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  • 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.
  • Nokso-Koivisto, Inka (2014)
    The microcosm-macrocosm analogy – the idea of man as a miniature of the surrounding reality or part of it – is a prevailing theme in Rasail Ikhwan as-Safa. This study examines the analogy primarily in this encyclopaedia completed during the tenth century and compares the views presented in it to those in certain other texts from the ninth to the thirteenth centuries: Sirr al-khaliqa, some texts attributed to Jabir ibn Hayyan, some Sufi writings of al-Ghazali (d. 1111), Suhrawardi (d. 1191) and Ibn Arabi (d. 1240), and al-Qazwini’s (d. 1283) Ajaib al-makhluqat. The aim is to explore the influence of microcosmism on the idea of man in these texts and to define the position of the Rasail in the development of the topic in mediaeval Islamic thought. Rudolf Allers’s classification of microcosmism is used as the main conceptual framework in this analysis. All Allers’s six varieties of the analogy receive various interpretations in the Islamic tradition. This study also proposes a threefold approach to the examination of microcosmism. Firstly, the analogy appears as a human-specific feature defining the cosmological position of the human species. In this form, microcosmism is used in all of the studied texts and often the role of the human being as an intermediate being in the universe is in focus. Secondly, attitudes towards the corporeal aspect of man are approached through the use of the analogy. In this form, the idea is closely related to the scientific worldview and sometimes the meaning given to the analogy can only be understood within the frames of a scientific theory. Thirdly, the normative aspect is included in the analogy and it is used in descriptions of epistemological and ethical ideals. Especially Sufi thinkers elaborate this form of the analogy and it is also in the key position of microcosmism in the Rasail. Microcosmism in the Rasail is a synthesis of various forms of the analogy developed earlier in the Islamic tradition and it anticipates many ideas that only become central in the later texts. Obvious thematic similarities between the texts can be found, but transmission of particular elements of microcosmism is possible to trace in only a few cases. For instance, some comparisons of the Rasail between the human body and the surrounding reality seem to be transmitted – directly or indirectly – even to the latest texts of the corpus.
  • Uddin, Mohammad Jasim (Helsingin yliopisto, 2013)
    This dissertation seeks to shed light on microcredit policies and practices, and attempts to contribute to the understanding of how microcredit relates to the lives of the borrowers in rural Bangladesh. More specifically, this study delves into whether the group-based micro-loans that are channeled through women facilitate social capital, reconstruct gender relations and provide a way out of poverty at the village level in Bangladesh. The empirical data of this study was collected from 151 married women microcredit borrowers of two project areas of the Grameen Bank (GB) and two project areas of the Bangladesh Rural Advancement Committee (BRAC) in the Sylhet District, Bangladesh where both the GB and the BRAC have been operating micro-loans over a period of several years. The ethnographic description relates to the questions of how NGOs use neoliberal policies and credit on the one hand, and also how local people appropriate credit on the other hand. I have endeavoured to contribute to understanding the focuses of microcredit initiatives within the context of Foucault s disciplinary power (1977) and governmentality (1978, 1979, 1988) against the background of neoliberalism. I stress the power relations, implications of enforced institutional discipline of microcredit organizations, and vulnerability of microcredit borrowers. By applying Scott s notions of hidden resistance (1990) and weapons of the weak (1985), I have focused on how microcredit borrowers criticize rules of programmes and procedures out of earshot and out of sight of the officials of the NGOs. Following the entitlement approach detailed by Sen (1981, 1987, 1999) I have also addressed the mechanism whereby poor people take credit year after year and get further mired in debt. Fundamentally, the objective of this study has rotated around some complex questions: To what extent microcredit programmes, a product of neoliberalism and the capitalist world system, intersect and connect with the local women borrowers to facilitate social capital, women s empowerment and poverty alleviation in rural Bangladesh? Does the group lending tenet really work, or is it only a process of getting access to credit for the borrowers or does it fulfil a governing strategy of microcredit organizations to the borrowers in accordance with the market rationality? Are women microcredit borrowers really rational economic actors who invest credit themselves at the local level? Is credit through women a policy of women s empowerment or a project of covert regulative practice? Are the microcredit borrowers undergoing the win-win situation of increased financial outcomes and enhanced well-being that microcredit programmes have pledged to offer? My work draws attention to what microcredit NGOs aim to change, and the techniques they apply. My study is an analysis of what microcredit initiatives fail to do: mobilize social capital, reconstruct gender relations, and alleviate poverty. The study argues that microcredit can be regard as a form of governmentality that is exercised via a generalised control over people s behaviour and over their beliefs, and by spreading the values of entrepreneurship with the market as the solver of all ills. Whilst the much lauded microcredit organizations (such as GB, BRAC) push neoliberal ideologies onto rural borrowers, they have failed because many borrowers cannot yield sufficient profit and or use credit for the purposes for which it is supposed to be used for. The package of training and consciousness raising lessons that originally went alongside the GB or BRAC microcredit programme is now missing from their rural operations. Therefore, ensuring a win-win situation that microcredit originally promised has been failed. Microcredit organizations reinforce pre-existing kinship and gender structures, and there has been a wide scale mission creep, which has turned micro-lending NGOs into money-lending businesses or installment collecting organizations.
