Mycorrhizal fungi and nitrogen dynamics in drained peatland

Fungi have a fundamental role in carbon and nutrient transformations in the acids soils of boreal regions, such as peatlands, where high amounts of carbon (C) and nutrients are stored in peat, the pH is relatively low and the nutrient uptake of trees is highly dependent on mycorrhizae. In this thesis, the aim was to examine nitrogen (N) transformations and the availability of dissolved N compounds in forestry-drained peatlands, to compare the fungal community biomass and structure at various peat N levels, to investigate the growth of ectomycorrhizal fungi with variable P and K availability and to assess how the ectomycorrhizal fungi (ECM) affect N transformations. Both field and laboratory experiments were carried out. The peat N concentration did not affect the soil fungal community structure within a site. Phosphorus (P) and potassium (K) deficiency of the trees as well as the degree of decomposition and dissolved organic nitrogen (DON) concentration of the peat were shown to affect the fungal community structure and biomass of ECMs, highlighting the complexity of the below ground system on drained peatlands. The biomass of extrametrical mycorrhizal mycelia (EMM) was enhanced by P and/or K deficiency of the trees, and ECM biomass in the roots was increased by P deficiency. Thus, PK deficiency in drained peatlands may increase the allocation of C by the tree to ECMs. It was also observed that fungi can alter N mineralization processes in the rhizosphere but variously depending on fungal species and fertility level of peat. Gross N mineralization did not vary but the net N mineralization rate significantly increased along the N gradient in both field and laboratory experiments. Gross N immobilization also significantly increased when the peat N concentration increased. Nitrification was hardly detectable in either field or laboratory experiments. During the growing season, dissolved inorganic N (DIN) fluctuated much more than the relatively stable DON. Special methodological challenges associated with sampling and analysis in microbial studies on peatlands are discussed.

Microbial communities in Pb contaminated boreal forest soil

Lead contamination in the environment is of particular concern, as it is a known toxin. Until recently, however, much less attention has been given to the local contamination caused by activities at shooting ranges compared to large-scale industrial contamination. In Finland, more than 500 tons of Pb is produced each year for shotgun ammunition. The contaminant threatens various organisms, ground water and the health of human populations. However, the forest at shooting ranges usually shows no visible sign of stress compared to nearby clean environments. The aboveground biota normally reflects the belowground ecosystem. Thus, the soil microbial communities appear to bear strong resistance to contamination, despite the influence of lead. The studies forming this thesis investigated a shooting range site at Hälvälä in Southern Finland, which is heavily contaminated by lead pellets. Previously it was experimentally shown that the growth of grasses and degradation of litter are retarded. Measurements of acute toxicity of the contaminated soil or soil extracts gave conflicting results, as enchytraeid worms used as toxicity reporters were strongly affected, while reporter bacteria showed no or very minor decreases in viability. Measurements using sensitive inducible luminescent reporter bacteria suggested that the bioavailability of lead in the soil is indeed low, and this notion was supported by the very low water extractability of the lead. Nevertheless, the frequency of lead-resistant cultivable bacteria was elevated based on the isolation of cultivable strains. The bacterial and fungal diversity in heavily lead contaminated shooting sectors were compared with those of pristine sections of the shooting range area. The bacterial 16S rRNA gene and fungal ITS rRNA gene were amplified, cloned and sequenced using total DNA extracted from the soil humus layer as the template. Altogether, 917 sequenced bacterial clones and 649 sequenced fungal clones revealed a high soil microbial diversity. No effect of lead contamination was found on bacterial richness or diversity, while fungal richness and diversity significantly differed between lead contaminated and clean control areas. However, even in the case of fungi, genera that were deemed sensitive were not totally absent from the contaminated area: only their relative frequency was significantly reduced. Some operational taxonomic units (OTUs) assigned to Basidiomycota were clearly affected, and were much rarer in the lead contaminated areas. The studies of this thesis surveyed EcM sporocarps, analyzed morphotyped EcM root tips by direct sequencing, and 454-pyrosequenced fungal communities in in-growth bags. A total of 32 EcM fungi that formed conspicuous sporocarps, 27 EcM fungal OTUs from 294 root tips, and 116 EcM fungal OTUs from a total of 8 194 ITS2 454 sequences were recorded. The ordination analyses by non-parametric multidimensional scaling (NMS) indicated that Pb enrichment induced a shift in the EcM community composition. This was visible as indicative trends in the sporocarp and root tip datasets, but explicitly clear in the communities observed in the in-growth bags. The compositional shift in the EcM community was mainly attributable to an increase in the frequencies of OTUs assigned to the genus Thelephora, and to a decrease in the OTUs assigned to Pseudotomentella, Suillus and Tylospora in Pb-contaminated areas when compared to the control. The enrichment of Thelephora in contaminated areas was also observed when examining the total fungal communities in soil using DNA cloning and sequencing technology. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem remain undetermined. The results indicate that at the Hälvälä shooting range, lead influences the fungal communities but not the bacterial communities. The forest ecosystem shows apparent functional redundancy, since no significant effects were seen on forest trees. Recently, by means of 454 pyrosequencing , the amount of sequences in a single analysis run can be up to one million. It has been applied in microbial ecology studies to characterize microbial communities. The handling of sequence data with traditional programs is becoming difficult and exceedingly time consuming, and novel tools are needed to handle the vast amounts of data being generated. The field of microbial ecology has recently benefited from the availability of a number of tools for describing and comparing microbial communities using robust statistical methods. However, although these programs provide methods for rapid calculation, it has become necessary to make them more amenable to larger datasets and numbers of samples from pyrosequencing. As part of this thesis, a new program was developed, MuSSA (Multi-Sample Sequence Analyser), to handle sequence data from novel high-throughput sequencing approaches in microbial community analyses. The greatest advantage of the program is that large volumes of sequence data can be manipulated, and general OTU series with a frequency value can be calculated among a large number of samples.

