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  • Savola, Suvi (Helsingin yliopisto, 2009)
    Background: The Ewing sarcoma family of tumors (ESFT) are rare but highly malignant neoplasms that occur mainly in bone or but also in soft tissue. ESFT affects patients typically in their second decade of life, whereby children and adolescents bear the heaviest incidence burden. Despite recent advances in the clinical management of ESFT patients, their prognosis and survival are still disappointingly poor, especially in cases with metastasis. No targeted therapy for ESFT patients is currently available. Moreover, based merely on current clinical and biological characteristics, accurate classification of ESFT patients often fails at the time of diagnosis. Therefore, there is a constant need for novel molecular biomarkers to be applied in tandem with conventional parameters to further intensify ESFT risk-stratification and treatment selection, and ultimately to develop novel targeted therapies. In this context, a greater understanding of the genetics and immune characteristics of ESFT is needed. Aims: This study sought to open novel insights into gene copy number changes and gene expression in ESFT and, further, to enlighten the role of inflammation in ESFT. For this purpose, microarrays were used to provide gene-level information on a genomewide scale. In addition, this study focused on screening of 9p21.3 deletion sizes and frequencies in ESFT and, in another pediatric cancer, acute lymphocytic leukemia (ALL), in order to define more exact criteria for highrisk patient selection and to provide data for developing a more reliable diagnostic method to detect CDKN2A deletions. Results: In study I, 20 novel ESFT-associated suppressor genes and oncogenes were pinpointed using combined array CGH and expression analysis. In addition, interesting chromosomal rearrangements were identified: (1) Duplication of derivative chromosome der(22)(11;22) was detected in three ESFT patients. This duplication included the EWSR1-FLI1 fusion gene leading to increase in its copy number; (2) Cryptic amplifications on chromosomes 20 and 22 were detected, suggesting a novel translocation between chromosomes 20 and 22, which most probably produces a fusion between EWSR1 and NFATC2. In study II, bioinformatic analysis of ESFT expression profiles showed that inflammatory gene activation is detectable in ESFT patient samples and that the activation is characterized by macrophage gene expression. Most interestingly, ESFT patient samples were shown to express certain inflammatory genes that were prognostically significant. High local expression of C5 and JAK1 at the tumor site was shown to associate with favorable clinical outcome, whereas high local expression of IL8 was shown to be detrimental. Studies III and IV showed that the smallest overlapping region of deletion in 9p21.3 includes CDKN2A in all cases and that the length of this region is 12.2 kb in both Ewing sarcoma and ALL. Furthermore, our results showed that the most widely used commercial CDKN2A FISH probe creates false negative results in the narrowest microdeletion cases (<190 kb). Therefore, more accurate methods should be developed for the detection of deletions in the CDKN2A locus. Conclusions: This study provides novel insights into the genetic changes involved in the biology of ESFT, in the interaction between ESFT cells and immune system, and in the inactivation of CDKN2A. Novel ESFT biomarker genes identified in this study serve as a useful resource for future studies and in developing novel therapeutic strategies to improve the survival of patients with ESFT.
  • Jääskeläinen, Anne (Helsingin yliopisto, 2008)
    Currently, there are nine known human herpesviruses and these viruses appear to have been a very common companion of humans throughout the millenia. Of human herpesviruses, herpes simplex viruses 1 and 2 (HSV-1, HSV-2), causative agents of herpes labialis and genital herpes, and varicella-zoster virus (VZV), causative agent of chicken pox, are also common causes of central nervous system (CNS) infections. In addition, human cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7), all members of the herpesvirus family, can also be associated with encephalitis and meningitis. Accurate diagnostics and fast treatment are essential for patient recovery in CNS infections and therefore sensitive and effective diagnostic methods are needed. The aim of this thesis was to develop new potential detection methods for diagnosing of human herpesvirus infections, especially in immunocompetent patients, using the microarray technique. Therefore, methods based on microarrays were developed for simultaneous detection of HSV-1, HSV-2, VZV, CMV, EBV, HHV-6A, HHV-6B, and HHV-7 nucleic acids, and for HSV-1, HSV-2, VZV, and CMV antibodies from various clinical samples. The microarray methods developed showed potential for efficiently and accurately detecting human herpesvirus DNAs, especially in CNS infections, and for simultaneous detection of DNAs or antibodies for multiple different human herpesviruses from clinical samples. In fact, the microarray method revealed several previously unrecognized co-infections. The microarray methods developed were sensitive and provided rapid detection of human herpesvirus DNA, and therefore the method could be applied to routine diagnostics. The microarrays might also be considered as an economical tool for diagnosing human herpesvirus infections.