  • Verho, Jouko (Helsingfors universitet, 2008)
    This dissertation consists of four essays studying topics in empirical labour economics. The first essay evaluates the impact of an unemployment benefit reform in Finland. In 2003, the benefit level was increased in Finland for workers with long employment histories. The average benefit increase was 15% for the first 150 days of the unemployment spell. This study evaluates the effect of the benefit increase on the duration of unemployment by comparing the changes in the re-employment hazard profiles among the unemployed who became eligible for the increased benefits to the changes in a comparison group whose benefit structure remained unchanged. According to the results benefit increase reduced the re-employment hazards by on average 16%. The effect is largest at the beginning of the unemployment spell and disappears after the eligibility period for the increased benefits expires. The second essay analyses the long-term costs of unemployment in Finland by focusing on the deep recession period of 1991 - 1993. The number of plant closures increased sharply during the recession and the unemployment rate rose by more than 13 percentage points. In the analysis, prime working-age men who face unemployment due to plant closure are matched to those who remained employed during the recession. The effect of being unemployed during the recession is estimated for a 6 year follow-up period. In 1999, the unemployed individuals suffer a 25% loss in annual earnings, 10% reduction in employment and 14% wage scar. The third essay focuses on the recession of the early 1990s that caused a serious unemployment problem in Finland. This study analyses the determinants of unemployment duration using individual data from 1987 to 2000. Duration until employment is modelled using a proportional hazard model with piecewise constant baseline hazard. The main focus is on the relative contribution of compositional variation and macroeconomic conditions to unemployment duration. According to the results, the aggregate outflow effect dominates and the observed compositional variation implies only a small increasing trend in the average duration during the recession period. The last essay studies the effect of business cycle on the incidence of workplace accidents. Individual data covering Swedish inhospital care 1997 - 2005 is linked to the population database. These data allow studying if changes in the composition of workers or strategic worker behaviour are driving the cyclicality of accidents. The results show that the incidence of workplace accidents increases during economic upturns but only in specific subgroups. Some evidence is found that compositional changes in labour force may contribute to cyclicality for women. In the male population, on the other hand, only the less severe accidents are cyclical which would be consistent with strategic worker behaviour.
  • Razzauti Sanfeliu, Maria (Helsingin yliopisto, 2012)
    Puumala hantavirus (PUUV) is a zoonotic virus that in humans causes nephropathia epidemica (NE) in humans, a mild form of haemorrhagic fever with renal syndrome. An average of 10 000 cases are reported annually in Europe, many of which occur in Fennoscandia. The incidence of NE is connected to the distribution and population density of the the bank vole (Myodes glareolus), the main virus host. In Fennoscandia, high incidences of NE occur at 3-4 year intervals due to the characteristic population cycles of this woodland rodent. This study aimed to gain a better understanding of PUUV microevolution by examining genetic features of the virus in several bank vole populations of Finland and Latvia. Genetic variation in PUUV circulating in a bank vole population at Konnevesi in Central Finland was examined and monitored over five-years throughout a complete bank vole cycle, including two peak-phases in 2005 and 2008 and two population declines in 2006 and 2009 (i.e., viral bottlenecks). Altogether, 1369 bank voles were captured and 26.3% were detected PUUV-infected. Partial sequences of the three viral genome segments (Small, Medium and Large) were inspected from 365 PUUV genomes. Genetic diversity was 6.2% for the S segment, 4.8% for the M segment, and a surprisingly high 10.1% for the L segment. Each genome segment had accumulated mutations as a separate gene pool. The majority of nucleotide substitutions were synonymous and most of the deduced amino acid substitutions were conservative, suggesting a strong stabilizing selection operating at the protein level. Genetic markers found along the genome segments allowed for the recognition of two genogroups of PUUV co-circulating in the host population. Even though, one of the genogroups presented higher genetic diversity, no signs of completion were