A note on sampling chironomids for RNA-based studies of natural populations that retains critical morphological vouchers

The rapid uptake of transcriptomic approaches in freshwater ecology has seen a wealth of data produced concerning the ways in which organisms interact with their environment on a molecular level. Typically, such studies focus either at the community level and so don’t require species identifications, or on laboratory strains of known species identity or natural populations of large, easily identifiable taxa. For chironomids, impediments still exist for applying these technologies to natural populations because they are small-bodied and often require time-consuming secondary sorting of stream material and morphological voucher preparation to confirm species diagnosis. These procedures limit the ability to maintain RNA quantity and quality in such organisms because RNA degrades rapidly and gene expression can be altered rapidly in organisms; thereby limiting the inclusion of such taxa in transcriptomic studies. Here, we demonstrate that these limitations can be overcome and outline an optimised protocol for collecting, sorting and preserving chironomid larvae that enables retention of both morphological vouchers and RNA for subsequent transcriptomics purposes. By ensuring that sorting and voucher preparation are completed within <4 hours after collection and that samples are kept cold at all times, we successfully retained both RNA and morphological vouchers from all specimens. Although not prescriptive in specific methodology, we anticipate that this paper will assist in promoting transcriptomic investigations of the sublethal impact on chironomid gene expression of changes to aquatic environments.

Clays Can Decrease Gaseous Nutrient Losses from Soil-Applied Livestock Manures

Clays could underpin a viable agricultural greenhouse gas (GHG) abatement technology given their affinity for nitrogen and carbon compounds. We provide the first investigation into the efficacy of clays to decrease agricultural nitrogen GHG emissions (i.e., N2O and NH3). Via laboratory experiments using an automated closed-vessel analysis system, we tested the capacity of two clays (vermiculite and bentonite) to decrease N2O and NH3 emissions and organic carbon losses from livestock manures (beef, pig, poultry, and egg layer) incorporated into an agricultural soil. Clay addition levels varied, with a maximum of 1:1 to manure (dry weight). Cumulative gas emissions were modeled using the biological logistic function, with 15 of 16 treatments successfully fitted (P < 0.05) by this model. When assessing all of the manures together, NH3 emissions were lower (×2) at the highest clay addition level compared with no clay addition, but this difference was not significant (P = 0.17). Nitrous oxide emissions were significantly lower (×3; P < 0.05) at the highest clay addition level compared with no clay addition. When assessing manures individually, we observed generally decreasing trends in NH3 and N2O emissions with increasing clay addition, albeit with widely varying statistical significance between manure types. Most of the treatments also showed strong evidence of increased C retention with increasing clay additions, with up to 10 times more carbon retained in treatments containing clay compared with treatments containing no clay. This preliminary assessment of the efficacy of clays to mitigate agricultural GHG emissions indicates strong promise.

EU Multi-level Governance in the Making - The Community Initiative LEADER+ in Finland and Germany

In order to fully understand the process of European integration it is of paramount importance to consider developments at the sub-national and local level. EU integration scholars shifted their attention to the local level only at the beginning of the 1990s with the concept of multi-level governance (MLG). While MLG is the first concept to scrutinise the position of local levels of public administration and other actors within the EU polity, I perceive it as too optimistic in the degree of influence it ascribes to local levels. Thus, learning from and combining MLG with other concepts, such as structural constructivism, helps to reveal some of the hidden aspects of EU integration and paint a more realistic picture of multi-level interaction. This thesis also answers the call for more case studies in order to conceptualise MLG further. After a critical study of theories and concepts of European integration, above all, MLG, I will analyse sub-national and local government in Finland and Germany. I show how the sub-national level and local governments are embedded in the EU s multi-level structure of governance and how, through EU integration, those levels have been empowered but also how their scope of action has partially decreased. After theoretical and institutional contextualisation, I present the results of my empirical study of the EU s Community Initiative LEADER+. LEADER stands for Liaison Entre Actions de Développement de l'Économie Rurale , and aims at improving the economic conditions in Europe s rural areas. I was interested in how different actors construct and shape EU financed rural development, especially in how local actors organised in so-called local action groups (LAGs) cooperate with other administrative units within the LEADER+ administrative chain. I also examined intra-institutional relations within those groups, in order to find out who are the most influential and powerful actors within them. Empirical data on the Finnish and German LAGs was first gathered through a survey, which was then supplemented and completed by interviewing LAG members, LAG-managers, several civil servants from Finnish and German decision-making and managing authorities and a civil servant from the EU Commission. My main argument is that in both Germany and Finland, the Community Initiative LEADER+ offered a space for multi-level interaction and local-level involvement, a space that on the one hand consists of highly motivated people actively contributing to the improvement of the quality of life and economy in Europe s countryside but which is dependent and also restricted by national administrative practices, implementation approaches and cultures on the other. In Finland, the principle of tri-partition (kolmikantaperiaatte) in organising the executive committees of LAGs is very noticeable. In comparison to Germany, for instance, the representation of public administration in those committees is much more limited due to this principle. Furthermore, the mobilisation of local residents and the bringing together of actors from the local area with different social and institutional backgrounds to become an active part of LEADER+ was more successful in Finland than in Germany. Tri-partition as applied in Finland should serve as a model for similar policies in other EU member states. EU integration changed the formal and informal inter-institutional relations linking the different levels of government. The third sector including non-governmental institutions and interest groups gained access to policy-making processes and increasingly interact with government institutions at all levels of public administration. These developments do not necessarily result in the empowering of the local level.