  • Veckman, Ville (Helsingin yliopisto, 2007)
    Human body is in continuous contact with microbes. Although many microbes are harmless or beneficial for humans, pathogenic microbes possess a threat to wellbeing. Antimicrobial protection is provided by the immune system, which can be functionally divided into two parts, namely innate and adaptive immunity. The key players of the innate immunity are phagocytic white blood cells such as neutrophils, monocytes, macrophages and dendritic cells (DCs), which constantly monitor the blood and peripheral tissues. These cells are armed for rapid activation upon microbial contact since they express a variety of microbe-recognizing receptors. Macrophages and DCs also act as antigen presenting cells (APCs) and play an important role in the development of adaptive immunity. The development of adaptive immunity requires intimate cooperation between APCs and T lymphocytes and results in microbe-specific immune responses. Moreover, adaptive immunity generates immunological memory, which rapidly and efficiently protects the host from reinfection. Properly functioning immune system requires efficient communication between cells. Cytokines are proteins, which mediate intercellular communication together with direct cell-cell contacts. Immune cells produce inflammatory cytokines rapidly following microbial contact. Inflammatory cytokines modulate the development of local immune response by binding to cell surface receptors, which results in the activation of intracellular signalling and modulates target cell gene expression. One class of inflammatory cytokines chemokines has a major role in regulating cellular traffic. Locally produced inflammatory chemokines guide the recruitment of effector cells to the site of inflammation during microbial infection. In this study two key questions were addressed. First, the ability of pathogenic and non-pathogenic Gram-positive bacteria to activate inflammatory cytokine and chemokine production in different human APCs was compared. In these studies macrophages and DCs were stimulated with pathogenic Steptococcus pyogenes or non-pathogenic Lactobacillus rhamnosus. The second aim of this thesis work was to analyze the role of pro-inflammatory cytokines in the regulation of microbe-induced chemokine production. In these studies bacteria-stimulated macrophages and influenza A virus-infected lung epithelial cells were used as model systems. The results of this study show that although macrophages and DCs share several common antimicrobial functions, these cells have significantly distinct responses against pathogenic and non-pathogenic Gram-positive bacteria. Macrophages were activated in a nearly similar fashion by pathogenic S. pyogenes and non-pathogenic L. rhamnosus. Both bacteria induced the production of similar core set of inflammatory chemokines consisting of several CC-class chemokines and CXCL8. These chemokines attract monocytes, neutrophils, dendritic cells and T cells. Thus, the results suggest that bacteria-activated macrophages efficiently recruit other effector cells to the site of inflammation. Moreover, macrophages seem to be activated by all bacteria irrespective of their pathogenicity. DCs, in contrast, were efficiently activated only by pathogenic S. pyogenes, which induced DC maturation and production of several inflammatory cytokines and chemokines. In contrast, L. rhamnosus-stimulated DCs matured only partially and, most importantly, these cells did not produce inflammatory cytokines or chemokines. L. rhamnosus-stimulated DCs had a phenotype of "semi-mature" DCs and this type of DCs have been suggested to enhance tolerogenic adaptive immune responses. Since DCs have an essential role in the development of adaptive immune response the results suggest that, in contrast to macrophages, DCs may be able to discriminate between pathogenic and non-pathogenic bacteria and thus mount appropriate inflammatory or tolerogenic adaptive immune response depending on the microbe in question. The results of this study also show that pro-inflammatory cytokines can contribute to microbe-induced chemokine production at multiple levels. S. pyogenes-induced type I interferon (IFN) was found to enhance the production of certain inflammatory chemokines in macrophages during bacterial stimulation. Thus, bacteria-induced chemokine production is regulated by direct (microbe-induced) and indirect (pro-inflammatory cytokine-induced) mechanisms during inflammation. In epithelial cells IFN- and tumor necrosis factor- (TNF-) were found to enhance the expression of PRRs and components of cellular signal transduction machinery. Pre-treatment of epithelial cells with these cytokines prior to virus infection resulted in markedly enhanced chemokine response compared to untreated cells. In conclusion, the results obtained from this study show that pro-inflammatory cytokines can enhance microbe-induced chemokine production during microbial infection by providing a positive feedback loop. In addition, pro-inflammatory cytokines can render normally low-responding cells to high chemokine producers via enhancement of microbial detection and signal transduction.
  • Pietilä, Taija (Helsingin yliopisto, 2010)
    Two types of antigen-presenting cells (APCs), macrophages and dendritic cells (DCs), function at the interface of innate and adaptive immunity. Through recognition of conserved microbial patterns, they are able to detect the invading pathogens. This leads to activation of signal transduction pathways that in turn induce gene expression of various molecules required for immune responses and eventually pathogen clearance. Cytokines are among the genes induced upon detection of microbes. They play an important role in regulating host immune responses during microbial infection. Chemotactic cytokines, chemokines, are involved in migratory events of immune cells. Cytokines also promote the differentiation of distinct T cell responses. Because of the multiple roles of cytokines in the immune system, the cytokine network needs to be tightly regulated. In this work, the induction of innate immune responses was studied using human primary macrophages or DCs as cell models. Salmonella enterica serovar Typhimurium served as a model for an intracellular bacterium, whereas Sendai virus was used in virus experiments. The starting point of this study was that DCs of mouse origin had recently been characterized as host cells for Salmonella. However, only little was known about the immune responses initiated in Salmonella-infected human DCs. Thus, cellular responses of macrophages and DCs, in particular the pattern of cytokine production, to Salmonella infection were compared. Salmonella-induced macrophages and DCs were found to produce multiple cytokines including interferon (IFN) -gamma, which is conventionally produced by T and natural killer (NK) cells. Both macrophages and DCs also promoted the intracellular survival of the bacterium. Phenotypic maturation of DCs as characterized by upregulation of costimulatory and human leukocyte antigen (HLA) molecules, and production of CCL19 chemokine, were also detected upon infection with Salmonella. Another focus of this PhD work was to unravel the regulatory events controlling the expression of cytokine genes encoding for CCL19 and type III IFNs, which are central to DC biology. We found that the promoters of CCL19 and type III IFNs contain similar regulatory elements that bind nuclear factor kappaB (NF-kappaB) and interferon regulatory factors (IRFs), which could mediate transcriptional activation of the genes. The regulation of type III IFNs in virus infection resembled that of type I IFNs a cytokine class traditionally regarded as antiviral. The induction of type I and type III IFNs was also observed in response to bacterial infection. Taken together, this work identifies new details about the interaction of Salmonella with its phagocytic host cells of human origin. In addition, studies provide information on the regulatory events controlling the expression of CCL19 and the most recently identified IFN family genes, type III IFN genes.