Self-assembly of hydrophobin proteins from the fungus Trichoderma reesei

Hydrophobins are small surface active proteins that are produced by filamentous fungi. The surface activity of hydrophobin proteins leads to the formation of a film at the air-water interface and adsorption to surfaces. The formation of these hydrophobin films and coatings is important in many stages of fungal development. Furthermore, these properties make hydrophobins interesting for potential use in technical applications. The surfactant-like properties of hydrophobins from Trichoderma reesei were studied at the air-water interface, at solid surfaces, and in solution. The hydrophobin HFBI was observed to spontaneously form a cohesive film on a water drop. The film was imaged using atomic force microscopy from both sides, revealing a monomolecular film with a defined molecular structure. The use of hydrophobins as surface immobilization carriers for enzymes was studied using fusion proteins of HFBI or HFBII and an enzyme. Furthermore, sitespecifically modified variants of HFBI were shown to retain their ability to selfassemble at interfaces and to be able to bind a second layer of proteins by biomolecular recognition. In order to understand the function of hydrophobins at interfaces, an understanding of their overall behavior and self-assembly is needed. HFBI and HFBII were shown to associate in solution into dimers and tetramers in a concentration-dependent manner. The association dynamics and protein-protein interactions of HFBI and HFBII were studied using Förster resonance energy transfer and size exclusion chromatography. It was shown that the surface activity of HFBI is not directly dependent on the formation of multimers in solution.

Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.