Below-ground processes in meadow soil under elevated ozone and carbon dioxide : - Greenhouse gas fluxes, N cycling and microbial communities

This thesis focuses on how elevated CO2 and/or O3 affect the below-ground processes in semi-natural vegetation, with an emphasis on greenhouse gases, N cycling and microbial communities. Meadow mesocosms mimicking lowland hay meadows in Jokioinen, SW Finland, were enclosed in open-top chambers and exposed to ambient and elevated levels of O3 (40-50 ppb) and/or CO2 (+100 ppm) for three consecutive growing season, while chamberless plots were used as chamber controls. Chemical and microbiological analyses as well as laboratory incubations of the mesocosm soils under different treatments were used to study the effects of O3 and/or CO2. Artificially constructed mesocosms were also compared with natural meadows with regards to GHG fluxes and soil characteristics. In addition to research conducted at the ecosystem level (i.e. the mesocosm study), soil microbial communities were also examined in a pot experiment with monocultures of individual species. By comparing mesocosms with similar natural plant assemblage, it was possible to demonstrate that artificial mesocosms simulated natural habitats, even though some differences were found in the CH4 oxidation rate, soil mineral N, and total C and N concentrations in the soil. After three growing seasons of fumigations, the fluxes of N2O, CH4, and CO2 were decreased in the NF+O3 treatment, and the soil NH4+-N and mineral N concentrations were lower in the NF+O3 treatment than in the NF control treatment. The mesocosm soil microbial communities were affected negatively by the NF+O3 treatment, as the total, bacterial, actinobacterial, and fungal PLFA biomasses as well as the fungal:bacterial biomass ratio decreased under elevated O3. In the pot survey, O3 decreased the total, bacterial, actinobacterial, and mycorrhizal PLFA biomasses in the bulk soil and affected the microbial community structure in the rhizosphere of L. pratensis, whereas the bulk soil and rhizosphere of the other monoculture, A. capillaris, remained unaffected by O3. Elevated CO2 caused only minor and insignificant changes in the GHG fluxes, N cycling, and the microbial community structure. In the present study, the below-ground processes were modified after three years of moderate O3 enhancement. A tentative conclusion is that a decrease in N availability may have feedback effects on plant growth and competition and affect the N cycling of the whole meadow ecosystem. Ecosystem level changes occur slowly, and multiplication of the responses might be expected in the long run.

Aerobic carbon-cycle related microbial communities in boreal peatlands: responses to water-level drawdown

