Electron and proton transfer in NADH:ubiquinone oxidoreductase (Complex I) from Escherichia coli

Cells of every living organism on our planet − bacterium, plant or animal − are organized in such a way that despite differences in structure and function they utilize the same metabolic energy represented by electrochemical proton gradient across a membrane. This gradient of protons is generated by the series of membrane bound multisubunit proteins, Complex I, II, III and IV, organized in so-called respiratory or electron transport chain. In the eukaryotic cell it locates in the inner mitochondrial membrane while in the bacterial cell it locates in the cytoplasmic membrane. The function of the respiratory chain is to accept electrons from NADH and ubiquinol and transfer them to oxygen resulting in the formation of water. The free energy released upon these redox reactions is converted by respiratory enzymes into an electrochemical proton gradient, which is used for synthesis of ATP as well as for many other energy dependent processes. This thesis is focused on studies of the first member of the respiratory chain − NADH:ubiquinone oxidoreductase or Complex I. This enzyme has a boot-shape structure with hydrophilic and hydrophobic domains, the former of which has all redox groups of the protein, the flavin and eight to nine iron-sulfur clusters. Complex I serves as a proton pump coupling transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the membrane. So far the mechanism of energy transduction by Complex I is unknown. In the present study we applied a set of different methods to study the electron and proton transfer reactions in Complex I from Escherichia coli. The main achievement was the experiment that showed that the electron transfer through the hydrophilic domain of Complex I is unlikely to be coupled to proton transfer directly or to conformational changes in the protein. In this work for the first time properties of all redox centers of Complex I were characterized in the intact purified bacterial enzyme. We also probed the role of several conserved amino acid residues in the electron transfer of Complex I. Finally, we found that highly conserved amino acid residues in several membrane subunits form a common pattern with a very prominent feature – the presence of a few lysines within the membrane. Based on the experimental data, we suggested a tentative principle which may govern the redox-coupled proton pumping in Complex I.

Impacts of agriculture on amphibians at multiple scales

Agriculture-mediated habitat loss and degradation together with climate change are among the greatest global threats to species, communities, and ecosystem functioning. During the last century, more than 50% of the world's wetlands have been lost and agricultural activities have subjected wetland species to increased isolation and decreased quality of habitats. Likewise, as a part of agricultural intensification, the use of pesticides has increased notably, and pesticide residues occur frequently in wetlands making the exposure of wetland organisms to pesticides highly probable. In this thesis, a set of ecotoxicological and landscape ecological studies were carried out to investigate pesticide-effects on tadpoles, and species-habitat relationships of amphibians in agricultural landscapes. The results show that the fitness of R. temporaria tadpoles can be negatively affected by sublethal pesticide concentrations, and that pesticides may increase the costs of response to natural environmental stressors. However, tadpoles may also be able to compensate for some of the negative effects of pesticides. The results further demonstrate that both historic and current-day agricultural land use can negatively impact amphibians, but that in some cases the costs of living in agricultural habitats may only become apparent when amphibians face other environmental stressors, such as drought. Habitat heterogeneity may, however, increase the persistence of amphibians in agricultural landscapes. Hence, the results suggest that amphibians are likely to be affected by agricultural processes that operate at several spatial and temporal scales, and that it is probable that various processes related to current-day agriculture will affect both larval and adult amphibians. The results imply that maintaining dense wetland patterns could enhance persistence of amphibian populations in agricultural habitats, and indicate that heterogeneous landscapes may lower the risk of regional amphibian population declines under extreme weather perturbations.

