Development of regulatory T cells in the human thymus

The role of the immune system is to protect an organism against pathogens while maintaining tolerance against self. T cells are an essential component of the immune system and they develop in the thymus. The AIRE (autoimmune regulator) gene product plays an important role in T cell development, as it promotes expression of peripheral tissue antigens in the thymus. Developing T cells, thymocytes, which recognize self-antigens with high affinity are deleted. However, this deletion process is not perfect and not all autoreactive T cells are destroyed. When the distinction between self and non-self fails, tolerance breaks and the immune system attacks the host s own tissues. This results in autoimmunity. Regulatory T cells contribute to the maintenance of self-tolerance. They can actively suppress the function of autoreactive cells. Several populations of cells with regulatory properties have been described, but the best characterized population is the natural regulatory T cells (Treg cells), which develop in the thymus and express the transcription factor FOXP3. The thymic development of Treg cells in humans is the subject of this thesis. Thymocytes at different developmental stages were analyzed using flow cytometry. The CD4-CD8- double-negative (DN) thymocytes are the earliest T cell precursors in the T cell lineage. My results show that the Treg cell marker FOXP3 is up-regulated already in a subset of these DN thymocytes. FOXP3+ cells were also found among the more mature CD4+CD8+ double-positive (DP) cells and among the CD4+ and CD8+ single-positive (SP) thymocytes. The different developmental stages of the FOXP3+ thymocytes were isolated and their gene expression examined by quantitative PCR. T cell receptor (TCR) repertoire analysis was used to compare these different thymocyte populations. My data show that in humans commitment to the Treg cell lineage is an early event and suggest that the development of Treg cells follows a linear developmental pathway, FOXP3+ DN precursors evolving through the DP stage to become mature CD4+ Treg cells. Most T cells have only one kind of TCR on their cell surface, but a small fraction of cells expresses two different TCRs. My results show that the expression of two different TCRs is enriched among Treg cells. Furthermore, both receptors were capable of transmitting signals when bound by a ligand. By extrapolating flow cytometric data, it was estimated that the majority of peripheral blood Treg cells are indeed dual-specific. The high frequency of dual-specific cells among human Treg cells suggests that dual-specificity has a role in directing these cells to the Treg cell lineage. It is known that both genetic predisposition and environmental factors influence the development of autoimmunity. It is also known that the dysfunction or absence of Treg cells leads to the development of autoimmune manifestations. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare monogenic autoimmune disease, caused by mutations in the AIRE gene. In the absence of AIRE gene product, deletion of self-specific T cells is presumably disturbed and autoreactive T cells escape to the periphery. I examined whether Treg cells are also affected in APECED. I found that the frequency of FOXP3+ Treg cells and the level of FOXP3 expression were significantly lower in APECED patients than in controls. Additionally, when studied in cell cultures, the suppressive capacity of the patients' Treg cells was impaired. Additionally, repertoire analysis showed that the TCR repertoire of Treg cells was altered. These results suggest that AIRE contributes to the development of Treg cells in humans and the selection of Treg cells is impaired in APECED patients. In conclusion, my thesis elucidates the developmental pathway of Treg cells in humans. The differentiation of Tregs begins early during thymic development and both the cells dual-specificity and AIRE probably affect the final commitment of Treg cells.

Modern diagnosis of Epstein-Barr virus infections and post-transplant lymphoproliferative disease

