ORP1L, a new Rab7 effector and regulator of late endosome functions

Oxysterol binding protein (OSBP) homologues have been found in eukaryotic organisms ranging from yeast to humans. These evolutionary conserved proteins have in common the presence of an OSBP-related domain (ORD) which contains the fully conserved EQVSHHPP sequence motif. The ORD forms a barrel structure that binds sterols in its interior. Other domains and sequence elements found in OSBP-homologues include pleckstrin homology domains, ankyrin repeats and two phenylalanines in an acidic tract (FFAT) motifs, which target the proteins to distinct subcellular compartments. OSBP homologues have been implicated in a wide range of intracellular processes, including vesicle trafficking, lipid metabolism and cell signaling, but little is known about the functional mechanisms of these proteins. The human family of OSBP homologues consists of twelve OSBP-related proteins (ORP). This thesis work is focused on one of the family members, ORP1, of which two variants were found to be expressed tissue-specifically in humans. The shorter variant, ORP1S contains an ORD only. The N-terminally extended variant, ORP1L, comprises a pleckstrin homology domain and three ankyrin repeats in addition to the ORD. The two ORP1 variants differ in intracellular localization. ORP1S is cytosolic, while the ankyrin repeat region of ORP1L targets the protein to late endosomes/lysosomes. This part of ORP1L also has profound effects on late endosomal morphology, inducing perinuclear clustering of late endosomes. A central aim of this study was to identify molecular interactions of ORP1L on late endosomes. The morphological changes of late endosomes induced by overexpressed ORP1L implies involvement of small Rab GTPases, regulators of organelle motility, tethering, docking and/or fusion, in generation of the phenotype. A direct interaction was demonstrated between ORP1L and active Rab7. ORP1L prolongs the active state of Rab7 by stabilizing its GTP-bound form. The clustering of late endosomes/lysosomes was also shown to be linked to the minus end-directed microtubule-based dynein-dynactin motor complex through the ankyrin repeat region of ORP1L. ORP1L, Rab7 and the Rab7-interacting lysosomal protein (RILP) were found to be part of the same effector complex recruiting the dynein-dynactin complex to late endosomes, thereby promoting minus end-directed movement. The proteins were found to be physically close to each other on late endosomes and RILP was found to stabilize the ORP1L-Rab7 interaction. It is possible that ORP1L and RILP bind to each other through their C-terminal and N-terminal regions, respectively, when they are bridged by Rab7. With the results of this study we have been able to place a member of the uncharacterized OSBP-family, ORP1L, in the endocytic pathway, where it regulates motility and possibly fusion of late endosomes through interaction with the small GTPase Rab7.

Sterol-binding proteins in late endosomes : Regulation of endosome motility and lipid metabolism

Despite its bad reputation in the mass media, cholesterol is an indispensable constituent of cellular membranes and vertebrate life. It is, however, also potentially lethal as it may accumulate in the arterial intima causing atherosclerosis or elsewhere in the body due to inherited conditions. Studying cholesterol in cells, and research on how the cell biology of cholesterol affects on system level is essential for a better understanding of the disease states associated with cholesterol and for the development of new therapies for these conditions. On its way to the cell, exogenous cholesterol traverses through endosomes, transport vesicles involved in internalizing material to cells, and needs to be transported out of this compartment. This endosomal pool of cholesterol is important for understanding both the common disorders of metabolism and the more rare hereditary disorders of cholesterol metabolism. The study of cholesterol in cells has been hampered by the lack of bright fluorescent sterol analogs that would resemble cholesterol enough to be used in cellular studies. In the first study of my thesis, we present a new sterol analog, Boron-Dipyrromethene (BODIPY)-cholesterol for visualizing sterols in living cells and organism. This fluorescent cholesterol derivative is shown to behave similarly to cholesterol both by atomic scale computer simulations and biochemical experiments. We characterize its localization inside different types of living cells and show that it can be used to study sterol trafficking in living organisms. Two sterol binding proteins associated with the endosomal membrane; the Niemann-Pick type C disease protein 1 (NPC1) and the Oxysterol Binding Protein Related Protein 1 (ORP1) are the subjects of the rest of this study. Sensing cholesterol on endosomes, transporting lipids away from this compartment and the effects these lipids play on cellular metabolism are considered. In the second study we characterize how the NPC1 protein affects lipid metabolism. We show that this cholesterol binding protein affects synthesis of triglycerides and that genetic polymorphisms or a genetic defect in the NPC1 gene affect triglyceride on the whole body level. These effects take place via regulation of carbon fluxes to different lipid classes in cells. In the third part we characterize the effects of another endosomal sterol binding protein, ORP1L on the function and motility of endosomes. Specifically we elucidate how a mutation in the ability of ORP1L to bind sterols affects its behavior in cells, and how a change in ORP1L levels in cells affects the localization, degradative capacity and motility of endosomes. In addition we show that ORP1L manipulations affect cholesterol balance also in macrophages, a cell type important for the development of atherosclerosis.