From environmental concerns towards sustainable food provisioning. : Material flow and food consumption scenario studies on sustainability of agri-food systems

Agriculture is an economic activity that heavily relies on the availability of natural resources. Through its role in food production agriculture is a major factor affecting public welfare and health, and its indirect contribution to gross domestic product and employment is significant. Agriculture also contributes to numerous ecosystem services through management of rural areas. However, the environmental impact of agriculture is considerable and reaches far beyond the agroecosystems. The questions related to farming for food production are, thus, manifold and of great public concern. Improving environmental performance of agriculture and sustainability of food production, sustainabilizing food production, calls for application of wide range of expertise knowledge. This study falls within the field of agro-ecology, with interphases to food systems and sustainability research and exploits the methods typical of industrial ecology. The research in these fields extends from multidisciplinary to interdisciplinary and transdisciplinary, a holistic approach being the key tenet. The methods of industrial ecology have been applied extensively to explore the interaction between human economic activity and resource use. Specifically, the material flow approach (MFA) has established its position through application of systematic environmental and economic accounting statistics. However, very few studies have applied MFA specifically to agriculture. The MFA approach was used in this thesis in such a context in Finland. The focus of this study is the ecological sustainability of primary production. The aim was to explore the possibilities of assessing ecological sustainability of agriculture by using two different approaches. In the first approach the MFA-methods from industrial ecology were applied to agriculture, whereas the other is based on the food consumption scenarios. The two approaches were used in order to capture some of the impacts of dietary changes and of changes in production mode on the environment. The methods were applied at levels ranging from national to sector and local levels. Through the supply-demand approach, the viewpoint changed between that of food production to that of food consumption. The main data sources were official statistics complemented with published research results and expertise appraisals. MFA approach was used to define the system boundaries, to quantify the material flows and to construct eco-efficiency indicators for agriculture. The results were further elaborated for an input-output model that was used to analyse the food flux in Finland and to determine its relationship to the economy-wide physical and monetary flows. The methods based on food consumption scenarios were applied at regional and local level for assessing feasibility and environmental impacts of relocalising food production. The approach was also used for quantification and source allocation of greenhouse gas (GHG) emissions of primary production. GHG assessment provided, thus, a means of crosschecking the results obtained by using the two different approaches. MFA data as such or expressed as eco-efficiency indicators, are useful in describing the overall development. However, the data are not sufficiently detailed for identifying the hot spots of environmental sustainability. Eco-efficiency indicators should not be bluntly used in environmental assessment: the carrying capacity of the nature, the potential exhaustion of non-renewable natural resources and the possible rebound effect need also to be accounted for when striving towards improved eco-efficiency. The input-output model is suitable for nationwide economy analyses and it shows the distribution of monetary and material flows among the various sectors. Environmental impact can be captured only at a very general level in terms of total material requirement, gaseous emissions, energy consumption and agricultural land use. Improving environmental performance of food production requires more detailed and more local information. The approach based on food consumption scenarios can be applied at regional or local scales. Based on various diet options the method accounts for the feasibility of re-localising food production and environmental impacts of such re-localisation in terms of nutrient balances, gaseous emissions, agricultural energy consumption, agricultural land use and diversity of crop cultivation. The approach is applicable anywhere, but the calculation parameters need to be adjusted so as to comply with the specific circumstances. The food consumption scenario approach, thus, pays attention to the variability of production circumstances, and may provide some environmental information that is locally relevant. The approaches based on the input-output model and on food consumption scenarios represent small steps towards more holistic systemic thinking. However, neither one alone nor the two together provide sufficient information for sustainabilizing food production. Environmental performance of food production should be assessed together with the other criteria of sustainable food provisioning. This requires evaluation and integration of research results from many different disciplines in the context of a specified geographic area. Foodshed area that comprises both the rural hinterlands of food production and the population centres of food consumption is suggested to represent a suitable areal extent for such research. Finding a balance between the various aspects of sustainability is a matter of optimal trade-off. The balance cannot be universally determined, but the assessment methods and the actual measures depend on what the bottlenecks of sustainability are in the area concerned. These have to be agreed upon among the actors of the area

Spatial ecology of food webs : herbivore-parasitoid communities on the pedunculate oak