Boreal peatlands represent a considerable portion of the global carbon (C) pool. Water-level drawdown (WLD) causes peatland drying and induces a vegetation change, which affects the decomposition of soil organic matter and the release of greenhouse gases (CO2 and CH4). The objective of this thesis was to study the microbial communities related to the C cycle and their response to WLD in two boreal peatlands. Both sampling depth and site type had a strong impact on all microbial communities. In general, bacteria dominated the deeper layers of the nutrient-rich fen and the wettest surfaces of the nutrient-poor bog sites, whereas fungi seemed more abundant in the drier surfaces of the bog. WLD clearly affected the microbial communities but the effect was dependent on site type. The fungal and methane-oxidizing bacteria (MOB) community composition changed at all sites but the actinobacterial community response was apparent only in the fen after WLD. Microbial communities became more similar among sites after long-term WLD. Litter quality had a large impact on community composition, whereas the effects of site type and WLD were relatively minor. The decomposition rate of fresh organic matter was influenced slightly by actinobacteria, but not at all by fungi. Field respiration measurements in the northern fen indicated that WLD accelerates the decomposition of soil organic matter. In addition, a correlation between activity and certain fungal sequences indicated that community composition affects the decomposition of older organic matter in deeper peat layers. WLD had a negative impact on CH4 oxidation, especially in the oligotrophic fen. Fungal sequences were matched to taxa capable of utilizing a broad range of substrates. Most of the actinobacterial sequences could not be matched to characterized taxa in reference databases. This thesis represents the first investigation of microbial communities and their response to WLD among a variety of boreal peatland habitats. The results indicate that microbial community responses to WLD are complex but dependent on peatland type, litter quality, depth, and variable among microbes.

Plasmids and aromatic degradation in Sphingomonas for bioremediation : Aromatic ring cleavage genes in soil and rhizosphere

Microbial degradation pathways play a key role in the detoxification and the mineralization of polyaromatic hydrocarbons (PAHs), which are widespread pollutants in soil and constituents of petroleum hydrocarbons. In microbiology the aromatic degradation pathways are traditionally studied from single bacterial strains with capacity to degrade certain pollutant. In soil the degradation of aromatics is performed by a diverse community of micro-organisms. The aim of this thesis was to study biodegradation on different levels starting from a versatile aromatic degrader Sphingobium sp. HV3 and its megaplasmid, extending to revelation of diversity of key catabolic enzymes in the environment and finally studying birch rhizoremediation in PAH-polluted soil. To understand biodegradation of aromatics on bacterial species level, the aromatic degradation capacity of Sphingobium sp. HV3 and the role of the plasmid pSKY4, was studied. Toluene, m-xylene, biphenyl, fluorene, phenanthrene were detected as carbon and energy sources of the HV3 strain. Tn5 transposon mutagenesis linked the degradation capacity of toluene, m-xylene, biphenyl and naphthalene to the pSKY4 plasmid and qPCR expression analysis showed that plasmid extradiol dioxygenases genes (bphC and xylE) are inducted by phenanthrene, m-xylene and biphenyl whereas the 2,4-dichlorophenoxyacetic acid herbicide induced the chlorocatechol 1,2-dioxygenase gene (tfdC) from the ortho-pathway. A method to study upper meta-pathway extradiol dioxygenase gene diversity in soil was developed. The extradiol dioxygenases catalyse cleavage of the aromatic ring between a hydroxylated carbon and an adjacent non-hydroxylated carbon (meta-cleavage). A high diversity of extradiol dioxygenases were detected from polluted soils. The detected extradiol dioxygenases showed sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Five groups of extradiol dioxygenases contained sequences with no close homologues in the database, representing novel genes. In rhizoremediation experiment with birch (Betula pendula) treatment specific changes of extradiol dioxygenase communities were shown. PAH pollution changed the bulk soil extradiol dioxygenase community structure and birch rhizosphere contained a more diverse extradiol dioxygenase community than the bulk soil showing a rhizosphere effect. The degradation of pyrene in soil was enhanced with birch seedlings compared to soil without birch. The complete 280,923 kb nucleotide sequence of pSKY4 plasmid was determined. The open reading frames of pSKY4 were divided into putative conjugative transfer, aromatic degradation, replication/maintaining and transposition/integration function-encoding proteins. Aromatic degradation orfs shared high similarity to corresponding genes in pNL1, a plasmid from the deep subsurface strain Novosphingobium aromaticivorans F199. The plasmid backbones were considerably more divergent with lower similarity, which suggests that the aromatic pathway has functioned as a plasmid independent mobile genetic element. The functional diversity of microbial communities in soil is still largely unknown. Several novel clusters of extradiol dioxygenases representing catabolic bacteria, whose function, biodegradation pathways and phylogenetic position is not known were amplified with single primer pair from polluted soils. These extradiol dioxygenase communities were shown to change upon PAH pollution, which indicates that their hosts function in PAH biodegradation in soil. Although the degradation pathways of specific bacterial species are substantially better depicted than pathways in situ, the evolution of degradation pathways for the xenobiotic compounds is largely unknown. The pSKY4 plasmid contains aromatic degradation genes in putative mobile genetic element causing flexibility/instability to the pathway. The localisation of the aromatic biodegradation pathway in mobile genetic elements suggests that gene transfer and rearrangements are a competetive advantage for Sphingomonas bacteria in the environment.