Infection by Epstein-Barr virus (EBV) occurs in approximately 95% of the world s population. EBV was the first human virus implicated in oncogenesis. Characteristic for EBV primary infection are detectable IgM and IgG antibodies against viral capsid antigen (VCA). During convalescence the VCA IgM disappears while the VCA IgG persists for life. Reactivations of EBV occur both among immunocompromised and immunocompetent individuals. In serological diagnosis, measurement of avidity of VCA IgG separates primary from secondary infections. However, in serodiagnosis of mononucleosis it is quite common to encounter, paradoxically, VCA IgM together with high-avidity VCA IgG, indicating past immunity. We determined the etiology of this phenomenon and found that, among patients with cytomegalovirus (CMV) primary infection a large proportion (23%) showed antibody profiles of EBV reactivation. In contrast, EBV primary infection did not appear to induce immunoreactivation of CMV. EBV-associated post-transplant lymphoproliferative disease (PTLD) is a life threatening complication of allogeneic stem cell or solid organ transplantation. PTLD may present with a diverse spectrum of clinical symptoms and signs. Due to rapidity of PTLD progression especially after stem cell transplantation, the diagnosis must be obtained quickly. Pending timely detection, the evolution of the fatal disease may be halted by reduction of immunosuppression. A promising new PTLD treatment (also in Finland) is based on anti-CD-20 monoclonal antibodies. Diagnosis of PTLD has been demanding because of immunosuppression, blood transfusions and the latent nature of the virus. We set up in 1999 to our knowledge first in Finland for any microbial pathogen a real-time quantitative PCR (qPCR) for detection of EBV DNA in blood serum/plasma. In addition, we set up an in situ hybridisation assay for EBV RNA in tissue sections. In collaboration with a group of haematologists at Helsinki University Central Hospital we retrospectively determined the incidence of PTLD among 257 allogenic stem cell transplantations (SCT) performed during 1994-1999. Post-mortem analysis revealed 18 cases of PTLD. From a subset of PTLD cases (12/18) and a series of corresponding controls (36), consecutive samples of serum were studied by the new EBV-qPCR. All the PTLD patients were positive for EBV-DNA with progressively rising copy numbers. In most PTLD patients EBV DNA became detectable within 70 days of SCT. Of note, the appearance of EBV DNA preceded the PTLD symptoms (fever, lymphadenopathy, atypical lymphocytes). Among the SCT controls, EBV DNA occurred only sporadically, and the EBV-DNA levels remained relatively low. We concluded that EBV qPCR is a highly sensitive (100%) and specific (96%) new diagnostic approach. We also looked for and found risk factors for the development of PTLD. Together with a liver transplantation group at the Transplantation and Liver Surgery Clinic we wanted to clarify how often and how severely do EBV infections occur after liver transplantation. We studied by the EBV qPCR 1284 plasma samples obtained from 105 adult liver transplant recipients. EBV DNA was detected in 14 patients (13%) during the first 12 months. The peak viral loads of 13 asymptomatic patients were relatively low (<6600/ml), and EBV DNA subsided quickly from circulation. Fatal PTLD was diagnosed in one patient. Finally, we wanted to determine the number and clinical significance of EBV infections of various types occurring among a large, retrospective, nonselected cohort of allogenic SCT recipients. We analysed by EBV qPCR 5479 serum samples of 406 SCT recipients obtained during 1988-1999. EBV DNA was seen in 57 (14%) patients, of whom 22 (5%) showed progressively rising and ultimately high levels of EBV DNA (median 54 million /ml). Among the SCT survivors, EBV DNA was transiently detectable in 19 (5%) asymptomatic patients. Thereby, low-level EBV-DNA positivity in serum occurs relatively often after SCT and may subside without specific treatment. However, high molecular copy numbers (>50 000) are diagnostic for life-threatening EBV infection. We furthermore developed a mathematical algorithm for the prediction of development of life-threatening EBV infection.

Human parvovirus B19: tissue persistence and prevalence of prototypic and new variants

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.

Genetic aspects of outcome in kidney transplantation: cytokine and thrombosis associated candidate genes and gene expression biomarkers