Herbivorous insects, their host plants and natural enemies form the largest and most species-rich communities on earth. But what forces structure such communities? Do they represent random collections of species, or are they assembled by given rules? To address these questions, food webs offer excellent tools. As a result of their versatile information content, such webs have become the focus of intensive research over the last few decades. In this thesis, I study herbivore-parasitoid food webs from a new perspective: I construct multiple, quantitative food webs in a spatially explicit setting, at two different scales. Focusing on food webs consisting of specialist herbivores and their natural enemies on the pedunculate oak, Quercus robur, I examine consistency in food web structure across space and time, and how landscape context affects this structure. As an important methodological development, I use DNA barcoding to resolve potential cryptic species in the food webs, and to examine their effect on food web structure. I find that DNA barcoding changes our perception of species identity for as many as a third of the individuals, by reducing misidentifications and by resolving several cryptic species. In terms of the variation detected in food web structure, I find surprising consistency in both space and time. From a spatial perspective, landscape context leaves no detectable imprint on food web structure, while species richness declines significantly with decreasing connectivity. From a temporal perspective, food web structure remains predictable from year to year, despite considerable species turnover in local communities. The rate of such turnover varies between guilds and species within guilds. The factors best explaining these observations are abundant and common species, which have a quantitatively dominant imprint on overall structure, and suffer the lowest turnover. By contrast, rare species with little impact on food web structure exhibit the highest turnover rates. These patterns reveal important limitations of modern metrics of quantitative food web structure. While they accurately describe the overall topology of the web and its most significant interactions, they are disproportionately affected by species with given traits, and insensitive to the specific identity of species. As rare species have been shown to be important for food web stability, metrics depicting quantitative food web structure should then not be used as the sole descriptors of communities in a changing world. To detect and resolve the versatile imprint of global environmental change, one should rather use these metrics as one tool among several.

Mechanisms of azole resistance and carcinogenic acetaldehyde production in chronic mucosal candidosis

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS1) is an autoimmune disease caused by a loss-of function mutation in the autoregulator gene (AIRE). Patients with APECED suffer from chronic mucocutaneous candidosis (CMC) of the oral cavity and oesophagus often since early childhood. The patients are mainly colonized with Candida albicans and decades of exposure to antifungal agents have lead to the development of clinical and microbiological resistance in the treatment of CMC in the APECED patient population in Finland. A high incidence of oral squamous cell carcinoma is associated with oral CMC lesions in the APECED patients over the age of 25. The overall aim of this study was firstly, to investigate the effect of long-term azole exposure on the metabolism of oral C. albicans isolates from APECED patients with CMC and secondly, to analyse the specific molecular mechanisms that are responsible for these changes. The aim of the first study was to examine C. albicans strains from APECED patients and the level of cross-resistance to miconazole, the recommended topical compound for the treatment of oral candidosis. A total of 16% of the strains had decreased susceptibility to miconazole and all of these isolates had decreased susceptibility to fluconazole. Miconazole MICs also correlated with MICs to voriconazole and posaconazole. A significant positive correlation between the years of miconazole exposure and the MICs to azole antifungal agents was also found. These included azoles the patients had not been exposed to. The aim of our second study was to determine if the APECED patients are continuously colonized with the same C. albicans strains despite extensive antifungal treatment and to gain a deeper insight into the genetic changes leading to azole resistance. The strains were typed using MLST and our results confirmed that all patients were persistently colonized with the same or a genetically related strain despite antifungal treatment between isolations. No epidemic strains were found. mRNA expression was analysed by Northern blotting, protein level by western blotting, and TAC1 and ERG11 genes were sequenced. The main molecular mechanisms resulting in azole resistance were gain-of-function mutations in TAC1 leading to over expression of CDR1 and CDR2, genes linked to azole resistance. Several strains had also developed point mutations in ERG11, another gene linked to azole resistance. In the third study we used gas chromatography to test whether the level of carcinogenic acetaldehyde produced by C. albicans strains isolated from APECED patients were different from the levels produced by strains isolated from healthy controls and oral carcinoma patients. Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast, acetaldehyde is a by-product of the pyruvate bypass that converts pyruvate into acetyl-CoA during fermentation. Our results showed that strains isolated from APECED patients produced mutagenic levels of acetaldehyde in the presence of glucose (100mM, 18g/l) and the levels produced were significantly higher than those from strains isolated from controls and oral carcinoma patients. All strains in the study, however, were found to produce mutagenic levels of acetaldehyde in the presence of ethanol (11mM). The glucose and ethanol levels used in this study are equivalent to those found in food and beverages and our results highlight the role of dietary sugars and ethanol on carcinogenesis. The aims of our fourth study were to research the effect of growth conditions in the levels of acetaldehyde produced by C. albicans and to gain deeper insight into the role of different genes in the pyruvate-bypass in the production of high acetaldehyde levels. Acetaldehyde production in the presence of glucose increased by 17-fold under moderately hypoxic conditions compared to the levels produced under normoxic conditions. Under moderately hypoxic conditions acetaldehyde levels did not correlate with the expression of ADH1 and ADH2, genes catalyzing the oxidation of ethanol to acetaldehyde, or PDC11, the gene catalyzing the oxidation of pyruvate to acetaldehyde but correlated with the expression of down-stream genes ALD6 and ACS1. Our results highlight a problem where indiscriminate use of azoles may influence azole susceptibility and lead to the development of cross-resistance. Despite clinically successful treatment leading to relief of symptoms, colonization by C. albicans strains is persistent within APECED patients. Microevolution and point mutations that occur in strains may lead to the development of azole-resistant isolates and metabolic changes leading to increased production of carcinogenic acetaldehyde.