Kestävä kehitys globaalin ajan hyvinvointiyhteiskunnan haasteena

The objective of the dissertation was to determine the concept of sustainable development according to current understanding and to analyze the structuration of sustainable daily life and how it varies between different groups. The present dissertation is both a literature-based theoretical study and data-based empirical research. The theoretical framework of the study was a greated model of the Structuration of Sustainability in Everyday Life. The model is based on a synthesis of Giddens Theory of Structuration (1984), Spaargaren JA van Vliet's Theory of Consumption as Social Practices (2000) and principles of sustainable development. According to the model created, sustainable everyday life is generated in a context of internal and external factors compromising the interests of ecosystems, society and business. The literature used in the thesis included international and national statements on sustainable development and research into sustainability and the transition to sustainable societies. The data were collected at Helsinki Metropolia University of Applied Sciences. The discretionary sample consisted of students of social services (n = 210) and were collected using the semantic differential technique. The data were analyzed using quantitative and qualitative methods. The results showed that the value placed on ecological, economic and social sustainability increased with age. Activity in non governmental organizations was associated with the acceptance of sustainable development as a whole and especially with global responsibility. Women's everyday life promoted sustainability more than men´s. People living in Helsinki had more sustainable ways of living than those living in the surrounding municipalities because of greater recycling and the low importance given to ownership. Prefering vegetarian food turned out to be a real opportunity for a more sustainable way of living because there were few barriers identified. Contradictory human behavior occurred when advanced sustainable consumer were ready to risk their health. The importance of communality was high and it was considered an aspect of health. The most significant obstacles to sustainable development in daily life were high costs, lack of knowledge and busyness. Similar attitudes towards sustainable development translate into different people´s behavior, which indicates complexities of the behaviour change in the context of sustainable development. The role of non governmental organizations is significant in increasing global responsibility. Education presents an opportunity to increase sustainability, but there are challenges to face from system thinking and in understanding entities in a state of transition towards sustainable everyday life. The responsibility of policy makers is paramount because high costs create a barrier to a sustainable way of living. The implementation of the concept of sustainable development should be focused on the planetary ethics which cover humans, animals, plants and ecosystems. Keywords: Sustainable development, sustainable thinking, behaviour change  

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.