  • Laitila, Arja (Helsingin yliopisto, 2007)
    Microbes have a decisive role in the barley-malt-beer chain. A major goal of this thesis was to study the relationships between microbial communities and germinating grains during malting. Furthermore, the study provided a basis for tailoring of malt properties with natural, malt-derived microbes. The malting ecosystem is a dynamic process, exhibiting continous change. The first hours of steeping and kilning were the most important steps in the process with regard to microbiological quality. The microbial communities consisting of various types of bacteria, yeasts and filamentous fungi formed complex biofilms in barley tissues and were well-protected. Inhibition of one microbial population within the complex ecosystem led to an increase of non-suppressed populations, which must be taken into account because a shift in microbial community dynamics may be undesirable. Both bacterial and fungal communities should be monitored simultaneously. Using different molecular approaches we showed that the diversity of microbes in the malting ecosystem was greater than expected. Even some new microbial groups were found in the malting ecosystem. Suppression of Gram-negative bacteria during steeping was advanategous for grain germination and malt brewhouse performance. Fungal communities including both filamentous fungi and yeasts significantly contributed to the production of microbial beta-glucanases and xylanases, and were also involved in proteolysis. Well-characterized lactic acid bacteria (Lactobacillus plantarum VTT E-78076 and Pediococcus pentosaceus VTT E-90390) proved to be an effective way of balancing the microbial communities in malting. Furthermore, they had positive effects on malt characteristics and notably improved wort separation. Previously the significance of yeasts in the malting ecosystem has been largely underestimated. This study showed that yeast community was an important part of the industrial malting ecosystem. Yeasts produced extracellular hydrolytic enzymes with a potentially positive contribution to malt processability. Furthermore, several yeasts showed strong antagonistic activity against field and storage moulds. Addition of a selected yeast culture (Pichia anomala VTT C-04565) into steeping restricted Fusarium growth and hydrophobin production and thus prevented beer gushing. Addition of P. anomala C565 into steeping water tended to retard wort filtration, but the filtration was improved when the yeast culture was combined with L. plantarum E76. The combination of different microbial cultures offers a possibility to use ther different properties, thus making the system more robust. Improved understanding of complex microbial communities and their role in malting enables a more controlled process management and the production of high quality malt with tailored properties
  • Kurola, Jukka (Helsingin yliopisto, 2006)
    This thesis deals with the response of biodegradation of selected anthropogenic organic contaminants and natural autochthonous organic matter to low temperature in boreal surface soils. Furthermore, the thesis describes activity, diversity and population size of autotrophic ammonia-oxidizing bacteria (AOB) in a boreal soil used for landfarming of oil-refinery wastes, and presents a new approach, in which the particular AOB were enriched and cultivated in situ from the landfarming soil onto cation exchange membranes. This thesis demonstrates that rhizosphere fraction of natural forest humus soil and agricultural clay loam soil from Helsinki Metropolitan area were capable of degrading of low to moderate concentrations (0.2 50 µg cm-3) of PCP, phenanthrene and 2,4,5-TCP at temperatures realistic to boreal climate (-2.5 to +15 °C). At the low temperatures, the biodegradation of PCP, phenanthrene and 2,4,5-TCP was more effective (Q10-values from 1.6 to 7.6) in the rhizosphere fraction of the forest soil than in the agricultural soil. Q10-values of endogenous soil respiration (carbon dioxide evolution) and selected hydrolytic enzyme activities (acetate-esterase, butyrate-esterase and β-glucosidase) in acid coniferous forest soil were 1.6 to 2.8 at temperatures from -3 to +30 °C. The results indicated that the temperature dependence of decomposition of natural autochthonous soil organic matter in the studied coniferous forest was only moderate. The numbers of AOB in the landfarming (sandy clay loam) soil were determined with quantitative polymerase chain reaction (real-time PCR) and with Most Probable Number (MPN) methods, and potential ammonium oxidation activity was measured with the chlorate inhibition technique. The results indicated presence of large and active AOB populations in the heavily oil-contaminated and urea-fertilised landfarming soil. Assessment of the populations of AOB with denaturing gradient gel electrophoresis (DGGE) profiling and sequence analysis of PCR-amplified 16S rRNA genes showed that Nitrosospira-like AOB in clusters 2 and 3 were predominant in the oily landfarming soil. This observation was supported by fluorescence in situ hybridization (FISH) analysis of the AOB grown on the soil-incubated cation-exchange membranes. The results of this thesis expand the suggested importance of Nitrosospira-like AOB in terrestrial environments to include chronically oil-contaminated soils.
  • Hui, Nan (Helsingin yliopisto, 2010)
    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.