Mapping the risk of soil-transmitted helminthic infections in the Philippines

Background In order to increase the efficient allocation of soil-transmitted helminth (STH) disease control resources in the Philippines, we aimed to describe for the first time the spatial variation in the prevalence of A. lumbricoides, T. trichiura and hookworm across the country, quantify the association between the physical environment and spatial variation of STH infection and develop predictive risk maps for each infection. Methodology/Principal Findings Data on STH infection from 35,573 individuals across the country were geolocated at the barangay level and included in the analysis. The analysis was stratified geographically in two major regions: 1) Luzon and the Visayas and 2) Mindanao. Bayesian geostatistical models of STH prevalence were developed, including age and sex of individuals and environmental variables (rainfall, land surface temperature and distance to inland water bodies) as predictors, and diagnostic uncertainty was incorporated. The role of environmental variables was different between regions of the Philippines. This analysis revealed that while A. lumbricoides and T. trichiura infections were widespread and highly endemic, hookworm infections were more circumscribed to smaller foci in the Visayas and Mindanao. Conclusions/Significance This analysis revealed significant spatial variation in STH infection prevalence within provinces of the Philippines. This suggests that a spatially targeted approach to STH interventions, including mass drug administration, is warranted. When financially possible, additional STH surveys should be prioritized to high-risk areas identified by our study in Luzon.

Critical test for Altshuler-Aronov theory: Evolution of the density of states singularity in double perovskite Sr2FeMoO6 with controlled disorder

With high-resolution photoemission spectroscopy measurements, the density of states (DOS) near the Fermi level (E-F) of double perovskite Sr2FeMoO6 having different degrees of Fe/Mo antisite disorder has been investigated with varying temperature. The DOS near E-F showed a systematic depletion with increasing degree of disorder, and recovered with increasing temperature. Altshuler-Aronov (AA) theory of disordered metals well explains the dependences of the experimental results. Scaling analysis of the spectra provides experimental indication for the functional form of the AA DOS singularity.

Long-term effects of physical activity level on changes in healthy body mass index over 12 years in young adult women

Objectives To examine the effects of overall level and timing of physical activity (PA) on changes from a healthy body mass index (BMI) category over 12 years in young adult women. Patients and Methods Participants in the Australian Longitudinal Study on Women's Health (younger cohort, born 1973-1978) completed surveys between 2000 (age 22-27 years) and 2012 (age 34-39 years). Physical activity was measured in 2000, 2003, 2006, and 2009 and was categorized as very low, low, active, or very active at each survey, and a cumulative PA score for this 9-year period was created. Logistic regression was used to examine relationships between PA accumulated across all surveys (cumulative PA model) and PA at each survey (critical periods PA model), with change in BMI category (from healthy to overweight or healthy to obese) from 2000 to 2012. Results In women with a healthy BMI in 2000, there were clear dose-response relationships between accumulated PA and transition to overweight (P=.03) and obesity (P<.01) between 2000 and 2012. The critical periods analysis indicated that very active levels of PA at the 2006 survey (when the women were 28-33 years old) and active or very active PA at the 2009 survey (age 31-36 years) were most protective against transitioning to overweight and obesity. Conclusion These findings confirm that maintenance of very high PA levels throughout young adulthood will significantly reduce the risk of becoming overweight or obese. There seems to be a critical period for maintaining high levels of activity at the life stage when many women face competing demands of caring for infants and young children.

Carbon dynamics in peatlands under changing hydrology : Effects of water level drawdown on litter quality, microbial enzyme activities and litter decomposition rates