A systematic-ecological approach to Baltic Sea ice studies of algae and protists

The seasonal occurrence of sea ice that annually covers almost half the Baltic Sea area provides a unique habitat for halo- and cold temperature-tolerant extremophiles. Baltic Sea ice biology has more than 100 years of tradition that began with the floristic observation of species by the early pioneers using light microscopic techniques that were the only thing available at the time. Since the discovery of life within sea ice, more technologies have become available for taxonomy. Electron microscopy and genetic evidence have been used to identify sea ice biota revealing increased numbers of taxa. Meanwhile ecologists have used light microscopic cell enumeration in addition to the chemical and physical properties of sea ice in attempts to explain the food web structure of sea ice and its functions. Thus, during the Baltic winter, the sea ice hosts more abundant and diverse microbial communities than the water column beneath it. These communities are typically dominated by autotrophic diatoms together with a diverse assortment of dinoflagellates, auto- and heterotrophic flagellates, ciliates, metazoan rotifers and bacteria, which are mostly responsible for the recycling of nutrients. This thesis comprises ecological and systematic studies. In addition to the results of the previous studies carried out on landfast ice, the data presented here provide new insight into the spatial distribution of pelagial sea ice, which has remained largely unexplored. The studies reveal spatial heterogeneity in the pelagial sea ice of the Gulf of Bothnia. There were mismatches in chlorophyll-a concentrations and in photosynthetic efficiencies of the communities studied. The temporal succession was followed and experimental studies performed investigating the community responses towards increased or decreased light in landfast ice in the Gulf of Finland. The systematic studies carried out with established dinoflagellate cultures revealed a new resting cyst belonging to common sea ice dinoflagellate, Scrippsiella hangoei (Schiller) Larsen 1995. The cyst can be used to explain the overwintering of this species during prolonged periods of darkness. The dissimilarities and similarities in the material isolated from the sea ice called for description of a new subspecies Heterocapsa arctica ssp. frigida. The cells obtained in the cultured material were unlike those of the previously described species, necessitating description of ssp. frigida. As a result of its own unique habitus, the subspecies had been noted by Finnish taxonomists during the past three decades and thus its annual occurrence and geographical distribution in the Baltic Sea. This illustrates how combining ecology and systematics increases our understanding of organisms.

The role of cyclase-associated protein (CAP) in actin dynamics during cell motility and morphogenesis

The highly dynamic remodeling of the actin cytoskeleton is responsible for most motile and morphogenetic processes in all eukaryotic cells. In order to generate appropriate spatial and temporal movements, the actin dynamics must be under tight control of an array of actin binding proteins (ABPs). Many proteins have been shown to play a specific role in actin filament growth or disassembly of older filaments. Very little is known about the proteins affecting recycling i.e. the step where newly depolymerized actin monomers are funneled into new rounds of filament assembly. A central protein family involved in the regulation of actin turnover is cyclase-associated proteins (CAP, called Srv2 in budding yeast). This 50-60 kDa protein was first identified from yeast as a suppressor of an activated RAS-allele and a factor associated with adenylyl cyclase. The CAP proteins harbor N-terminal coiled-coil (cc) domain, originally identified as a site for adenylyl cyclase binding. In the N-terminal half is also a 14-3-3 like domain, which is followed by central proline-rich domains and the WH2 domain. In the C-terminal end locates the highly conserved ADP-G-actin binding domain. In this study, we identified two previously suggested but poorly characterized interaction partners for Srv2/CAP: profilin and ADF/cofilin. Profilins are small proteins (12-16 kDa) that bind ATP-actin monomers and promote the nucleotide exchange of actin. The profilin-ATP-actin complex can be directly targeted to the growth of the filament barbed ends capped by Ena/VASP or formins. ADF/cofilins are also small (13-19 kDa) and highly conserved actin binding proteins. They depolymerize ADP-actin monomers from filament pointed ends and remain bound to ADP-actin strongly inhibiting nucleotide exchange. We revealed that the ADP-actin-cofilin complex is able to directly interact with the 14-3-3 like domain at the N-terminal region of Srv2/CAP. The C-terminal high affinity ADP-actin binding site of Srv2/CAP competes with cofilin for an actin monomer. Cofilin can thus be released from Srv2/CAP for the subsequent round of depolymerization. We also revealed that profilin interacts with the first proline-rich region of Srv2/CAP and that the binding occurs simultaneously with ADP-actin binding to C-terminal domain of Srv2/CAP. Both profilin and Srv2/CAP can promote nucleotide exchange of actin monomer. Because profilin has much higher affinity to ATP-actin than Srv2/CAP, the ATP-actin-profilin complex is released for filament polymerization. While a disruption of cofilin binding in yeast Srv2/CAP produces a severe phenotype comparable to Srv2/CAP deletion, an impairment of profilin binding from Srv2/CAP results in much milder phenotype. This suggests that the interaction with cofilin is essential for the function of Srv2/CAP, whereas profilin can also promote its function without direct interaction with Srv2/CAP. We also show that two CAP isoforms with specific expression patterns are present in mice. CAP1 is the major isoform in most tissues, while CAP2 is predominantly expressed in muscles. Deletion of CAP1 from non-muscle cells results in severe actin phenotype accompanied with mislocalization of cofilin to cytoplasmic aggregates. Together these studies suggest that Srv2/CAP recycles actin monomers from cofilin to profilin and thus it plays a central role in actin dynamics in both yeast and mammalian cells.