Multifunctional RNA silencing pathway polymerase QDE-1 of Neurospora crassa

Double-stranded RNA and associated proteins are known to regulate the gene expression of most eukaryotic organisms. These regulation pathways have different components, outcomes and distinct nomenclature depending on the model system, and often they are referred to collectively as RNA silencing. In many cases, RNA-dependent RNA polymerases (RdRPs) are found to be involved in the RNA silencing, but their targets, activities, interaction partners and reaction products remain enigmatic. In the filamentous fungus Neurospora crassa, the RdRP QDE-1 is critical for silencing of transgenes a phenomenon known as quelling. In this thesis the structure, biochemical activities and biological functions of QDE-1 were extensively studied. This dimeric RdRP was shown to possess five distinct catalytic in vitro activities that could be dissected by mutagenesis and by altering reaction conditions. The biochemical characterization implied that QDE-1 is actually an active DNA-dependent RNA polymerase that has additional RdRP activity. It also provided a structural explanation for the dimerization and suggested a biological framework for the functions of QDE-1 in vivo. (I) QDE-1 was also studied in a broader context along with the other components of the quelling pathway. It was shown that DNA damage in Neurospora causes a dramatic increase in the expression level of the Argonaute protein QDE-2 as well as the synthesis of a novel class of small RNAs known as qiRNAs. The accumulation of qiRNAs was shown to be dependent on several quelling components, and particularly to be derived from an aberrant ssRNA (aRNA) molecule that is synthesized by QDE-1 in the nucleus. The genomic distribution of qiRNA targets was analyzed and the possible biological significance of qiRNAs was studied. Importantly, qiRNAs are the first class of small RNAs that are induced by DNA damage. (II) After establishing that QDE-1 is a multifunctional RNA polymerase with several activities, template specificities and subcellular locations, the focus was turned onto its interaction partners. It had been previously known that QDE-1 associates with Replication Protein A (RPA), but the RecQ helicase QDE-3 was now shown to regulate this interaction. RPA was also observed to promote QDE-1 dependent dsRNA synthesis in vitro. By characterizing the interplay between QDE-1, QDE-3 and RPA, a working model of quelling and qiRNA pathways in Neurospora was presented. (III) This work sheds light on the complexity of the various RNA silencing pathways of a fungal model system. It shows how an RdRP can regulate gene expression on many levels, and suggests novel lines of research in other eukaryotic organisms.

Comparative and functional genome analysis of fungi for development of the protein production host Trichoderma reesei

Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies.

The effect of lignin content and lignin modification on Norway spruce wood properties and decay resistance

Three different Norway spruce cutting clones growing in three environments with different soil and climatic conditions were studied. The purpose was to follow variation in the radial growth rate, wood properties and lignin content and to modify wood lignin with a natural monolignol, coniferyl alcohol, by making use of inherent wood peroxidases. In addition, the incorporation of chlorinated anilines into lignin was studied with synthetic model compounds and synthetic lignin preparations to show whether unnatural compounds originating from pesticides could be bound in the lignin polymer. The lignin content of heartwood, sapwood and earlywood was determined by applying Fourier transform infrared (FTIR) spectroscopy and a principal component regression (PCR) technique. Wood blocks were treated with coniferyl alcohol by using a vacuum impregnation method. The effect of impregnation was assessed by FTIR and by a fungal decay test. Trees from a fertile site showed the highest growth rate and sapwood lignin content and the lowest latewood proportion, weight density and modulus of rupture (MOR). Trees from a medium fertile site had the lowest growth rate and the highest latewood proportion, weight density, modulus of elasticity (MOE) and MOR. The most rapidly growing clone showed the lowest latewood proportion, weight density, MOE and MOR. The slowest growing clone had the lowest sapwood lignin content and the highest latewood proportion, weight density, MOE and MOR. Differences between the sites and clones were small, while fairly large variation was found between the individual trees and growing seasons. The cutting clones maintained clone-dependent wood properties in the different growing sites although variation between trees was high and climatic factors affected growth. The coniferyl alcohol impregnation increased the content of different lignin-type phenolic compounds in the wood as well as wood decay resistance against a white-rot fungus, Coriolus versicolor. During the synthetic lignin preparation 3,4-dichloroaniline became bound by a benzylamine bond to β-O-4 structures in the polymer and it could not be released by mild acid hydrolysis. The natural monolignol, coniferyl alcohol, and chlorinated anilines could be incorporated into the lignin polymer in vivo and in vitro, respectively.