Kidney transplantation (Tx) is the treatment of choice for end stage renal disease. Immunosuppressive medications are given to prevent an immunological rejection of the transplant. However, immunosuppressive drugs increase e.g. the risk of infection, cancer or nephrotoxicity. A major genetic contributors to immunological acceptance of the graft are human leukocyte antigen (HLA) genes. Also other non-HLA gene polymorphisms may predict the future risk of complications before Tx, possibly enabling individualised immunotherapy. Graft function after Tx is monitored using non-specific clinical symptoms and laboratory markers. The definitive diagnosis of graft rejection however relies on a biopsy of the graft. In the acute rejection (AR) diagnostics there is a need for an alternative to biopsy that would be an easily repeatable and simple method for regular use. Frequent surveillance of acute or subclinical rejection (SCR) may improve long-term function. In this thesis, associations between cytokine and thrombosis associated candidate genes and the outcome of kidney Tx were studied. Cytotoxic and co-stimulatory T lymphocyte molecule gene expression biomarkers for the diagnosis of the AR and the SCR were also investigated. We found that polymorphisms in the cytokine genes tumor necrosis factor and interleukin 10 (IL10) of the recipients were associated with AR. In addition, certain IL10 gene polymorphisms of the donors were associated with the incidence of cytomegalovirus infection and occurrence of later infection in a subpopulation of recipients. Further, polymorphisms in genes related to the risk of thrombosis and those of certain cytokines were not associated with the occurrence of thrombosis, infarction, AR or graft survival. In the study of biomarkers for AR, whole blood samples were prospectively collected from adult kidney Tx patients. With real-time quantitative PCR (RT-QPCR) gene expression quantities of CD154 and ICOS differentiated the patients with AR from those without, but not from the patients with other causes of graft dysfunction. Biomarkers for SCR were studied in paediatric kidney Tx patients. We used RT-QPCR to quantify the gene expression of immunological candidate genes in a low-density array format. In addition, we used RT-QPCR to validate the results of the microarray analysis. No gene marker differentiated patients with SCR from those without SCR. This research demonstrates the lack of robust markers among polymorphisms or biomarkers in investigated genes that could be included in routine analysis in a clinical laboratory. In genetic studies, kidney Tx can be regarded as a complex trait, i.e. several environmental and genetic factors may determine its outcome. A number of currently unknown genetic factors probably influence the results of Tx.

Prognostic importance of the tumour microenvironment in follicular lymphoma patients treated with immunochemotherapy

Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma. It is an indolent and clinically heterogeneous disease, which is generally considered incurable. Currently, immunochemotherapy has significantly improved the outcome of FL patients. This is based on the combination of rituximab, a monoclonal anti-CD20 antibody, with chemotherapy, and is used at present as a standard first-line therapy in FL. Thus far, however, patients have been selected for treatment based on clinical risk factors and indices that were developed before the rituximab era. Therefore, there is a growing need to understand the molecular mechanisms underlying the disease, which would not only provide information to predict survival in the rituximab era, but also enable the design of more targeted therapeutic strategies. In this study, our aim was to identify genes predicting the outcome in FL patients treated with immunochemotherapy. Thus, we performed a cDNA microarray with 24 FL patients. When gene expression differences from diagnostic tumour samples were related to the clinical outcome, we identified novel genes with a prognostic impact on survival. The expression of selected genes was further characterized with quantitative PCR and immunohistochemistry (IHC). Interestingly, the prognostic influence of these genes was often associated with their expression in non-malignant cells instead of tumour cells. Based on the observed gene expression patterns, we analyzed the abundance and prognostic value of non-malignant immune cells in 95-98 FL patients treated with immunochemotherapy. We observed that a high content of tumour-associated macrophages was a marker of a favourable prognosis. In contrast, the accumulation of mast cells correlated with a poor outcome and was further associated with tumour vascularity. Increased microvessel density also correlated with an inferior outcome. In addition, we used the same microarray data with a systems biology approach to identify signalling pathways or groups of genes capable of separating patients with favourable or adverse outcomes. Among the transcripts, there were many genes associated with signal transducers and activators of the transcription (STAT5a) pathway. When IHC was used as validation, STAT5a expression was mostly observed in T-cells and follicular dendritic cells, and expression was found to predict a favourable outcome. In cell cultures, rituximab was observed to induce the expression of STAT5a-associated interleukins in human lymphoma cell lines, which might provide a possible link for the cross-talk between rituximab-induced FL cells and their microenvironment. In conclusion, we have demonstrated that the microenvironment has a prognostic role in FL patients treated with immunochemotherapy. The results also address the importance of re-evaluating the prognostic markers in the rituximab era of lymphoma therapies.

Molecular characterization of sediment bacterial communities affected by fish farming