Non-enzymatic Degradation of (1→3)(1→4)-β-D-glucan in Aqueous Processing of Oats

Cereal water-soluble β-glucan [(1→3)(1→4)-β-D-glucan] has well-evidenced health benefits and it contributes to the texture properties of foods. These functions are characteristically dependent on the excellent viscosity forming ability of this cell wall polysaccharide. The viscosity is affected by the molar mass, solubility and conformation of β-glucan molecule, which are further known to be altered during food processing. This study focused on demonstrating the degradation of β-glucan in water solutions following the addition of ascorbic acid, during heat treatments or high pressure homogenisation. Furthermore, the motivation of this study was in the non-enzymatic degradation mechanisms, particularly in oxidative cleavage via hydroxyl radicals. The addition of ascorbic acid at food-related concentrations (2-50 mM), autoclaving (120°C) treatments, and high pressure homogenisation (300-1000 bar) considerably cleaved the β-glucan chains, determined as a steep decrease in the viscosity of β-glucan solutions and decrease in the molar mass of β-glucan. The cleavage was more intense in a solution of native β-glucan with co-extracted compounds than in a solution of highly purified β-glucan. Despite the clear and immediate process-related degradation, β-glucan was less sensitive to these treatments compared to other water-soluble polysaccharides previously reported in the literature. In particular, the highly purified β-glucan was relatively resistant to the autoclaving treatments without the addition of ferrous ions. The formation of highly oxidative free radicals was detected at the elevated temperatures, and the formation was considerably accelerated by added ferrous ions. Also ascorbic acid pronounced the formation of these oxidative radicals, and oxygen was simultaneously consumed by ascorbic acid addition and by heating the β-glucan solutions. These results demonstrated the occurrence of oxidative reactions, most likely the metal catalysed Fenton-like reactions, in the β-glucan solutions during these processes. Furthermore, oxidized functional groups (carbonyls) were formed along the β-glucan chain by the treatments, including high pressure homogenisation, evidencing the oxidation of β-glucan by these treatments. The degradative forces acting on the particles in the high pressure homogenisation are generally considered to be the mechanical shear, but as shown here, carbohydrates are also easily degraded during the process, and oxidation may have a role in the modification of polysaccharides by this technique. In the present study, oat β-glucan was demonstrated to be susceptible to degradation during aqueous processing by non-enzymatic degradation mechanisms. Oxidation was for the first time shown to be a highly relevant degradation mechanism of β-glucan in food processing.