  • Yu, Dan (Helsingin yliopisto, 2014)
    An enormous quantity of solid waste is generated annually all over the world. Solid waste can be divided into three main categories: municipal waste, industrial waste and agricultural waste. The focus of the research presented in this thesis was on the biodegradable fraction of municipal solid waste (MSW), and particularly on the biowaste and sewage sludge generated in the Nordic countries. In general, there are two major options for processing biodegradable MSW in a sustainable manner: aerobic treatment (e.g. composting) and anaerobic treatment (e.g. anaerobic digestion). The key interest of this study was in analysing the microbial community composition in composting and anaerobic co-digestion using various types of biodegradable MSW as feedstock. In addition, the aims of this study were to: 1) understand the connections between the microbial communities and the capacities of disease-suppressive composts against the soil-borne plant pathogens Pythium and Fusarium; 2) investigate the effects of pH and microbial composition on odour emission in biowaste composting in Nordic countries; 3) study the connections between microbial communities and the key methanogenesis intermediates under various conditions (e.g. temperature, OLR) in the anaerobic digestion process; and 4) find a functional compromise between the waste treatment capacity of anaerobic digestion, biogas production and a stable microbial community. To achieve the above-mentioned goals, DNA-based microbiological techniques (e.g. DNA extraction, PCR, qPCR, DGGE and cloning) and sequencing techniques (i.e. Sanger sequencing and high-throughput pyrosequencing) were applied. The results are summarized in five articles/manuscripts enclosed with this thesis. Studies on composting (articles I IV) illustrated that the microbial communities were abundant and diverse, with Proteobacteria and Ascomycota as the dominant bacterial and fungal candidates, respectively. The presence of bacterial Acidobacteria Gp14 and fungal Cystobasidiomycetes in Pythium-suppressive composts indicated their possible roles in the suppression of Pythium wilt disease (article I). Actinobacterium and non-pathogenic relatives of the pathogen Fusarium may suppress Fusarium disease on tomato plants (article II). In food waste composting (articles III and IV), results indicated that LAB (e.g. Lactobacillus) together with Clostridia were responsible for the high odour emission in the studied composts. The results suggest that a potential odour reduction strategy would be to rapidly overcome the low pH phase through high initial aeration rates and the use of additives such as bulking material, as well as pre-treatment of the composting feedstock in anaerobic digestion. A study on anaerobic digestion (article V) demonstrated a rather limited methanogenic archaeal community that was dominated by Methanobacteriales and Methanosarcina in both meso- and thermophilic processes, even with Methanothermobacter as an additional abundant genus in the thermophilic production cycle. Key factors such as the acetate concentration and OLR, as well as substrates such as propionate apparently contributed to the dominance of Methanosarcina. Biogas production was greater in the thermophilic process; when the OLR increased to 5 kg VS m-3 d-1, the efficiency maximum was reached. The microbial groups within the microbial community usually remain largely unexplored. In this thesis, the key microbial candidates that are involved in plant disease suppression, composting odour emission and anaerobic co-digestion were revealed. As the rapid progress of high-throughput sequencing approaches continues, better coverage of the microbial community will be achieved.
  • Hultman, Jenni (Helsingin yliopisto, 2009)
    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.
  • Ritari, Jarmo (Helsingin yliopisto, 2012)
    Microbes in natural and artificial environments as well as in the human body are a key part of the functional properties of these complex systems. The presence or absence of certain microbial taxa is a correlate of functional status like risk of disease or course of metabolic processes of a microbial community. As microbes are highly diverse and mostly notcultivable, molecular markers like gene sequences are a potential basis for detection and identification of key types. The goal of this thesis was to study molecular methods for identification of microbial DNA in order to develop a tool for analysis of environmental and clinical DNA samples. Particular emphasis was placed on specificity of detection which is a major challenge when analyzing complex microbial communities. The approach taken in this study was the application and optimization of enzymatic ligation of DNA probes coupled with microarray read-out for high-throughput microbial profiling. The results show that fungal phylotypes and human papillomavirus genotypes could be accurately identified from pools of PCR amplicons generated from purified sample DNA. Approximately 1 ng/μl of sample DNA was needed for representative PCR amplification as measured by comparisons between clone sequencing and microarray. A minimum of 0,25 amol/μl of PCR amplicons was detectable from amongst 5 ng/μl of background DNA, suggesting that the detection limit of the test comprising of ligation reaction followed by microarray read-out was approximately 0,04%. Detection from sample DNA directly was shown to be feasible with probes forming a circular molecule upon ligation followed by PCR amplification of the probe. In this approach, the minimum detectable relative amount of target genome was found to be 1% of all genomes in the sample as estimated from 454 deep sequencing results. Signal-to-noise of contact printed microarrays could be improved by using an internal microarray hybridization control oligonucleotide probe together with a computational algorithm. The algorithm was based on identification of a bias in the microarray data and correction of the bias as shown by simulated and real data. The results further suggest semiquantitative detection to be possible by ligation detection, allowing estimation of target abundance in a sample. However, in practise, comprehensive sequence information of full length rRNA genes is needed to support probe design with complex samples. This study shows that DNA microarray has the potential for an accurate microbial diagnostic platform to take advantage of increasing sequence data and to replace traditional, less efficient methods that still dominate routine testing in laboratories. The data suggests that ligation reaction based microarray assay can be optimized to a degree that allows good signal-tonoise and semiquantitative detection.