Novel Molecular Mechanisms of Arabidopsis Disease Resistance

Plants are capable of recognizing phytopathogens through the perception of pathogen-derived molecules or plant cell-wall degradation products due to the activities of pathogen-secreted enzymes. Such elicitor recognition events trigger an array of inducible defense responses involving signal transduction networks and massive transcriptional re-programming. The outcome of a pathogen infection relies on the balance between different signaling pathways, which are integrated by regulatory proteins. This thesis characterized two key regulatory components: a damage control enzyme, chlorophyllase 1 (AtCHL1), and a transcription factor, WRKY70. Their roles in defense signaling were then investigated. The Erwinia-derived elicitors rapidly activated the expression of AtCLH1 and WRKY70 through different signaling pathways. The expression of the AtCHL1 gene was up-regulated by jasmonic acid (JA) but down-regulated by salicylic acid (SA), whereas WRKY70 was activated by SA and repressed by JA. In order to elucidate the functions of AtCLH1 and WRKY70 in plant defense, stable transgenic lines were produced where these genes were overexpressed or silenced. Additionally, independent knockout lines were also characterized. Bacterial and fungal pathogens were then used to assess the contribution of these genes to the Arabidopsis disease resistance. The transcriptional modulation of AtCLH1 by either the constitutive over-expression or RNAi silencing caused alterations in the chlorophyll-to-chlorophyllide ratio, supporting the claim that chlorophyllase 1 has a role in the chlorophyll degradation pathway. Silencing of this gene led to light-dependent over-accumulation of the reactive oxygen species (ROS) in response to infection by Erwinia carotovora subsp. carotovora SCC1. This was followed by an enhanced induction of SA-dependent defense genes and an increased resistance to this pathogen. Interestingly, little effect on the pathogen-induced SA accumulation at the early infection was observed, suggesting that action of ROS might potentiate SA signaling. In contrast, the pathogen-induced JA production was significantly reduced in the RNAi silenced plants. Moreover, JA signaling and resistance to Alternaria brassicicola were impaired. These observations provide support for the argument that the ROS generated in chloroplasts might have a negative impact on JA signaling. The over-expression of WRKY70 resulted in an enhanced resistance to E. carotovora subsp. carotovora SCC1, Pseudomonas syringae pv. tomato DC3000 and Erysiphe cichoracearum UCSC1, whilst an antisense suppression or an insertional inactivation of WRKY70 led to a compromised resistance to E. carotovora subsp. carotovora SCC1 and to E. cichoracearum UCSC1 but not to P. syringae pv. tomato DC3000. Gene expression analysis revealed that WRKY70 activated many known defense-related genes associated with the SAR response but suppressed a subset of the JA-responsive genes. In particular, I was able to show that both the basal and the induced expression of AtCLH1 was enhanced by the antisense silencing or the insertional inactivation of WRKY70, whereas a reduction in AtCLH1 expression was observed in the WRKY70 over-expressors following an MeJA application or an A. brassicicola infection. Moreover, the SA-induced suppression of AtCLH1 was relieved in wrky70 mutants. These results indicate that WRKY70 down-regulates AtCLH1. An epistasis analysis suggested that WRKY70 functions downstream of the NPR1 in an SA-dependent signaling pathway. When challenged with A. brassicicola, WRKY70 over-expressing plants exhibited a compromised disease resistance while wrky70 mutants had the opposite effect. These results confirmed the WRKY70-mediated inhibitory effects on JA signaling. Furthermore, the WRKY70-controlled suppression of A. brassicicola resistance was mainly through an NPR1-dependent mechanism. Taking all the data together, I suggest that the pathogen-responsive transcription factor WRKY70 is a common component in both SA- and JA-dependent pathways and plays a crucial role in the SA-mediated suppression of JA signaling.

Damp building moulds: Assessment of sensitization in patients and studies into mechanisms of airway inflammation using experimental models