Immunochemical analysis of prolamins in gluten-free foods

People with coeliac disease have to maintain a gluten-free diet, which means excluding wheat, barley and rye prolamin proteins from their diet. Immunochemical methods are used to analyse the harmful proteins and to control the purity of gluten-free foods. In this thesis, the behaviour of prolamins in immunological gluten assays and with different prolamin-specific antibodies was examined. The immunoassays were also used to detect residual rye prolamins in sourdough systems after enzymatic hydrolysis and wheat prolamins after deamidation. The aim was to characterize the ability of the gluten analysis assays to quantify different prolamins in varying matrices in order to improve the accuracy of the assays. Prolamin groups of cereals consist of a complex mixture of proteins that vary in their size and amino acid sequences. Two common characteristics distinguish prolamins from other cereal proteins. Firstly, they are soluble in aqueous alcohols, and secondly, most of the prolamins are mainly formed from repetitive amino acid sequences containing high amounts of proline and glutamine. The diversity among prolamin proteins sets high requirements for their quantification. In the present study, prolamin contents were evaluated using enzyme-linked immunosorbent assays based on ω- and R5 antibodies. In addition, assays based on A1 and G12 antibodies were used to examine the effect of deamidation on prolamin proteins. The prolamin compositions and the cross-reactivity of antibodies with prolamin groups were evaluated with electrophoretic separation and Western blotting. The results of this thesis research demonstrate that the currently used gluten analysis methods are not able to accurately quantify barley prolamins, especially when hydrolysed or mixed in oats. However, more precise results can be obtained when the standard more closely matches the sample proteins, as demonstrated with barley prolamin standards. The study also revealed that all of the harmful prolamins, i.e. wheat, barley and rye prolamins, are most efficiently extracted with 40% 1-propanol containing 1% dithiothreitol at 50 °C. The extractability of barley and rye prolamins was considerably higher with 40% 1-propanol than with 60% ethanol, which is typically used for prolamin extraction. The prolamin levels of rye were lowered by 99.5% from the original levels when an enzyme-active rye-malt sourdough system was used for prolamin degradation. Such extensive degradation of rye prolamins suggest the use of sourdough as a part of gluten-free baking. Deamidation increases the diversity of prolamins and improves their solubility and ability to form structures such as emulsions and foams. Deamidation changes the protein structure, which has consequences for antibody recognition in gluten analysis. According to the resuts of the present work, the analysis methods were not able to quantify wheat gluten after deamidation except at very high concentrations. Consequently, deamidated gluten peptides can exist in food products and remain undetected, and thus cause a risk for people with gluten intolerance. The results of this thesis demonstrate that current gluten analysis methods cannot accurately quantify prolamins in all food matrices. New information on the prolamins of rye and barley in addition to wheat prolamins is also provided in this thesis, which is essential for improving gluten analysis methods so that they can more accurately quantify prolamins from harmful cereals.