  • Kanto Öqvist, Charlotta (Helsingfors universitet, 2008)
    This thesis deals with the roles of microorganisms and different chemicals in the formation of deposits and biofilms at paper and board machines. "Deposit" in this thesis means solid matter that accumulates and immobilizes on machine areas or interfaces meant for unhindered flow of slurries, liquids or air. The deposit is a "biofilm" when microorganisms, or substances produced by them, are its major or otherwise significant building block. The work in this thesis builds on the hypothesis that i.) knowledge on the biotic and abiotic components of the deposit, and ii.) understanding their roles in the build-up, architecture, biological, physico-chemical and technical properties of the deposit will guide the researcher towards preventing or reversing the formation of unwanted deposits in a sustainable way. Multiple analytical tools were used for documenting the buildup of the deposit, including electron microscopy, confocal laser scanning microscopy (CLSM), energy dispersive X-ray (EDX) analysis, pyrolysis gas chromatography - mass spectrometry (Py-GCMS), ion exchange chromatography, gas liquid chromatography and microbiological analyses. I took actively part in developing innovative tools, based on back-scattered light sensoring that can be used for on-line measurement of biofilm in the water loops and containers of paper machines. In the thesis work it was discovered that many of the paper making chemicals interacted forming organic, adhesive layers on steel surfaces in machine circuits. Structures were found, by light microscopy originally judged as microbes, but electron microscopy revealed that they were alum that precipitated as aluminum hydroxide at pH 6.8 in the white water of recycled fiber using machines. Alum is still used as a fixative today among many paper makers, even if the process conditions have changed from acid to neutral pH. It is considered to be the “aspirin” for paper makers; the risk of this was clearly seen in this thesis work. Organic deposits were found, soaps (calcium soaps) of different compounds, like pitch, at the base of the deposit of many paper/board machines. Bacteria, morphologically resembling Deinococcus geothermalis, were demonstrated to grow as colonies firmly attached to the surface of clean stainless steel coupons, immersed in circulation waters at paper machines. Such deinococcal colonies could function as a pedestal, adhesion aid, for later massive attachment of other microorganisms, explaining why deposits frequently contain deinococci as a small, but never as the major building block of paper machine deposits. For assessing the quality of water (raw water, warm water or bio-water) at the paper machines, the measurement method is important. The correlation between cultivation methods and possible contamination of bacteria seen on the coupon surfaces was low, especially when filamentous bacteria were part of the contamination. Environmental concerns have forced paper and board machines to close their circuits. The circulation and reuse of the process waters lead to higher processing temperatures and increase of the colloidal and dissolved material in the circulations. The chemical composition of three different mills was studied, with different final discharges of, 0 m3, 0.5 m3 and 4 m3 waste water per ton product produced, one with reuse of bio-water. The zero discharge mill accumulated high amounts of organic carbon in the circulation waters (> 10 g L-1), including volatile acids (lactic, acetic, propionic and butyric). Contents of sulfate, chloride, sodium and calcium were also high, > 1 g L-1 of each. The major part (40 %) of all identifiable bacterial 16S rRNA gene sequences were closest but yet distantly related (<96 %) to Enterococcus cecorum. In the 4 m3 per ton product discharging mill, additionally Bacillus thermoamylovorans and Bacillus coagulans were found. Slimes and deposits contained high amounts, ≥108 g-1, of archaea, but only one genus, Methanothrix had a sequence match close enough for identification. The results showed that closing the water circuits strongly limited the diversity of the mill microbiota but allowed efficient mineralization of the dissolved and suspended matter.
  • Poutanen, Marjo (Helsingin yliopisto, 2008)
    Proteomics provides detailed descriptions of the structure and function of biological systems in different biological conditions. Although proteomics as a field is relatively new, many methodologies in proteomics have been under development for decades. Proteome analyses are often accomplished by a combination of two-dimensional gel electrophoresis (2-DE) for protein separation and visualization, and mass spectrometry (MS) for protein identification. A proteomic experiment often aims to analyse as many proteins as possible in a proteome. The separation of proteins and peptides is therefore a key element in proteomic analyses. In proteomic studies, developments and improvements in MS-based techniques have revolutionized the identification of proteins. However, the identification of the proteins present in a sample is only the first step in the process of understanding their functions. Thus, a successful model of protein function and regulation pathways in the cell requires a broad understanding of protein interaction with other proteins and a comprehensive understanding of cellular metabolism. In this thesis, proteomic methods including different forms of 2-DE based separation and MS-based identification techniques were adapted and validated. These techniques were used to reveal the metabolic responses of the recombinant xylose-fermenting Saccharomyces cerevisiae and stress responses of the probiotic Lactobacillus gasseri and the mastitis pathogen Streptococcus uberis. In the first study the methods for protein separation and identification for S. cerevisiae were adapted and validated. These methods were used and further optimized in the second publication, which was the first proteome wide study of a recombinant xylose-fermenting S. cerevisiae. In the background of these yeast studies lies increasing concern about global climate warming, that has accelerated the development of alternative energy sources. Bioethanol production from cellulosic biomass by fermentation with baker s yeast S. cerevisiae is one of the most studied areas in this field. D-xylose is abundant in the biomass, thus the metabolic engineering of S. cerevisiae for utilisation of pentose sugars is of particular interest. Because S. cerevisiae does not naturally utilize xylose or other pentose sugars, the introduction of an active xylose utilization pathway into S. cerevisiae was likely to have a major effect on cellular metabolism. As a result we found altered metabolic fluxes especially in the acetate and glycerol pathways in cells growing on xylose compared to cells growing on glucose. In the third study, improved proteomic methods were applied to investigate heat-stress response of the probiotic Lactobacillus gasseri. Probiotics are microbes that are known to confer health benefits in the host. The use of food products containing probiotic microorganisms is of increasing economic importance and during the manufacturing processes and digestion these microorganisms are exposed to technological and digestive stresses. The study of heat shock response of L. gasseri revealed an increased abundance of four Clp ATPases, which are a family of stress proteins that are known as regulators of several biological processes and virulence factors in a number of pathogenic bacteria. We also showed that a functional clpL gene is essential for the development of constitutive and induced thermotolerance in L. gasseri. The adapted proteomic methods were applied in the fourth publication to investigate the global changes in the proteomes of Streptococcus uberis in response to mutagenesis-inducing ciprofloxacin challenge, and to elucidate the mechanisms by which resistance to ciprofloxacin is developed. S. uberis is an environmental mastitis pathogen and it is known that the fluoroquinolone antibiotic ciprofloxacin induces a mutagenic response in S. uberis. This proteomic study revealed activation of the oxidative damage response, reduction in NADH generation and changes in the pool of deoxyribonucleotides, potentially providing S. uberis time to stimulate mutagenesis and adapt to changes in its environment.