Substance flow analysis in Finland : Four case studies on N and P flows

Nitrogen (N) and phosphorus (P) are essential elements for all living organisms. However, in excess, they contribute to several environmental problems such as aquatic and terrestrial eutrophication. Globally, human action has multiplied the volume of N and P cycling since the onset of industrialization. The multiplication is a result of intensified agriculture, increased energy consumption and population growth. Industrial ecology (IE) is a discipline, in which human interaction with the ecosystems is investigated using a systems analytical approach. The main idea behind IE is that industrial systems resemble ecosystems, and, like them, industrial systems can then be described using material, energy and information flows and stocks. Industrial systems are dependent on the resources provided by the biosphere, and these two cannot be separated from each other. When studying substance flows, the aims of the research from the viewpoint of IE can be, for instance, to elucidate the ways how the cycles of a certain substance could be more closed and how the flows of a certain substance could be decreased per unit of production (= dematerialization). In Finland, N and P are studied widely in different ecosystems and environmental emissions. A holistic picture comparing different societal systems is, however, lacking. In this thesis, flows of N and P were examined in Finland using substance flow analysis (SFA) in the following four subsystems: I) forest industry and use of wood fuels, II) food production and consumption, III) energy, and IV) municipal waste. A detailed analysis at the end of the 1990s was performed. Furthermore, historical development of the N and P flows was investigated in the energy system (III) and the municipal waste system (IV). The main research sources were official statistics, literature, monitoring data, and expert knowledge. The aim was to identify and quantify the main flows of N and P in Finland in the four subsystems studied. Furthermore, the aim was to elucidate whether the nutrient systems are cyclic or linear, and to identify how these systems could be more efficient in the use and cycling of N and P. A final aim was to discuss how this type of an analysis can be used to support decision-making on environmental problems and solutions. Of the four subsystems, the food production and consumption system and the energy system created the largest N flows in Finland. For the creation of P flows, the food production and consumption system (Paper II) was clearly the largest, followed by the forest industry and use of wood fuels and the energy system. The contribution of Finland to N and P flows on a global scale is low, but when compared on a per capita basis, we are one of the largest producers of these flows, with relatively high energy and meat consumption being the main reasons. Analysis revealed the openness of all four systems. The openness is due to the high degree of internationality of the Finnish markets, the large-scale use of synthetic fertilizers and energy resources and the low recycling rate of many waste fractions. Reduction in the use of fuels and synthetic fertilizers, reorganization of the structure of energy production, reduced human intake of nutrients and technological development are crucial in diminishing the N and P flows. To enhance nutrient recycling and replace inorganic fertilizers, recycling of such wastes as wood ash and sludge could be promoted. SFA is not usually sufficiently detailed to allow specific recommendations for decision-making to be made, but it does yield useful information about the relative magnitude of the flows and may reveal unexpected losses. Sustainable development is a widely accepted target for all human action. SFA is one method that can help to analyse how effective different efforts are in leading to a more sustainable society. SFA's strength is that it allows a holistic picture of different natural and societal systems to be drawn. Furthermore, when the environmental impact of a certain flow is known, the method can be used to prioritize environmental policy efforts.