Fish farming introduces nutrients, microbes and a wide variety of chemicals such as heavy metals, antifoulants and antibiotics to the surrounding environment. Introduction of antibiotics has been linked with the increased incidence of antibiotic resistant pathogenic bacteria in the farm vicinities. In this thesis molecular methods such as quantitative PCR and DNA sequencing were applied to analyze bacterial communities in sediments from fish farms and pristine locations. Altogether four farms and four pristine sites were sampled in the Baltic Sea. Two farm and two pristine locations were sampled over a surveillance period of four years. Furthermore, a new methodology was developed as a part of the study that permits amplifying single microbial genomes and capturing them according to any genetic traits, including antibiotic resistance genes. The study revealed that several resistance genes for tetracycline were found at the sediment underneath the aquaculture farms. The copy number of these genes remained elevated even at a farm that had not used any antibiotics since year 2000, six years before this study started. Similarly, an increase in the amount of mercury resistance gene merA was observed at the aquaculture sediment. The persistence of the resistance genes in absence of any selection pressure from antibiotics or heavy metals suggests that the genes may be introduced to the sediment by the farming process. This is also supported by the diversity pattern of the merA gene between farm and pristine sediments. The bacterial community-level changes in response to fish farming were very complex and no single phylogenetic groups were found that would be typical to fish farm sediments. However, the community structures had some correlation with the exposure to fish farming. Our studies suggest that the established approaches to deal with antibiotic resistance at the aquaculture, such as antibiotic cycling, are fundamentally flawed because they cannot prevent the introduction of the resistance genes and resistant bacteria to the farm area by the farming process. Further studies are required to study the entire fish farming process to identify the sources of the resistance genes and the resistant bacteria. The results also suggest that in order to prevent major microbiological changes in the surrounding aquatic environment, the farms should not be founded in shallow water where currents do not transport sedimenting matter from the farms. Finally, the technique to amplify and select microbial genomes will potentially have a considerable impact in microbial ecology and genomics.

Optimization of sample preparation for next-generation sequencing with Illumina Genome Analyzer II

The growing interest for sequencing with higher throughput in the last decade has led to the development of new sequencing applications. This thesis concentrates on optimizing DNA library preparation for Illumina Genome Analyzer II sequencer. The library preparation steps that were optimized include fragmentation, PCR purification and quantification. DNA fragmentation was performed with focused sonication in different concentrations and durations. Two column based PCR purification method, gel matrix method and magnetic bead based method were compared. Quantitative PCR and gel electrophoresis in a chip were compared for DNA quantification. The magnetic bead purification was found to be the most efficient and flexible purification method. The fragmentation protocol was changed to produce longer fragments to be compatible with longer sequencing reads. Quantitative PCR correlates better with the cluster number and should thus be considered to be the default quantification method for sequencing. As a result of this study more data have been acquired from sequencing with lower costs and troubleshooting has become easier as qualification steps have been added to the protocol. New sequencing instruments and applications will create a demand for further optimizations in future.

Merkel cell carcinoma : Epidemiological study with special reference to polyomavirus and vascular factors in pathogenesis

Merkel cell carcinoma (MCC) is a rare cutaneous malignancy that occurs predominantly on sun exposed skin areas. A new polyomavirus (MCPyV) was identified in MCC tumor tissues in 2008 suggesting that a viral infection might be an etiological factor. A typical MCC is a rapidly growing painless purple nodule. In its early stage it can be misjudged by its appearance as a cyst or abscess. Recurrences are common and approximately half of the patients will develop lymph node metastases and third of the patents will have distant metastases. It affects mostly elderly persons at an average age of 70 at the time of diagnosis. MCC was first described in 1972 and the first MCC patient in Finland was identified in 1983. MCC has been poorly recognized, but increased awareness and better diagnostic accuracy has increased the incidence since the early years. In this study, all cases with a notation of MCC during 1979 2008 were obtained from the Finnish Cancer Registry. Based on this data, the incidence is 0.11 for men and 0.12 for women. It is similar than that of other Nordic countries, but lower than in the USA. For clinical series, the files of patients diagnosed with MCC during 1983 2004 were reviewed, and the tissue samples were re-evaluated, if available (n=181). Third of the patients were men, and the most common site of the primary tumor was the head and neck (53%). The majority of the patients (86%) presented with a clinically node-negative (Stage I or II) disease, but the disease recurred in 38% of them. The treatment schemes were heterogeneous. No additional benefit from a wide margin (≥2 cm) was found compared to a margin of 0.1-1.9 cm, but intralesional excision was more often associated with local recurrence. None of the patients with Stage I-II disease who had received postoperative radiotherapy had local recurrence during the follow-up period. The 5-year relative survival ratio for Stage I disease was 68%, for Stage II 67%, for Stage III 16%, and for Stage IV 0%. The relative excess risk of death was significantly lower among women than among men. Some of these tissue samples were further analyzed for vascular invasion (n=126) by immunohistochemistry using vascular endothelial markers CD-31 and D2-40. Vascular invasion was seen in 93% of the samples and it was observed already in very small, <5mm tumors. The tissue samples were also analyzed for the presence of MCPyV by using a polymerase chain reaction (PCR) and quantitative PCR. MCPyV DNA was present in 80% of 114 samples studied. The patients with virus-positive tumors had better overall survival than patients with virus-negative tumors. Immunohistochemical analyses were performed for the expression of VEGFR-2 (n=21) and endostatin (n=19), but they had no prognostic value. Our results support the concept of treating MCC with margin-negative excision and radiotherapy to the tumor bed to reduce local recurrence. The finding of a high frequency of lymphovascular invasion reduces its value as a prognostic factor, but emphasizes the role of sentinel node biopsy even in very small primary MCC.