  • Nykter, Minna (Helsingin yliopisto, 2006)
    Flax and hemp have traditionally been used mainly for textiles, but recently interest has also been focused on non-textile applications. Microbial quality throughout the whole processing chain of bast fibres has not previously been studied. This study concentrates on the microbial quality and possible microbial risks in the production chain of hemp and flax fibres and fibrous thermal insulations. In order to be able to utilize hemp and flax fibres, the bast fibres must be separated from the rest of the plant. Non-cellulosic components can be removed with various pretreatment processes, which are associated with a certain risk of microbial contamination. In this study enzymatic retting and steam explosion (STEX) were examined as pretreatment processes. On the basis of the results obtained in this study, the microbial contents on stalks of both plants studied increased at the end of the growing season and during the winter. However, by processing and mechanical separation it is possible to produce fibres containing less moulds and bacteria than the whole stem. Enzymatic treatment encouraged the growth of moulds in fibres. Steam explosion reduced the amount of moulds in fibres. Dry thermal treatment used in this study did not markedly reduce the amount of microbes. In this project an emission measurement chamber was developed which was suitable for measurements of emissions from both mat type and loose fill type insulations, and capable of interdisciplinary sampling. In this study, the highest amounts of fungal emissions were in the range of 10^3 10^5 cfu/m^3 from the flax and hemp insulations at 90% RH of air. The fungal emissions from stone wool, glass wool and recycled paper insulations were below 10^2 cfu/m^3 even at 90% RH. Equally low values were obtained from bast fibrous materials in lower humidities (at 30% and 80% RH of air). After drying of moulded insulations at 30% RH, the amounts of emitted moulds were in all cases higher compared to the emissions at 90% RH before drying. The most common fungi in bast fibres were Penicillium and Rhizopus. The widest variety of different fungi was in the untreated hemp and linseed fibres and in the commercial loose-fill flax insulation. Penicillium, Rhizopus and Paecilomyces were the most tolerant to steam explosion. According to the literature, the most common fungi in building materials and indoor air are Penicillium, Aspergillus and Cladosporium, which were all found in some of the bast fibre materials in this study. As organic materials, hemp and flax fibres contain high levels of nutrients for microbial growth. The amount of microbes can be controlled and somewhat decreased by the processing methods presented.
  • Wallenius, Kaisa (Helsingin yliopisto, 2011)
    The study of soil microbiota and their activities is central to the understanding of many ecosystem processes such as decomposition and nutrient cycling. The collection of microbiological data from soils generally involves several sequential steps of sampling, pretreatment and laboratory measurements. The reliability of results is dependent on reliable methods in every step. The aim of this thesis was to critically evaluate some central methods and procedures used in soil microbiological studies in order to increase our understanding of the factors that affect the measurement results and to provide guidance and new approaches for the design of experiments. The thesis focuses on four major themes: 1) soil microbiological heterogeneity and sampling, 2) storage of soil samples, 3) DNA extraction from soil, and 4) quantification of specific microbial groups by the most-probable-number (MPN) procedure. Soil heterogeneity and sampling are discussed as a single theme because knowledge on spatial (horizontal and vertical) and temporal variation is crucial when designing sampling procedures. Comparison of adjacent forest, meadow and cropped field plots showed that land use has a strong impact on the degree of horizontal variation of soil enzyme activities and bacterial community structure. However, regardless of the land use, the variation of microbiological characteristics appeared not to have predictable spatial structure at 0.5-10 m. Temporal and soil depth-related patterns were studied in relation to plant growth in cropped soil. The results showed that most enzyme activities and microbial biomass have a clear decreasing trend in the top 40 cm soil profile and a temporal pattern during the growing season. A new procedure for sampling of soil microbiological characteristics based on stratified sampling and pre-characterisation of samples was developed. A practical example demonstrated the potential of the new procedure to reduce the analysis efforts involved in laborious microbiological measurements without loss of precision. The investigation of storage of soil samples revealed that freezing (-20 °C) of small sample aliquots retains the activity of hydrolytic enzymes and the structure of the bacterial community in different soil matrices relatively well whereas air-drying cannot be recommended as a storage method for soil microbiological properties due to large reductions in activity. Freezing below -70 °C was the preferred method of storage for samples with high organic matter content. Comparison of different direct DNA extraction methods showed that the cell lysis treatment has a strong impact on the molecular size of DNA obtained and on the bacterial community structure detected. An improved MPN method for the enumeration of soil naphthalene degraders was introduced as an alternative to more complex MPN protocols or the DNA-based quantification approach. The main advantage of the new method is the simple protocol and the possibility to analyse a large number of samples and replicates simultaneously.
