahtumia ovat uni, muisti, oppiminen ja epileptiset poissaolokohtaukset. Näiden lisäksi T-tyypin kalsiumkanavalla on myös periferaalisia vaikutuksia, mutta tämä tutkielma keskittyy sen neuronaalisiin toimintoihin. Tämän matalan jännitteen säätelemän kanavan toiminta neurogeneesin aikana on vähemmän tutkittua ja tunnettua kuin sen vaikutukset kehittyneissä aivoissa. T-tyypin kalsiumkanavan tiedetään edistävän kantasolujen proliferaatiota ja erilaistumista neurogeneesiksen aikana, mutta vaikutukset niiden migraatioon ovat vähemmän tunnetut. Tämä tutkimus näyttää T-tyypin kalsiumkanavan todennäköisesti osallistuvan neuronaaliseen migraatioon hiiren alkion subventrikkeli alueelta eristetyillä kanta- tai progeniittorisoluilla tehdyissä kokeissa. Selektiiviset T-tyypin kalsiumkanavan antagonistit, etosuksimidi, nikkeli ja skorpionitoksiini, kurtoxin hidastivat migraatiota erilaistuvissa progeniittorisoluissa. Tämä tutkimus koostuu kirjallisuuskatsauksesta ja kokeellisesta osasta. Tämän tutkimuksen toinen tarkoitus oli esitellä vaihtoehtoinen lähestymistapa invasiiviselle kantasoluterapialle, joka vaatii kantasolujen viljelyä ja siirtämistä ihmiseen. Tämä toinen tapa on endogeenisten kantasolujen eiinvasiivinen stimulointi, jolla ne saadaan migratoitumaan kohdekudokseen, erilaistumaan siellä ja tehtävänsä suoritettuaan lopettamaan jakaantumisen. Non-invasiivinen kantasoluterapia on vasta tiensä alussa, ja tarvitsee farmakologista osaamista kehittyäkseen. Joitain onnistuneita ei-invasiivisia hoitoja on jo tehty selkärangan vaurioiden korjaamisessa. Vastaavanlaisia menetelmiä voitaisiin käyttää myös keskushermoston vaurioiden ja neurodegeneratiivisten sairauksien hoidossa. Näiden menetelmien kehittäminen vaatii endogeenisten kantasoluja inhiboivien ja indusoivien mekanismien tuntemista. Yksi tärkeä kantasolujen erilaistumista stimuloiva tekijä on kalsiumioni. Jänniteherkät kalsiumkanavat osallistuvat kaikkiin neurogeneesiksen eri vaiheisiin. T-tyypin kalsiumkanava, joka ekspressoituu suuressa määrin keskushermoston kehityksen alkuvaiheessa ja vähenee neuronaalisen kehityksen edetessä, saattaa olla oleellisessa asemassa progeniittorisolujen ohjaamisessa.

Molecular profiling of indoor microbial communities in moisture damaged and non-damaged buildings

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.