Trafficking and ethanol-induced inhibition of AMPA receptors

AMPA receptors are an important class of ionotropic glutamate receptors which participate in fast excitatory synaptic transmission in most brain areas. They have a pivotal role in adjustment of cell membrane excitability as their cell membrane expression levels is altered in brain physiology such as in learning and memory formation. AMPA receptor function and trafficking is regulated by several proteins, such as transmembrane AMPA receptor regulatory proteins (TARPs). NMDA-type glutamate receptors are important target molecules of ethanol. The role of AMPA receptors in the actions of ethanol has not been clarified as thoroughly. Furthermore, the regulation of AMPA receptor synthesis and their possible adaptation in neurons with altered inhibitory mechanisms are poorly understood. In this thesis work AMPA receptor pharmacology, trafficking and synaptic localization was studied using patch-clamp electrophysiology. Both native and recombinant AMPA receptors were studied. Hippocampal slices from transgenic Thy1alfa6 mice with altered inhibition were used to study adaptation of AMPA receptors. Ethanol was found to inhibit AMPA receptor function by increasing desensitization of the receptor, as the steady-state current was inhibited more than the peak current. Ethanol inhibition was reduced when cyclothiazide was used to block desensitization and when non-desensitizing mutant receptors were studied. Ethanol also increased the rate of desensitization, which was increased further by the coexpression of TARP-proteins. We found that the agonist binding capability is important for trafficking AMPA receptors from endoplasmic reticulum to the cell membrane. TARP rescues the surface expression of non-binding AMPA receptor mutants in HEK293 cells, but not in native neurons. Studies with Thy1alfa6 mice revealed that decreased inhibition decrease AMPA receptor mediated excitation keeping the neurotransmission in balance. Thy1alfa6 mice also had lower sensitivity to electroshock convulsions, presumably due to the decreased AMPA receptor function. The results suggest that during alcohol intoxication ethanol may inhibit AMPA receptors by increasing the rate and the extent of desensitization. TARPs appear to enhance ethanol inhibition. TARPs also participate in trafficking of AMPA receptors upon their synthesis in the cell. AMPA receptors mediate also long-term adaptation to altered neuronal excitability, which adds to their well-known role in synaptic plasticity.

Water-ethanol mixtures by molecular dynamics and x-ray Compton scattering

Water-ethanol mixtures are commonly used in industry and house holds. However, quite surprisingly their molecular-level structure is still not completely understood. In particular, there is evidence that the local intermolecular geometries depend significantly on the concentration. The aim of this study was to gain information on the molecular-level structures of water-ethanol mixtures by two computational methods. The methods are classical molecular dynamics (MD), where the movement of molecules can be studied, and x-ray Compton scattering, in which the scattering cross section is sensitive to the electron momentum density. Firstly, the water-ethanol mixtures were studied with MD simulations, with the mixture concentration ranging from 0 to 100%. For the simulations well-established force fields were used for the water and ethanol molecules (TIP4P and OPLS-AA, respectively). Moreover, two models were used for ethanol, rigid and non-rigid. In the rigid model the intramolecular bond lengths are fixed, whereas in the non-rigid model the lengths are determined by harmonic potentials. Secondly, mixtures with three different concentrations employing both ethanol models were studied by calculating the experimentally observable x-ray quantity, the Compton profile. In the MD simulations a slight underestimation in the density was observed as compared to experiment. Furthermore, a positive excess of hydrogen bonding with water molecules and a negative one with ethanol was quantified. Also, the mixture was found more structured when the ethanol concentration was higher. Negligible differences in the results were found between the two ethanol models. In contrast, in the Compton scattering results a notable difference between the ethanol models was observed. For the rigid model the Compton profiles were similar for all the concentrations, but for the non-rigid model they were distinct. This leads to two possibilities of how the mixing occurs. Either the mixing is similar in all concentrations (as suggested by the rigid model) or the mixing changes for different concentrations (as suggested by the non-rigid model). Either way, this study shows that the choice of the force field is essential in the microscopic structure formation in the MD simulations. When the sources of uncertainty in the calculated Compton profiles were analyzed, it was found that more statistics needs to be collected to reduce the statistical uncertainty in the final results. The obtained Compton scattering results can be considered somewhat preliminary, but clearly indicative of the behaviour of the water-ethanol mixtures when the force field is modified. The next step is to collect more statistics and compare the results with experimental data to decide which ethanol model describes the mixture better. This way, valuable information on the microscopic structure of water-ethanol mixtures can be found. In addition, information on the force fields in the MD simulations and on the ability of the MD simulations to reproduce the microscopic structure of binary liquids is obtained.

Dental Service Utilization, Dental Health Production and Equity in Dental Care: the Finnish Experience