  • Kostamo, Katriina (Helsingin yliopisto, 2006)
    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.
  • Östman, Pekka (Helsingin yliopisto, 2007)
    With the increasing demand for faster and more cost-effective analytical methods, miniaturization of analytical instruments is becoming ever more popular. The major motivations of miniaturization are increased speed of analysis, lower sample and reagent consumption, and reduced waste production. These miniaturized analytical instruments can be highly automated and with the small dimensions, many systems can be constructed in parallel, providing increasing sample throughput. Decreased sample volumes require an adequate, specific, and highly sensitive detection method, like mass spectrometry (MS). In this work, two miniaturized ion sources for atmospheric pressure ionization ionization mass spectrometry (API-MS) were investigated. First, a novel microchip heated nebulizer for atmospheric pressure chemical ionization mass spectrometry (APCI-MS) was developed and evaluated. The microchip APCI provided flow rates down to 50 nl/min and high ionization efficiency (mass flow sensitivity about 100-200 times better than conventional interface) thus enabling, for the first time, the use of low-flow rate separation techniques with APCI-MS. The microchip APCI provides easy connection of the microchip to any MS equipped with API source, temperature optimization for individual analytes, robust analysis, and cost efficient manufacturing. Use of the microchip APCI was demonstrated with microfluidics, capillary liquid chromatography (capillary LC) and gas chromatography (GC). The feasibility of the microchip APCI for qualitative and quantitative analysis was evaluated for capillary LC-APCI-MS and GC-APCI-MS. In quantitative analysis the microchip APCI showed good linearity and repeatability with detection limits down to fmol range. Next, the feasibility of atmospheric pressure desorption/ionization on silicon-mass spectrometry (AP-DIOS-MS) for drug analysis was investigated. DIOS is a relatively new matrix-assisted laser desorption/ionization (MALDI) related technique, in which bare porous silicon is used to assist ionization of the sample molecules instead of the matrix compounds used in MALDI. Since a matrix compound is not used in DIOS, mass spectra with significantly lower background at low mass range can be produced. The main advantage of AP-DIOS relative to conventional vacuum DIOS is that delay between the sample plate introduction to the ion source and the measurement is minimized enabling higher sample throughput. It was observed that only compounds with relatively high proton affinity, above a threshold value of 920-950 kJ/mol, are efficiently ionized under AP-DIOS conditions. The good linearity, linear dynamic range of 3 orders of magnitude, and repeatability showed that the method was suitable for quantitative analysis.
  • Nordman, Nina (Helsingin yliopisto, 2015)
    Analytical microsystems are attractive in modern bioanalysis where sample amounts often are low and fast analyses are required. Microsystems also provide the prospect of integrating several functional elements on a single platform. The aim of this work was to develop analytical microsystems for fast analysis of bio- and drug molecules. For this, microchips with separation- and injection channels and monolithically integrated electrospray ionization (ESI) emitter were fabricated of epoxy photoresist SU-8 by photolithography and adhesive bonding. For peptide mass fingerprinting and protein sequencing characteristic tryptic peptides were fast and easily separated and detected by microchip capillary electrophoresis (MCE)-ESI/mass spectrometry (MS). Additionally, protein identification based on tandem MS fragmentation data of a single tryptic peptide was achieved. Finally, this rapid (total analysis time below ten minutes) microchip method permitted analysis of human muscle cell lysates. For online coupling of microchip capillary isoelectric focusing (cIEF) to ESI/MS a bilateral sheath flow interface or a two-dimensional separation unit was integrated on-chip. Rapid focusing of peptides by their isoelectric points (pI) was achieved without pretreatment of the SU-8 surface. After focusing the peptides were electrokinetically mobilized toward ESI/MS. The two-dimensional chip design enabled unique separation selectivity for peptides based on both pI values and intrinsic electrophoretic mobilities by multiplex-cIEF-transient-isotachophoresis. Rapid metabolic profiling was demonstrated from urine after intake of tramadol or paracetamol. Both phase I- and II metabolites were separated and detected by MCE-ESI/MS within 35 s. In addition, Michaelis-Menten kinetics was successfully determined for the CYP450-mediated oxidation of bufuralol to 1-hydroxybufuralol. Sample preconcentration (pretreatment) was integrated on-chip by solid-phase extraction (SPE) and liquid-phase microextraction (LPME). For SPE, a monolith zone was firmly anchored at the injection cross of the MCE-ESI/MS microchip by laser induced photopolymerization. The monolith was selective toward hydrophobic and hydrophilic sample molecules and enrichment factors as high as 23-fold was achieved with a loading time as short as 25 s. In addition, LPME was easily downscaled to low volume applications and offered selectivity in the analysis of phase I metabolites compared to SPE. In contrast to previous research in the same field this work offers bioanalysis with several on-chip integrated steps (preconcentration, injection, separation, and analysis) without considerably increasing the short analysis times characteristic of microchip assays.