Exposure to water-damaged buildings and the associated health problems have evoked concern and created confusion during the past 20 years. Individuals exposed to moisture problem buildings report adverse health effects such as non-specific respiratory symptoms. Microbes, especially fungi, growing on the damp material have been considered as potential sources of the health problems encountered in these buildings. Fungi and their airborne fungal spores contain allergens and secondary metabolites which may trigger allergic as well as inflammatory types of responses in the eyes and airways. Although epidemiological studies have revealed an association between damp buildings and health problems, no direct cause-and-effect relationship has been established. Further knowledge is needed about the epidemiology and the mechanisms leading to the symptoms associated with exposure to fungi. Two different approaches have been used in this thesis in order to investigate the diverse health effects associated with exposure to moulds. In the first part, sensitization to moulds was evaluated and potential cross-reactivity studied in patients attending a hospital for suspected allergy. In the second part, one typical mould known to be found in water-damaged buildings and to produce toxic secondary metabolites was used to study the airway responses in an experimental model. Exposure studies were performed on both naive and allergen sensitized mice. The first part of the study showed that mould allergy is rare and highly dependent on the atopic status of the examined individual. The prevalence of sensitization was 2.7% to Cladosporium herbarum and 2.8% to Alternaria alternata in patients, the majority of whom were atopic subjects. Some of the patients sensitized to mould suffered from atopic eczema. Frequently the patients were observed to possess specific serum IgE antibodies to a yeast present in the normal skin flora, Pityrosporum ovale. In some of these patients, the IgE binding was partly found to be due to binding to shared glycoproteins in the mould and yeast allergen extracts. The second part of the study revealed that exposure to Stachybotrys chartarum spores induced an airway inflammation in the lungs of mice. The inflammation was characterized by an influx of inflammatory cells, mainly neutrophils and lymphocytes, into the lungs but with almost no differences in airway responses seen between the satratoxin producing and non-satratoxin producing strain. On the other hand, when mice were exposed to S. chartarum and sensitized/challenged with ovalbumin the extent of the inflammation was markedly enhanced. A synergistic increase in the numbers of inflammatory cells was seen in BAL and severe inflammation was observed in the histological lung sections. In conclusion, the results in this thesis imply that exposure to moulds in water damaged buildings may trigger health effects in susceptible individuals. The symptoms can rarely be explained by IgE mediated allergy to moulds. Other non-allergic mechanisms seem to be involved. Stachybotrys chartarum is one of the moulds potentially responsible for health problems. In this thesis, new reaction models for the airway inflammation induced by S. chartarum have been found using experimental approaches. The immunological status played an important role in the airway inflammation, enhancing the effects of mould exposure. The results imply that sensitized individuals may be more susceptible to exposure to moulds than non-sensitized individuals.

Microbiological, environmental and proteolytic aspects in chronic rhinosinusitis with nasal polyposis

Chronic rhinosinusitis is one of the most common chronic respiratory tract diseases affecting up to 15% of the adult population in the Western world. It may be perpetuated by factors predisposing to sinus ostial obstruction together with inflammatory changes in the sinus mucosa. Chronic rhinosinusitis is associated with asthma, and it may represent the same disease process. Chronic rhinosinusitis with nasal polyposis (CRSwNP) and asthma share also the characteristic inflammatory features and histopathologic feature of airway remodelling. Remodelling is considered as a key event in the pathogenesis of asthma. It is controlled by a delicate balance between the matrix metalloproteinases (MMPs) and their regulators. The purpose of the present study was to evaluate the microbiological findings, inflammatory features and MMP and tissue inhibitor of metalloproteinases-1 (TIMP-1) expression in CRSwNP. The results were related to the patient history, exposure to moisture and clinical outcome in order to find out possible explanations for the etiology and chronicity of CRSwNP. Bacterial culture results were similar in patients and in controls and do not explain the chronic course of CRSwNP. The presence of fungi seems to be more common in CRSwNP than chronic rhinosinusitis in general, and they should be actively searched for using microbiological as well as histological methods. Typical outdoor fungal species were found in nasal lavage samples taken from controls in the autumn but not in the winter, reflecting environmental exposure. Exposure to moisture was reported by 46% of the CRSwNP patients, which is in accordance to the Finnish general population. Exposed patients did not differ significantly from non-exposed subjects with regards to microbiological findings, tissue eosinophilia and clinical outcome. Significantly elevated levels of collagenase-2 (MMP-8) and interleukin (IL)-8 but not tumour necrosis factor-α were found in CRSwNP patients. In particular, the activation of mesenchymal-type MMP-8 but not polymorphonuclear-type MMP-8 was associated with elevated IL-8 levels. IL-8 and MMP-8 may form an inductive cytokine-proteinase cascade in CRSwNP pathogenesis and provide a target for novel therapies and a diagnostic tool for monitoring CRSwNP treatment. The proteolytic spectrum is different in eosinophilic and non-eosinophilic CRSwNP with the up-regulation of MMP-8 and MMP-9 in non-eosinophilic CRSwNP, suggesting different pathophysiology in these subgroups. The lack of MMP up-regulation was associated with a poor prognostic factor and worse clinical outcome, representing a possible synergic anti-inflammatory function of MMP-8 and MMP-9 in CRSwNP. This study provides new information about possible immunologic mechanisms in the pathogenesis of CRSwNP. The recently discovered anti-inflammatory/ defensive properties of MMP-8 and MMP-9 in animal models are reported for the first time in a clinical setting in human inflammatory diseases.