This thesis is grounded on four articles. Article I generally examines the factors affecting dental service utilization. Article II studies the factors associated with sector-specific utilization among young adults entitled to age-based subsidized dental care. Article III explores the determinants of dental ill-health as measured by the occurrence of caries and the relationship between dental ill-health and dental care use. Article IV measures and explains income-related inequality in utilization. Data employed were from the 1996 Finnish Health Care Survey (I, II, IV) and the 1997 follow-up study included in the longitudinal study of the Northern Finland 1966 Birth Cohort (III). Utilization is considered as a multi-stage decision-making process and measured as the number of visits to the dentist. Modified count data models and concentration and horizontal equity indices were applied. Dentist s recall appeared very efficient at stimulating individuals to seek care. Dental pain, recall, and the low number of missing teeth positively affected utilization. Public subvention for dental care did not seem to statistically increase utilization. Among young adults, a perception of insufficient public service availability and recall were positively associated with the choice of a private dentist, whereas income and dentist density were positively associated with the number of visits to private dentists. Among cohort females, factors increasing caries were body mass index and intake of alcohol, sugar, and soft drinks and those reducing caries were birth weight and adolescent school achievement. Among cohort males, caries was positively related to the metropolitan residence and negatively related to healthy diet and education. Smoking increased caries, whereas regular teeth brushing, regular dental attendance and dental care use decreased caries. We found equity in young adults utilization but pro-rich inequity in the total number of visits to all dentists and in the probability of visiting a dentist for the whole sample. We observed inequity in the total number of visits to the dentist and in the probability of visiting a dentist, being pro-poor for public care but pro-rich for private care. The findings suggest that to enhance equal access to and use of dental care across population and income groups, attention should focus on supply factors and incentives to encourage people to contact dentists more often. Lowering co-payments and service fees and improving public availability would likely increase service use in both sectors. To attain favorable oral health, appropriate policies aimed at improving dental health education and reducing the detrimental effects of common risk factors on dental health should be strengthened. Providing equal access with respect to need for all people ought to take account of the segmentation of the service system, with its two parallel delivery systems and different supplier incentives to patients and dentists.

Finnish and Austrian Wood Markets in a Changing Environment

At present the operating environment of sawmills in Europe is changing and there are uncertainties related in raw material supply in many countries. The changes in the operating environment of roundwood markets and the effects followed by these changes have brought up several interesting issues from the viewpoint of research. Lately new factors have been influencing the roundwood markets, such as increasing interest towards wood-based energy and implementation of new energy policies as well as changes in wood trade flows that affect the domestic markets in many countries. This Master’s thesis studies the adaptation ability of Finnish roundwood markets in a changing operating environment, aiming to produce an up-to-date analysis considering new development trends. The study concentrates on the roundwood markets from the viewpoint of sawmill industry since the industry is dependent on the functioning of the markets and sawmills are highly affected by the changes on the roundwood markets. To facilitate international comparison, the study is implemented by comparing Finnish and Austrian roundwood markets and analysing changes happening in the two countries. Finland and Austria share rather similar characteristics in the roundwood market structures, forest resources and forest ownership as well as production of roundwood and sawnwood. In addition they both are big exporters of forest industry products. In this study changes in the operating environment of sawmill industry both in Finland as well as in Austria are compared to each other aiming to recognise the main similarities and differences between the countries. In addition both development possibilities as well as challenges followed by the changes are discussed. The aim of the study is to define the main challenges and possibilities confronted by the actors on the markets and also to find new perspectives to approach these. The study is implemented as a qualitative study. The theoretical framework of the study describes the operating environment of wood markets from the viewpoint of the sawmill industry and represents the effects of supply and demand on the wood markets. The primary research material of the study was gathered by interviewing high level experts of forestry and sawmill industry in both Finland and Austria. The aim was to receive as extensive country specific viewpoint from the markets as possible, hence interviewees represented different parties of the markets. After creating country-specific profiles based on the theoretical framework a cross-country comparison was implemented. As a consequence the main similarities and differences in the operating environment and on the roundwood markets of Finland and Austria were recognized. In addition the main challenges and possibilites were identified. The results of the study offer a wide analysis regarding the main similarities and differences of the wood markets of Finland and Austria and their operating environments as well as concerning challenges and possibilities faced on the markets.

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.

Renewable Energy and Climate Policies: Studies in the Forest and Energy Sector

‪This dissertation examines the impacts of energy and climate policies on the energy and forest sectors, focusing on the case of Finland. The thesis consists of an introduction article and four separate studies. The dissertation was motivated by the climate concern and the increasing demand of renewable energy. In particular, the renewable energy consumption and greenhouse gas emission reduction targets of the European Union were driving this work. In Finland, both forest and energy sectors are in key roles in achieving these targets. In fact, the separation between forest and energy sector is diminishing as the energy sector is utilizing increasing amounts of wood in energy production and as the forest sector is becoming more and more important energy producer.‬ ‪The objective of this dissertation is to find out and measure the impacts of climate and energy policies on the forest and energy sectors. In climate policy, the focus is on emissions trading, and in energy policy the dissertation focuses on the promotion of renewable forest-based energy use. The dissertation relies on empirical numerical models that are based on microeconomic theory. Numerical partial equilibrium mixed complementarity problem models were constructed to study the markets under scrutiny. The separate studies focus on co-firing of wood biomass and fossil fuels, liquid biofuel production in the pulp and paper industry, and the impacts of climate policy on the pulp and paper sector.‬ ‪The dissertation shows that the policies promoting wood-based energy may have have unexpected negative impacts. When feed-in tariff is imposed together with emissions trading, in some plants the production of renewable electricity might decrease as the emissions price increases. The dissertation also shows that in liquid biofuel production, investment subsidy may cause high direct policy costs and other negative impacts when compared to other policy instruments. The results of the dissertation also indicate that from the climate mitigation perspective, perfect competition is the favored wood market competition structure, at least if the emissions trading system is not global.‬ ‪In conclusion, this dissertation suggests that when promoting the use of wood biomass in energy production, the favored policy instruments are subsidies that promote directly the renewable energy production (i.e. production subsidy, renewables subsidy or feed-in premium). Also, the policy instrument should be designed to be dependent on the emissions price or on the substitute price. In addition, this dissertation shows that when planning policies to promote wood-based renewable energy, the goals of the policy scheme should be clear before decisions are made on the choice of the policy instruments.‬