Bacteria colonizing paper machines

Bacteria growing in paper machines can cause several problems. Biofilms detaching from paper machine surfaces may lead to holes and spots in the end product or even break the paper web leading to expensive delays in production. Heat stable endospores will remain viable through the drying section of paper machine, increasing the microbial contamination of paper and board. Of the bacterial species regularly found in the end products, Bacillus cereus is the only one classified as a pathogen. Certain B. cereus strains produce cereulide, the toxin that causes vomiting disease in food poisonings connected to B. cereus. The first aim of this thesis was to identify harmful bacterial species colonizing paper machines and to assess the role of bacteria in the formation of end product defects. We developed quantitative PCR methods for detecting Meiothermus spp. and Pseudoxanthomonas taiwanensis. Using these methods I showed that Meiothermus spp. and Psx. taiwanensis are major biofoulers in paper machines. I was the first to be able to show the connection between end product defects and biofilms in the wet-end of paper machines. I isolated 48 strains of primary-biofilm forming bacteria from paper machines. Based on one of them, strain K4.1T, I described a novel bacterial genus Deinobacterium with Deinobacterium chartae as the type species. I measured the transfer of Bacillus cereus spores from packaging paper into food. To do this, we constructed a green fluorescent protein (GFP) labelled derivative of Bacillus thuringiensis and prepared paper containing spores of this strain. Chocolate and rice were the recipient foods when transfer of the labelled spores from the packaging paper to food was examined. I showed that only minority of the Bacillus cereus spores transferred into food from packaging paper and that this amount is very low compared to the amount of B. cereus naturally occurring in foods. Thus the microbiological risk caused by packaging papers is very low. Until now, the biological function of cereulide for the producer cell has remained unknown. I showed that B. cereus can use cereulide to take up K+ from environment where K+ is scarce: cereulide binds K+ ions outside the cell with high affinity and transports these ions across cell membrane into the cytoplasm. Externally added cereulide increased the growth rate of cereulide producing strains in medium where potassium was growth limiting. In addition, cereulide producing strains outcompeted cereulide non-producing B. cereus in potassium deficient environment, but not when the potassium concentration was high. I also showed that cereulide enhances biofilm formation of B. cereus.

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.

Die Eigenschaften der Benennungen und ihr Einfluss auf deren Verwendung am Beispiel der Euro-Währung : Eine quantitative und kontrastive Analyse der Terminologie im Finnischen und im Deutschen

This dissertation deals with the terminology of the Euro currency. Its aims are to determine the characteristics of the designations in a certain LSP and to discover whether the recommendations and rules that have been given to the formation of designations and 'ideal' designations have any influence on the usage of the designations. The characteristics analysed include length of the designation, part of speech, form, formation method, constancy, monosemy, suitability to a concept system and degree of specialty. The study analyses the actual usage of the designations in texts and the implementation of the designations. The study is an adaptation of a terminometric survey and uses concept analysis and quantitative analysis as its basic methods. The frequency of each characteristic is measured in terms of statistics. It is assumed that the 'ideality' of a designation influences its usage, for example that if a designation is short, it is used more than its longer rivals (synonyms). The results are analysed in a corpus consisting of a compilation of different texts concerning the Euro. The corpus is divided according to three features: year (1998-2003), genre (judicial texts, annual reports and brochures) and language (Finnish and German). Each analysis is performed according to each of these features and compared with the others. The results indicate that some of the characteristics of the designations indeed seem to have an influence on the usage of the designations. For example, monosemy and suitability to the concept system often lead to the implementation of the designation having the ideal or certain value in these characteristics in the analysed Finnish material. In German material, an 'ideal' value in the characteristics leads to the implementation of the designations more often than in Finnish. The contrastive study indicates that, for example, suitability to a concept system leads to implementation of a designation in judicial texts more often than in other genres. The recommendations given to an 'ideal' designation are thus often acceptable, but they cannot be generalized for all languages in the same extent.