Multiscale study on hydrogen mobility in metallic fusion divertor material

For achieving efficient fusion energy production, the plasma-facing wall materials of the fusion reactor should ensure long time operation. In the next step fusion device, ITER, the first wall region facing the highest heat and particle load, i.e. the divertor area, will mainly consist of tiles based on tungsten. During the reactor operation, the tungsten material is slowly but inevitably saturated with tritium. Tritium is the relatively short-lived hydrogen isotope used in the fusion reaction. The amount of tritium retained in the wall materials should be minimized and its recycling back to the plasma must be unrestrained, otherwise it cannot be used for fueling the plasma. A very expensive and thus economically not viable solution is to replace the first walls quite often. A better solution is to heat the walls to temperatures where tritium is released. Unfortunately, the exact mechanisms of hydrogen release in tungsten are not known. In this thesis both experimental and computational methods have been used for studying the release and retention of hydrogen in tungsten. The experimental work consists of hydrogen implantations into pure polycrystalline tungsten, the determination of the hydrogen concentrations using ion beam analyses (IBA) and monitoring the out-diffused hydrogen gas with thermodesorption spectrometry (TDS) as the tungsten samples are heated at elevated temperatures. Combining IBA methods with TDS, the retained amount of hydrogen is obtained as well as the temperatures needed for the hydrogen release. With computational methods the hydrogen-defect interactions and implantation-induced irradiation damage can be examined at the atomic level. The method of multiscale modelling combines the results obtained from computational methodologies applicable at different length and time scales. Electron density functional theory calculations were used for determining the energetics of the elementary processes of hydrogen in tungsten, such as diffusivity and trapping to vacancies and surfaces. Results from the energetics of pure tungsten defects were used in the development of an classical bond-order potential for describing the tungsten defects to be used in molecular dynamics simulations. The developed potential was utilized in determination of the defect clustering and annihilation properties. These results were further employed in binary collision and rate theory calculations to determine the evolution of large defect clusters that trap hydrogen in the course of implantation. The computational results for the defect and trapped hydrogen concentrations were successfully compared with the experimental results. With the aforedescribed multiscale analysis the experimental results within this thesis and found in the literature were explained both quantitatively and qualitatively.

Carbon dioxide and methane exchange between a boreal pristine lake and the atmosphere