Pristine peatlands are carbon (C) accumulating wetland ecosystems sustained by a high water level (WL) and consequent anoxia that slows down decomposition. Persistent WL drawdown as a response to climate and/or land-use change directly affects decomposition: increased oxygenation stimulates decomposition of the old C (peat) sequestered under prior anoxic conditions. Responses of the new C (plant litter) in terms of quality, production and decomposability, and the consequences for the whole C cycle of peatlands are not fully understood. WL drawdown induces changes in plant community resulting in shift in dominance from Sphagnum and graminoids to shrubs and trees. There is increasing evidence that the indirect effects of WL drawdown via the changes in plant communities will have more impact on the ecosystem C cycling than any direct effects. The aim of this study is to disentangle the direct and indirect effects of WL drawdown on the new C by measuring the relative importance of 1) environmental parameters (WL depth, temperature, soil chemistry) and 2) plant community composition on litter production, microbial activity, litter decomposition rates and, consequently, on the C accumulation. This information is crucial for modelling C cycle under changing climate and/or land-use. The effects of WL drawdown were tested in a large-scale experiment with manipulated WL at two time scales and three nutrient regimes. Furthermore, the effect of climate on litter decomposability was tested along a north-south gradient. Additionally, a novel method for estimating litter chemical quality and decomposability was explored by combining Near infrared spectroscopy with multivariate modelling. WL drawdown had direct effects on litter quality, microbial community composition and activity and litter decomposition rates. However, the direct effects of WL drawdown were overruled by the indirect effects via changes in litter type composition and production. Short-term (years) responses to WL drawdown were small. In long-term (decades), dramatically increased litter inputs resulted in large accumulation of organic matter in spite of increased decomposition rates. Further, the quality of the accumulated matter greatly changed from that accumulated in pristine conditions. The response of a peatland ecosystem to persistent WL drawdown was more pronounced at sites with more nutrients. The study demonstrates that the shift in vegetation composition as a response to climate and/or land-use change is the main factor affecting peatland ecosystem C cycle and thus dynamic vegetation is a necessity in any models applied for estimating responses of C fluxes to changes in the environment. The time scale for vegetation changes caused by hydrological changes needs to extend to decades. This study provides grouping of litter types (plant species and part) into functional types based on their chemical quality and/or decomposability that the models could utilize. Further, the results clearly show a drop in soil temperature as a response to WL drawdown when an initially open peatland converts into a forest ecosystem, which has not yet been considered in the existing models.

Probabilistic evaluation of seismic soil liquefaction potential based on SPT data

The performance-based liquefaction potential analysis was carried out in the present study to estimate the liquefaction return period for Bangalore, India, through a probabilistic approach. In this approach, the entire range of peak ground acceleration (PGA) and earthquake magnitudes was used in the evaluation of liquefaction return period. The seismic hazard analysis for the study area was done using probabilistic approach to evaluate the peak horizontal acceleration at bed rock level. Based on the results of the multichannel analysis of surface wave, it was found that the study area belonged to site class D. The PGA values for the study area were evaluated for site class D by considering the local site effects. The soil resistance for the study area was characterized using the standard penetration test (SPT) values obtained from 450 boreholes. These SPT data along with the PGA values obtained from the probabilistic seismic hazard analysis were used to evaluate the liquefaction return period for the study area. The contour plot showing the spatial variation of factor of safety against liquefaction and the corrected SPT values required for preventing liquefaction for a return period of 475 years at depths of 3 and 6 m are presented in this paper. The entire process of liquefaction potential evaluation, starting from collection of earthquake data, identifying the seismic sources, evaluation of seismic hazard and the assessment of liquefaction return period were carried out, and the entire analysis was done based on the probabilistic approach.

Litter quality and its response to water level drawdown in boreal peatlands at plant species and community level

Changes in the structure of plant communities may have much more impact on ecosystem carbon (C) cycling than any phenotypic responses to environmental changes. We studied these impacts via the response of plant litter quality, at the level of species and community, to persistent water-level (WL) drawdown in peatlands. We studied three sites with different nutrient regimes, and water-level manipulations at two time scales. The parameters used to characterize litter quality included extractable substances, cellulose, holocellulose, composition of hemicellulose (neutral sugars, uronic acids), Klason lignin, CuO oxidation phenolic products, and concentrations of C and several nutrients. The litters formed four chemically distinct groups: non-graminoid foliar litters, graminoids, mosses and woody litters. Direct effects of WL drawdown on litter quality at the species level were overruled by indirect effects via changes in litter type composition. The pristine conditions were characterized by Sphagnum moss and graminoid litters. Short-term (years) responses of the litter inputs to WL drawdown were small. In longterm (decades), total litter inputs increased, due to increased tree litter inputs. Simultaneously, the litter type composition and its chemical quality at the community level greatly changed. The changes that we documented will strongly affect soil properties and C cycle of peatlands.