Methodological and Empirical Advances in the Quantitative Analysis of Spontaneous Responses in Psychophysiological Time Series

The aim of the present study was to advance the methodology and use of time series analysis to quantify dynamic structures in psychophysiological processes and thereby to produce information on spontaneously coupled physiological responses and their behavioral and experiential correlates. Series of analyses using both simulated and empirical cardiac (IBI), electrodermal (EDA), and facial electromyographic (EMG) data indicated that, despite potential autocorrelated structures, smoothing increased the reliability of detecting response coupling from an interindividual distribution of intraindividual measures and that especially the measures of covariance produced accurate information on the extent of coupled responses. This methodology was applied to analyze spontaneously coupled IBI, EDA, and facial EMG responses and vagal activity in their relation to emotional experience and personality characteristics in a group of middle-aged men (n = 37) during the administration of the Rorschach testing protocol. The results revealed new characteristics in the relationship between phasic end-organ synchronization and vagal activity, on the one hand, and individual differences in emotional adjustment to novel situations on the other. Specifically, it appeared that the vagal system is intimately related to emotional and social responsivity. It was also found that the lack of spontaneously synchronized responses is related to decreased energetic arousal (e.g., depression, mood). These findings indicate that the present process analysis approach has many advantages for use in both experimental and applied research, and that it is a useful new paradigm in psychophysiological research. Keywords: Autonomic Nervous System; Emotion; Facial Electromyography; Individual Differences; Spontaneous Responses; Time Series Analysis; Vagal System

Expression of functional GABAc receptors in the brain

γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the nervous system and acts via three distinct receptor classes: A, B, and C. GABAC receptors are ionotropic receptors comprising ρ subunits. In this work, we aimed to elucidate the expression of ρ subunits in the postnatal brain, the characteristics of ρ2 homo-oligomeric receptors, and the function of GABAC receptors in the hippocampus. In situ hybridization on rat brain slices showed ρ2 mRNA expression from the newborn in the superficial grey layer of the superior colliculus, from the first postnatal week in the hippocampal CA1 region and the pretectal nucleus of the optic tract, and in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three ρ subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, ρ2 mRNA expression clearly dominated over ρ1 and ρ3. GABAC receptor protein expression was confirmed in the adult hippocampus, superior colliculus, and dorsal lateral geniculate nucleus by immunohistochemistry. From the selective distribution of ρ subunits, GABAC receptors may be hypothesized to be specifically involved in aspects of visual image motion processing in the rat brain. Although previous data had indicated a much higher expression level for ρ2 subunit transcripts than for ρ1 or ρ3 in the brain, previous work done on Xenopus oocytes had suggested that rat ρ2 subunits do not form functional homo-oligomeric GABAC receptors but need ρ1 or ρ3 subunits to form hetero-oligomers. Our results demonstrated, for the first time, that HEK 293 cells transfected with ρ2 cDNA displayed currents in whole-cell patch-clamp recordings. Homomeric rat ρ2 receptors had a decreased sensitivity to, but a high affinity for picrotoxin and a marked sensitivity to the GABAC receptor agonist CACA. Our results suggest that ρ2 subunits may contribute to brain function, also in areas not expressing other ρ subunits. Using extracellular electrophysiological recordings, we aimed to study the effects of the GABAC receptor agonists and antagonists on responses of the hippocampal neurons to electrical stimulation. Activation of GABAC receptors with CACA suppressed postsynaptic excitability and the GABAC receptor antagonist TPMPA inhibited the effects of CACA. Next, we aimed to display the activation of the GABAC receptors by synaptically released GABA using intracellular recordings. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation were prolonged by TPMPA. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABAC receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABAC receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA. Taken together with the restricted expression pattern of GABAC receptors in the brain and their distinctive pharmacological and biophysical properties, our findings supporting extrasynaptic localization of these receptors raise interesting possibilities for novel pharmacological therapies in the treatment of, for example, epilepsy and sleep disorders.