Characterization of hydrophobin proteins at interfaces and in solutions using x rays

Hydrophobins are a group of particularly surface active proteins. The surface activity is demonstrated in the ready adsorption of hydrophobins to hydrophobic/hydrophilic interfaces such as the air/water interface. Adsorbed hydrophobins self-assemble into ordered films, lower the surface tension of water, and stabilize air bubbles and foams. Hydrophobin proteins originate from filamentous fungi. In the fungi the adsorbed hydrophobin films enable the growth of fungal aerial structures, form protective coatings and mediate the attachment of fungi to solid surfaces. This thesis focuses on hydrophobins HFBI, HFBII, and HFBIII from a rot fungus Trichoderma reesei. The self-assembled hydrophobin films were studied both at the air/water interface and on a solid substrate. In particular, using grazing-incidence x-ray diffraction and reflectivity, it was possible to characterize the hydrophobin films directly at the air/water interface. The in situ experiments yielded information on the arrangement of the protein molecules in the films. All the T. reesei hydrophobins were shown to self-assemble into highly crystalline, hexagonally ordered rafts. The thicknesses of these two-dimensional protein crystals were below 30 Å. Similar films were also obtained on silicon substrates. The adsorption of the proteins is likely to be driven by the hydrophobic effect, but the self-assembly into ordered films involves also specific protein-protein interactions. The protein-protein interactions lead to differences in the arrangement of the molecules in the HFBI, HFBII, and HFBIII protein films, as seen in the grazing-incidence x-ray diffraction data. The protein-protein interactions were further probed in solution using small-angle x-ray scattering. Both HFBI and HFBII were shown to form mainly tetramers in aqueous solution. By modifying the solution conditions and thereby the interactions, it was shown that the association was due to the hydrophobic effect. The stable tetrameric assemblies could tolerate heating and changes in pH. The stability of the structure facilitates the persistence of these secreted proteins in the soil.

Species-specific and Indication-based Use of Antimicrobials in Dogs, Cats, Cattle and Horses in Finland : Data collected using three different methods

Increasing antimicrobial resistance in bacteria has led to the need for better understanding of antimicrobial usage patterns. In 1999, the World Organisation for Animal Health (OIE) recommended that an international ad hoc group should be established to address human and animal health risks related to antimicrobial resistance and the contribution of antimicrobial usage in veterinary medicine. In European countries the need for continuous recording of the usage of veterinary antimicrobials as well as for animal species-specific and indication-based data on usage has been acknowledged. Finland has been among the first countries to develop prudent use guidelines in veterinary medicine, as the Ministry of Agriculture and Forestry issued the first animal species-specific indication-based recommendations for antimicrobial use in animals in 1996. These guidelines have been revised in 2003 and 2009. However, surveillance on the species-specific use of antimicrobials in animals has not been performed in Finland. This thesis provides animal species-specific information on indication-based antimicrobial usage. Different methods for data collection have been utilized. Information on antimicrobial usage in animals has been gathered in four studies (studies A-D). Material from studies A, B and C have been used in an overlapping manner in the original publications I-IV. Study A (original publications I & IV) presents a retrospective cross-sectional survey on prescriptions for small animals at the Veterinary Teaching Hospital of the University of Helsinki. Prescriptions for antimicrobial agents (n = 2281) were collected and usage patterns, such as the indication and length of treatment, were reviewed. Most of the prescriptions were for dogs (78%), and primarily for the treatment of skin and ear infections most of which were treated with cephalexin for a median period of 14 days. Prescriptions for cats (18%) were most often for the treatment of urinary tract infections with amoxicillin for a median length of 10 days. Study B (original publication II) was a retrospective cross-sectional survey where prescriptions for animals were collected from 17 University Pharmacies nationwide. Antimicrobial prescriptions (n = 1038) for mainly dogs (65%) and cats (19%) were investigated. In this study, cephalexin and amoxicillin were also the most frequently used drugs for dogs and cats, respectively. In study C (original publications III & IV), the indication-based usage of antimicrobials of practicing veterinarians was analyzed by using a prospective questionnaire. Randomly selected practicing veterinarians in Finland (n = 262) recorded all their antimicrobial usage during a 7-day study period. Cattle (46%) with mastitis were the most common patients receiving antimicrobial treatment, generally intramuscular penicillin G or intramammary treatment with ampicillin and cloxacillin. The median length of treatment was four days, regardless of the route of administration. Antimicrobial use in horses was evaluated in study D, the results of which are previously unpublished. Firstly, data collected with the prospective questionnaire from the practicing veterinarians showed that horses (n = 89) were frequently treated for skin or wound infections by using penicillin G or trimethoprim-sulfadiazine. The mean duration of treatment was five to seven days. Secondly, according to retrospective data collected from patient records, horses (n = 74) that underwent colic surgery at the Veterinary Teaching Hospital of the University of Helsinki were generally treated according to national and hospital recommendations; penicillin G and gentamicin was administered preoperatively and treatment was continued for a median of three days postoperatively. In conclusion, Finnish veterinarians followed well the national prudent use guidelines. Narrow-spectrum antimicrobials were preferred and, for instance, fluoroquinolones were used sparingly. Prescription studies seemed to give good information on antimicrobials usage, especially when combined with complementary information from patient records. A prospective questionnaire study provided a fair amount of valuable data on several animal species. Electronic surveys are worthwhile exploiting in the future.

Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of β-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of β-casein resulted in the formation of high molecular mass (MM ca. 1 350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial β-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91 % of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of β-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked β-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked β-casein was more resistant to pepsin digestion when compared to that of non modified β-casein. The effects of enzymatic cross-linking of β-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native β-casein by 14 % when cross-linked by TrTyr and by 6 % after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.

The effects of abiotic and biotic factors on somatic embryogenesis and seedlings of Pinus sylvestris (L.)

Somatic embryogenesis (SE) is an asexual form of plant propagation that occurs in nature and mimics many of the events of sexual reproduction. Pinus sylvestris (L.) is an important source of timber in Northern Eurasia but it is recalcitrant to somatic embryogenesis. Several factors important for the success of the P. sylvestris embryogenic cultures have not been thoroughly investigated. In this study, we examined the effects of parental genotypes on the SE in P. sylvestris, the involvement of the gaseous plant growth regulator, ethylene in SE, and also biotic effects on somatic embryos as well as on seedlings. We tested parental effects on immature embryo initiation for different media, storage periods, and on the maturation process. Maternal effects were found to be crucial for SE in the absence of paternal effects. No maternal-paternal interaction was observed at any stage of somatic embryo production. Additionally the role of ethylene at different developmental stages of SE was investigated. Two ACC synthase genes, PsACS1 and PsACS2, were isolated and characterized. PsACS1 was expressed during the proliferation stage in all tested genotypes, whereas PsACS2 was only expressed in somatic embryos of each genotype. Ethylene production in embryos at stage 3 was significantly higher than the other stages. In a parallel study, the response of somatic embryos to fungal elicitors was investigated. Three fungi, a mutualistic ectomycorrhizal (ECM) fungus (Suillus bovinus), a weak Scots pine pathogen (Heterobasidion parviporum) and a strong pathogen (H. annosum) were used. The gene expression patterns for embryos exposed to the H. parviporum elicitor were found to be similar to that documented for S. bovinus among the tested genes. By contrast somatic embryos exposed to the H. annosum elicitor had a different pattern of regulation which was marked by a delayed response, and in some cases death of the embryos. Furthermore, interaction without direct contact between P. sylvestris seedlings and microbes (mutualistic and pathogenic fungus, cyanobacterium) were investigated. Several novel genes expressed in seedlings treated with ECM fungus were isolated which suggested that physical contact is not necessary for elicitation of host responses. The results suggest that somatic embryos and seedlings of P. sylvestris are genetically well equipped to respond to fungal elicitor/exudates and could serve as a suitable model for reproducible molecular studies in conifer tree patho- and symbiotic systems.