Response of fungal and actinobacterial communities to water-level drawdown in boreal peatland sites

"We used PCR-DGGE fingerprinting and direct sequencing to analyse the response of fungal and actinobacterial communities to changing hydrological conditions at 3 different sites in a boreal peatland complex in Finland. The experimental design involved a short-term (3 years; STD) and a long-term (43 years; LTD) water-level drawdown. Correspondence analyses of DGGE bands revealed differences in the communities between natural sites representing the nutrient-rich mesotrophic fen, the nutrient-poorer oligotrophic fen, and the nutrient-poor ombrotrophic bog. Still, most fungi and actinobacteria found in the pristine peatland seemed robust to the environmental variables. Both fungal and actinobacterial diversity was higher in the fens than in the bog. Fungal diversity increased significantly after STD whereas actinobacterial diversity did not respond to hydrology. Both fungal and actinobacterial communities became more similar between peatland types after LTD, which was not apparent after STD. Most sequences clustered equally between the two main fungal phyla Ascomycota and Basidiomycota. Sequencing revealed that basidiomycetes may respond more (either positively or negatively) to hydrological changes than ascomycetes. Overall, our results suggest that fungal responses to water-level drawdown depend on peatland type. Actinobacteria seem to be less sensitive to hydrological changes, although the response of some may similarly depend on peatland type. (C) 2009 Elsevier Ltd. All rights reserved."

High-risk patients and high-risk grafts in infrainguinal bypass for critical limb ischaemia

The main purpose of revascularization procedures for critical limb ischaemia (CLI) is to preserve the leg and sustain the patient s ambulatory status. Other goals are ischaemic pain relief and healing of ischaemic ulcers. Patients with CLI are usually old and have several comorbidities affecting the outcome. Revascularization for CLI is meaningless unless both life and limb are preserved. Therefore, the knowledge of both patient- and bypass-related risk factors is of paramount importance in clinical decision-making, patient selection and resource allocation. The aim of this study was to identify patient- and graft-related predictors of impaired outcome after infrainguinal bypass for CLI. The purpose was to assess the outcome of high-risk patients undergoing infrainguinal bypass and to evaluate the usefulness of specific risk scoring methods. The results of bypasses in the absence of optimal vein graft material were also evaluated, and the feasibility of the new method of scaffolding suboptimal vein grafts was assessed. The results of this study showed that renal insufficiency - not only renal failure but also moderate impairment in renal function - seems to be a significant risk factor for both limb loss and death after infrainguinal bypass in patients with CLI. Low estimated GFR (PIENEMPI KUIN 30 ml/min/1.73 m2) is a strong independent marker of poor prognosis. Furthermore, estimated GFR is a more accurate predictor of survival and leg salvage after infrainguinal bypass in CLI patients than serum creatinine level alone. We also found out that the life expectancy of octogenarians with CLI is short. In this patient group endovascular revascularization is associated with a better outcome than bypass in terms of survival, leg salvage and amputation-free survival especially in presence of coronary artery disease. This study was the first one to demonstrate that Finnvasc and modified Prevent III risk scoring methods both predict the long-term outcome of patients undergoing both surgical and endovascular infrainguinal revascularization for CLI. Both risk scoring methods are easy to use and might be helpful in clinical practice as an aid in preoperative patient selection and decision-making. Similarly than in previous studies, we found out that a single-segment great saphenous vein graft is superior to any other autologous vein graft in terms of mid-term patency and leg salvage. However, if optimal vein graft is lacking, arm vein conduits are superior to prosthetic grafts especially in infrapopliteal bypasses for CLI. We studied also the new method of scaffolding suboptimal quality vein grafts and found out that this method may enable the use of vein grafts of compromised quality otherwise unsuitable for bypass grafting. The remarkable finding was that patients with the combination of high operative risk due to severe comorbidities and risk graft have extremely poor survival, suggesting that only relatively fit patients should undergo complex bypasses with risk grafts. The results of this study can be used in clinical practice as an aid in preoperative patient selection and decision-making. In the future, the need of vascular surgery will increase significantly as the elderly and diabetic population increases, which emphasises the importance of focusing on those patients that will gain benefit from infrainguinal bypass. Therefore, the individual risk of the patient, ambulatory status, outcome expectations, the risk of bypass procedure as well as technical factors such as the suitability of outflow anatomy and the available vein material should all be assessed and taken into consideration when deciding on the best revascularization strategy.

Microbiological characterisation of soils : Evaluation of some critical steps in data collection and experimental design

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.