observed between them. Nearly 80% the variants exhibited a transient existence, and frequently occurring variants were integrated by most abundant segment genotypes suggesting a viral mutational robustness. A substantial portion (19.1%) of genomes appeared to be reassorted, with S and M typically being exchanged. Reassorted variants did not outcompete parental variants and were commonly transient. Reassortment was seasonal, occurring more frequently in autumn when recent infection risk increases. An imperceptible intra-genogroup reassortment could contribute to the steady state of the viral population, counteracting the effects of Muller s ratchet. Co-circulation and interaction of two distinct PUUV lineages (Finnish and North-Scandinavian) was monitored in a bank vole population at Pallasjärvi in Northern Finland. To date, seven genetic lineages have been detected, all of which exhibit geographic structure within the host distribution. Here, we present new evidence of two lineages circulating in the same bank vole phylogroup (Ural clade). Genetic diversity within each PUUV lineage was modest (up to 1.7%) and most substitutions were synonymous. However, genetic differences between the two lineages were as high as 18.9%. Phylogenetic analyses revealed that these distinct lineages naturally reassort with a frequency comparable to that genogroups circulating at Konnevesi, i.e., 32%. In contrast to Konnevesi, only M segment was exchanged between PUUV lineages at Pallasjärvi. Two distinct PUUV lineages were also found to co-circulate in Latvia. One (Russian) has been previously described and the other awaits formal description. The novel Latvian lineage is considerably divergent from other PUUV lineages and several amino acid markers made it easily distinguishable. Phylogenetic analysis suggested an independent evolutionary history for the segments of Latvian lineage. Similar to Pallasjärvi, both Russian and Latvian lineages were found in a single bank vole phylogroup (Carpathian clade), confirming earlier observations that PUUV lineages are not limited to a single host phylogroup.
  • Heino, Antti (Helsingin yliopisto, 2010)
    Milk microfiltration (0.05-0.2 um) is a membrane separation technique which divides milk components into casein-enriched and native whey fractions. Hitherto the effect of intensive microfiltration including a diafiltration step for both cheese and whey processing has not been studied. The microfiltration performance of skimmed milk was studied with polymeric and ceramic MF membranes. The changes caused by decreased concentration of milk lactose, whey protein and ash content for cheese milk quality and ripening were studied. The effects of cheese milk modification on the milk coagulation properties, cheese recovery yield, cheese composition, ripening and sensory quality as well as on the whey recovery yield and composition by microfiltration were studied. The functional properties of whey protein concentrate from native whey were studied and the detailed composition of whey protein concentrate powders made from cheese wheys after cheese milk pretreatments such as high temperature heat treatment (HH), microfiltration (MF) and ultrafiltration (UF) were compared. The studied polymeric spiral wound microfiltration membranes had 38.5% lower energy consumption, 30.1% higher retention of whey proteins to milk retentate and 81.9% lower permeate flux values compared to ceramic membranes. All studied microfiltration membranes were able to separate main whey proteins from skimmed milk. The optimal lactose content of Emmental cheese milk exceeded 3.2% and reduction of whey proteins and ash content of cheese milk with high concentration factor (CF) values increased the rate of cheese ripening. Reduction of whey protein content in cheese milk increased the concentration of caseinomacropeptide (CMP) of total proteins in cheese whey. Reduction of milk whey protein, lactose and ash content reduces milk rennet clotting time and increased the firmness of the coagulum. Cheese yield calculated from raw milk to cheese was lower with microfiltrated milks due to native whey production. Amounts of a-lactalbumin (a-LA) and b-lactoglobulin (b-LG) were significantly higher in the reference whey, indicating that HH, MF and UF milk pretreatments decrease the amounts of these valuable whey proteins in whey. Even low CF values in milk microfiltration (CF 1.4) reduced nutritional value of cheese whey. From the point of view of utilization of milk components it would be beneficial if the amount of native whey and the CMP content of cheese whey could be maximized. Whey protein concentrate powders made of native whey had excellent functional properties and their detailed amino acid composition differed from those of cheese whey protein concentrate powders.