  • Sallisalmi, Marko (Helsingin yliopisto, 2013)
    The study was a prospective observational and methodological study performed among thirty-six critically-ill patients and twenty-six healthy controls subjects in 2008-2012 in two Finnish intensive care units. In critically-ill patients the blood flow among the microcirculation becomes disturbed, which predisposes the patients to multiple organ failure and death. The endothelial glycocalyx layer, which is formed on the vascular surface of endothelial cells is implicated in microcirculatory events in animal studies, but the data among critically-ill patients is scarce and based on laboratory samples only. The viscosity of plasma determines shearing forces of blood, and thus, the shear induced excretion of nitric oxide by the endothelial cells, i.e. mechanosensing. In animal models, increased plasma viscosity is associated with increased microvascular flow. Futhermore, the technology of microvascular and glycocalyx research in humans is under development. The evidence of molecular mechanisms and causal relation between endothelial glycocalyx damage and microcvascular disturbance in critically-ill patients is lacking. The feasibility and reliability of a standardized technical quality analysis for video images obtained from human sublingual microcirculation was tested in the first part of the study. By analysis of a novel previously unvalidated set of technical criteria by two independent investigators, only 31% of the analyzed 240 video images qualified techically for further use in clinical studies. In the second part of the study, a semi-automatic computer algorithm was created and validated for the measurement of glycocalyx layer thickness. Use of the semi-automatic algorithm in microcirculatory video images obtained from human oral cavity diminished the coefficient of variation of measured glycocalyx layer thickness from 7.2% - 9.8% in manual measurements to 4.3% - 5.8% in the semi-automatic measurements. Two separate hypothesis were tested in the third and fourth parts of the study. A hypothesis that activation of vascular adhesion protein-1, a novel adhesion epitope and enzyme, coincides with shedding of the glycocalyx in sepsis, was supported. The second hypothesis, that the shedding of the endothelial glycocalyx layer in sepsis leads to increased plasma viscosity, was rejected. Contrary to the study hypothesis the viscosity of plasma in patients with sepsis was not increased, but was lower than controls values for four days. Ex vivo it was evident that the type of fluid therapy administered during the course of early treatment among critically-ill patients determines plasma viscosity and, thus, the shearing properies of blood in the microcirculation.
  • Teye, Frederick Kwame (Helsingin yliopisto, 2008)
    The aim of this thesis was to develop measurement techniques and systems for measuring air quality and to provide information about air quality conditions and the amount of gaseous emissions from semi-insulated and uninsulated dairy buildings in Finland and Estonia. Specialization and intensification in livestock farming, such as in dairy production, is usually accompanied by an increase in concentrated environmental emissions. In addition to high moisture, the presence of dust and corrosive gases, and widely varying gas concentrations in dairy buildings, Finland and Estonia experience winter temperatures reaching below -40 ºC and summer temperatures above +30 ºC. The adaptation of new technologies for long-term air quality monitoring and measurement remains relatively uncommon in dairy buildings because the construction and maintenance of accurate monitoring systems for long-term use are too expensive for the average dairy farmer to afford. Though the documentation of accurate air quality measurement systems intended mainly for research purposes have been made in the past, standardised methods and the documentation of affordable systems and simple methods for performing air quality and emissions measurements in dairy buildings are unavailable. In this study, we built three measurement systems: 1) a Stationary system with integrated affordable sensors for on-site measurements, 2) a Wireless system with affordable sensors for off-site measurements, and 3) a Mobile system consisting of expensive and accurate sensors for measuring air quality. In addition to assessing existing methods, we developed simplified methods for measuring ventilation and emission rates in dairy buildings. The three measurement systems were successfully used to measure air quality in uninsulated, semi-insulated, and fully-insulated dairy buildings between the years 2005 and 2007. When carefully calibrated, the affordable sensors in the systems gave reasonably accurate readings. The spatial air quality survey showed high variation in microclimate conditions in the dairy buildings measured. The average indoor air concentration for carbon dioxide was 950 ppm, for ammonia 5 ppm, for methane 48 ppm, for relative humidity 70%, and for inside air velocity 0.2 m/s. The average winter and summer indoor temperatures during the measurement period were -7º C and +24 ºC for the uninsulated, +3 ºC and +20 ºC for the semi-insulated and +10 ºC and +25 ºC for the fully-insulated dairy buildings. The measurement results showed that the uninsulated dairy buildings had lower indoor gas concentrations and emissions compared to fully insulated buildings. Although occasionally exceeded, the ventilation rates and average indoor air quality in the dairy buildings were largely within recommended limits. We assessed the traditional heat balance, moisture balance, carbon dioxide balance and direct airflow methods for estimating ventilation rates. The direct velocity measurement for the estimation of ventilation rate proved to be impractical for naturally ventilated buildings. Two methods were developed for estimating ventilation rates. The first method is applicable in buildings in which the ventilation can be stopped or completely closed. The second method is useful in naturally ventilated buildings with large openings and high ventilation rates where spatial gas concentrations are heterogeneously distributed. The two traditional methods (carbon dioxide and methane balances), and two newly developed methods (theoretical modelling using Fick s law and boundary layer theory, and the recirculation flux-chamber technique) were used to estimate ammonia emissions from the dairy buildings. Using the traditional carbon dioxide balance method, ammonia emissions per cow from the dairy buildings ranged from 7 g day-1 to 35 g day-1, and methane emissions per cow ranged from 96 g day-1 to 348 g day-1. The developed methods proved to be as equally accurate as the traditional methods. Variation between the mean emissions estimated with the traditional and the developed methods was less than 20%. The developed modelling procedure provided sound framework for examining the impact of production systems on ammonia emissions in dairy buildings.