Consumers' attitudes towards food prices

High food prices can be a barrier to healthy eating because some of the food products may be perceived as expensive. Understanding the role of price in food purchase situations is important, but only a few studies document attitudes towards expensiveness or cheapness in foods. In this thesis, the role of food price in food choice and consumers attitudes towards food prices were investigated and the aim was to measure the food price attitudes. Food price attitudes were hypothesized to have an impact on consumers willingness to pay judgements and their willingness to buy premium-priced food products. First, using qualitative data consisting of 40 thematic interviews the experiences of the expensiveness and cheapness in foods were explored by using functional food products as a target product category. Second, a Food Price Attitude Scale was developed using four quantitative surveys representing Finnish consumers (2001 N=1158; 2002 N=1156; 2004a N=1113; 2004b N=1027). Food price attitudes were confirmed to compose a multidimensional construct and consumers may perceive positive and negative attitudes towards both high and low food prices. Finnish consumers were clustered into four groups based on their food price attitudes. In the first group, 29% of respondents were negative towards high food prices and they were willing to seek low food prices, whereas respondents in another group (22%) were positive towards high food prices. Additionally, in the third group consumers (17%) were willing to pay for high quality but still looked for low food prices. In the fourth group, consumers (32%) were willing to look for low food prices, unwilling to pay for high quality, but high-priced food was appreciated if offered to others. It was found in qualitative data that consumers willingness to accept high prices in foods was connected to price fairness and to justifications. Feelings of fairness or unfairness might be a core element of food price attitudes. Using quantitative methods, it was confirmed that positive attitudes towards high food prices in terms of high quality enhanced consumers willingness to buy food products with certain benefits (e.g., a health claim). Additionally, the favourable attitude towards low food prices lowered the willingness to pay estimates. This type of tendency, however, can create a possible bias in small convenient samples. In the food price-related research, it is advisable to take into account food price attitudes as possible background variables. The Food Price Attitude Scale needs further development to increase construct validity even though, in the present study, it was shown to be a reliable measure with good predictive and discriminant validity. The theoretical and managerial implications of the results for a better understanding of the role of price in consumers food purchases are discussed.

The Effect of Food-Related Lifestyle on the Choices of Consumers of Five Food Products

The purpose of this study was to find out whether food-related lifestyle guides and explains product evaluations, specifically, consumer perceptions and choice evaluations of five different food product categories: lettuce, mincemeat, savoury sauce, goat cheese, and pudding. The opinions of consumers who shop in neighbourhood stores were considered most valuable. This study applies means-end chain (MEC) theory, according to which products are seen as means by which consumers attain meaningful goals. The food-related lifestyle (FRL) instrument was created to study lifestyles that reflect these goals. Further, this research has adopted the view that the FRL functions as a script which guides consumer behaviour. Two research methods were used in this study. The first was the laddering interview, the primary aim of which was to gather information for formulating the questionnaire of the main study. The survey consisted of two separate questionnaires. The first was the FRL questionnaire modified for this study. The aim of the other questionnaire was to determine the choice criteria for buying five different categories of food products. Before these analyses could be made, several data modifications were made following MEC analysis procedures. Beside forming FRL dimensions by counting sum-scores from the FRL statements, factor analysis was run in order to elicit latent factors underlying the dimensions. The lifestyle factors found were adventurous, conscientious, enthusiastic, snacking, moderate, and uninvolved lifestyles. The association analyses were done separately for each choice of product as well as for each attribute-consequence linkage with a non-parametric Mann-Whitney U test. The testing variables were FRL dimensions and the FRL lifestyle factors. In addition, the relation between the attribute-consequence linkages and the demographic variables were analysed. Results from this study showed that the choice of product is sequential, so that consumers first categorize products into groups based on specific criteria like health or convenience. It was attested that the food-related lifestyles function as a script in food choice and that the FRL instrument can be used to predict consumer buying behaviour. Certain lifestyles were associated with the choice of each product category. The actual product choice within a product category then appeared to be a different matter. In addition, this study proposes a modification to the FRL instrument. The positive towards advertising FRL dimension was modified to examine many kinds of information search including the internet, TV, magazines, and other people. This new dimension, which was designated as being open to additional information, proved to be very robust and reliable in finding differences in consumer choice behaviour. Active additional information search was linked to adventurous and snacking food-related lifestyles. The results of this study support the previous knowledge that consumers expect to get many benefits simultaneously when they buy food products. This study brought detailed information about the benefits sought, the combination of benefits differing between products and between respondents. Household economy, pleasure and quality were emphasized with the choice of lettuce. Quality was the most significant benefit in choosing mincemeat, but health related benefits were often evaluated as well. The dominant benefits linked to savoury sauce were household economic benefits, expected pleasurable experiences, and a lift in self-respect. The choice of goat cheese appeared not to be an economic decision, self-respect, pleasure, and quality being included in the choice criteria. In choosing pudding, the respondents considered the well-being of family members, and indulged their family members or themselves.