  • Islam, K M Zahidul (Helsingin yliopisto, 2011)
    The objectives of this study were to make a detailed and systematic empirical analysis of microfinance borrowers and non-borrowers in Bangladesh and also examine how efficiency measures are influenced by the access to agricultural microfinance. In the empirical analysis, this study used both parametric and non-parametric frontier approaches to investigate differences in efficiency estimates between microfinance borrowers and non-borrowers. This thesis, based on five articles, applied data obtained from a survey of 360 farm households from north-central and north-western regions in Bangladesh. The methods used in this investigation involve stochastic frontier (SFA) and data envelopment analysis (DEA) in addition to sample selectivity and limited dependent variable models. In article I, technical efficiency (TE) estimation and identification of its determinants were performed by applying an extended Cobb-Douglas stochastic frontier production function. The results show that farm households had a mean TE of 83% with lower TE scores for the non-borrowers of agricultural microfinance. Addressing institutional policies regarding the consolidation of individual plots into farm units, ensuring access to microfinance, extension education for the farmers with longer farming experience are suggested to improve the TE of the farmers. In article II, the objective was to assess the effects of access to microfinance on household production and cost efficiency (CE) and to determine the efficiency differences between the microfinance participating and non-participating farms. In addition, a non-discretionary DEA model was applied to capture directly the influence of microfinance on farm households production and CE. The results suggested that under both pooled DEA models and non-discretionary DEA models, farmers with access to microfinance were significantly more efficient than their non-borrowing counterparts. Results also revealed that land fragmentation, family size, household wealth, on farm-training and off farm income share are the main determinants of inefficiency after effectively correcting for sample selection bias. In article III, the TE of traditional variety (TV) and high-yielding-variety (HYV) rice producers were estimated in addition to investigating the determinants of adoption rate of HYV rice. Furthermore, the role of TE as a potential determinant to explain the differences of adoption rate of HYV rice among the farmers was assessed. The results indicated that in spite of its much higher yield potential, HYV rice production was associated with lower TE and had a greater variability in yield. It was also found that TE had a significant positive influence on the adoption rates of HYV rice. In article IV, we estimated profit efficiency (PE) and profit-loss between microfinance borrowers and non-borrowers by a sample selection framework, which provided a general framework for testing and taking into account the sample selection in the stochastic (profit) frontier function analysis. After effectively correcting for selectivity bias, the mean PE of the microfinance borrowers and non-borrowers were estimated at 68% and 52% respectively. This suggested that a considerable share of profits were lost due to profit inefficiencies in rice production. The results also demonstrated that access to microfinance contributes significantly to increasing PE and reducing profit-loss per hectare land. In article V, the effects of credit constraints on TE, allocative efficiency (AE) and CE were assessed while adequately controlling for sample selection bias. The confidence intervals were determined by the bootstrap method for both samples. The results indicated that differences in average efficiency scores of credit constrained and unconstrained farms were not statistically significant although the average efficiencies tended to be higher in the group of unconstrained farms. After effectively correcting for selectivity bias, household experience, number of dependents, off-farm income, farm size, access to on farm training and yearly savings were found to be the main determinants of inefficiencies. In general, the results of the study revealed the existence substantial technical, allocative, economic inefficiencies and also considerable profit inefficiencies. The results of the study suggested the need to streamline agricultural microfinance by the microfinance institutions (MFIs), donor agencies and government at all tiers. Moreover, formulating policies that ensure greater access to agricultural microfinance to the smallholder farmers on a sustainable basis in the study areas to enhance productivity and efficiency has been recommended. Key Words: Technical, allocative, economic efficiency, DEA, Non-discretionary DEA, selection bias, bootstrapping, microfinance, Bangladesh.
  • Pessi, Jenni (2013)
    Polymer microspheres hold great potential as oral drug delivery system for therapeutic proteins. Microspheres prepared with biocompatible and biodegredable polymers have been extensively studied, since the oral delivery of therapeutic proteins is challenging due to the conditions in the GI-tract. The aims of this research were to apply microfluidics on polymeric microsphere preparation process, to determine what kind of formulations are suitable for this technology, to establish a controlled preparation process that produces advanced particles and to create a template for oral protein drug delivery. With microfluidic fabrication it is possible to gain control over the process and content of each droplet. However, finding suitable formulations for microfluidics is demanding. In this study, biphasic flow was employed to successfully produce double (W/O/W) emulsion droplets with ultra thin shells. Once the process and formulation variables were optimized constant droplet production was achieved. Flow rates used were 500 μl/h in the inner and in the middle phase and 2500 μl/h in the outer phase, respectively. Two formulations were selected for further characterization: 5 % poly(vinyl alcohol) in water in the outer phase, 3 % polycaprolactone in ethyl acetate in the middle phase and either 10 % or 20 % poly(vinyl alcohol) and polyethylenglycol (1:4) in water in the inner phase. All the particles were found to be intact and contain the inner phase, as verified by confocal microscopy. Further, the particles were monodisperse and non-porous, as observed by scanning electron microscopy. Particle size was found to be around 20-40 μm, variation in the particle size within one batch was small and the particles were stable up to 4 weeks. The encapsulation efficiency of the particles was remarkable; as high as 85 % loading of the model compound, bovine serum albumin. Particles released 30 % of their content within 48 hours. In conlusion, developing functional formulations for micfoluidic technology was possible, the microparticles encapsulated the model protein extremely well and all in all microfluidic technology had a lot of potential for droplet manufacturing for pharmaceutical applications.
  • Markkanen, Tiina (Helsingin yliopisto, 2004)