Lakes serve as sites for terrestrially fixed carbon to be remineralized and transferred back to the atmosphere. Their role in regional carbon cycling is especially important in the Boreal Zone, where lakes can cover up to 20% of the land area. Boreal lakes are often characterized by the presence of a brown water colour, which implies high levels of dissolved organic carbon from the surrounding terrestrial ecosystem, but the load of inorganic carbon from the catchment is largely unknown. Organic carbon is transformed to methane (CH4) and carbon dioxide (CO2) in biological processes that result in lake water gas concentrations that increase above atmospheric equilibrium, thus making boreal lakes as sources of these important greenhouse gases. However, flux estimates are often based on sporadic sampling and modelling and actual flux measurements are scarce. Thus, the detailed temporal flux dynamics of greenhouse gases are still largely unknown. ----- One aim here was to reveal the natural dynamics of CH4 and CO2 concentrations and fluxes in a small boreal lake. The other aim was to test the applicability of a measuring technique for CO2 flux, i.e. the eddy covariance (EC) technique, and a computational method for estimation of primary production and community respiration, both commonly used in terrestrial research, in this lake. Continuous surface water CO2 concentration measurements, also needed in free-water applications to estimate primary production and community respiration, were used over two open water periods in a study of CO2 concentration dynamics. Traditional methods were also used to measure gas concentration and fluxes. The study lake, Valkea-Kotinen, is a small, humic, headwater lake within an old-growth forest catchment with no local anthropogenic disturbance and thus possible changes in gas dynamics reflect the natural variability in lake ecosystems. CH4 accumulated under the ice and in the hypolimnion during summer stratification. The surface water CH4 concentration was always above atmospheric equilibrium and thus the lake was a continuous source of CH4 to the atmosphere. However, the annual CH4 fluxes were small, i.e. 0.11 mol m-2 yr-1, and the timing of fluxes differed from that of other published estimates. The highest fluxes are usually measured in spring after ice melt but in Lake Valkea-Kotinen CH4 was effectively oxidised in spring and highest effluxes occurred in autumn after summer stratification period. CO2 also accumulated under the ice and the hypolimnetic CO2 concentration increased steadily during stratification period. The surface water CO2 concentration was highest in spring and in autumn, whereas during the stable stratification it was sometimes under atmospheric equilibrium. It showed diel, daily and seasonal variation; the diel cycle was clearly driven by light and thus reflected the metabolism of the lacustrine ecosystem. However, the diel cycle was sometimes blurred by injection of hypolimnetic water rich in CO2 and the surface water CO2 concentration was thus controlled by stratification dynamics. The highest CO2 fluxes were measured in spring, autumn and during those hypolimnetic injections causing bursts of CO2 comparable with the spring and autumn fluxes. The annual fluxes averaged 77 (±11 SD) g C m-2 yr-1. In estimating the importance of the lake in recycling terrestrial carbon, the flux was normalized to the catchment area and this normalized flux was compared with net ecosystem production estimates of -50 to 200 g C m-2 yr-1 from unmanaged forests in corresponding temperature and precipitation regimes in the literature. Within this range the flux of Lake Valkea-Kotinen yielded from the increase in source of the surrounding forest by 20% to decrease in sink by 5%. The free water approach gave primary production and community respiration estimates of 5- and 16-fold, respectively, compared with traditional bottle incubations during a 5-day testing period in autumn. The results are in parallel with findings in the literature. Both methods adopted from the terrestrial community also proved useful in lake studies. A large percentage of the EC data was rejected, due to the unfulfilled prerequisites of the method. However, the amount of data accepted remained large compared with what would be feasible with traditional methods. Use of the EC method revealed underestimation of the widely used gas exchange model and suggests simultaneous measurements of actual turbulence at the water surface with comparison of the different gas flux methods to revise the parameterization of the gas transfer velocity used in the models.

The prehistory of Suomussalmi, eastern Finland; the first billion years as revealed by isotopes and the composition of granitoid suites

The four papers summarized in this thesis deal with the Archean and earliest Paleoproterozoic granitoid suites observed in the Suomussalmi district, eastern Finland. Geologically, the area belongs to the Kianta Complex of the Western Karelian Terrane in the Karelian Province of the Fennoscandian shield. The inherited zircons up to 3440 Ma old together with Sm Nd and Pb Pb data confirm the existence of previously anticipated Paleoarchean protocrust in Suomussalmi. The general timeline of granitoid magmatism is similar to that of the surrounding areas. TTG magmatism occurred in three distinct phases: ca 2.95 Ga, 2.83 2.78 Ga and 2.76 2.74 Ga. In Suomussalmi the TTGs sensu stricto (K2O/Na2O less than 0.5) belong to the low-HREE type and are interpreted as partial melts of garnet amphibolites, which did not significantly interact with mantle peridotites. Transitional TTGs (K2O/Na2O more than 0.5), present in Suomussalmi and absent from surrounding areas, display higher LILE concentrations, but otherwise closely resemble the TTGs sensu stricto and indicate that recycling of felsic crust commenced in Suomussalmi 200 Ma earlier than in surrounding areas. The youngest TTG phase was coeval with the intrusion of the Likamännikkö quartz alkali feldspar syenite (2741 ± 2 Ma) complex. The complex contains angular fragments of ultrabasic rock, which display considerable compositional heterogeneity and are interpreted as cumulates containing clinopyroxene (generally altered to actinolite), apatite, allanite, epidote, and albite. The quartz alkali feldspar syenite cannot be regarded as alkaline sensu stricto, despite clear alkaline affinities. Within Likamännikkö there are also calcite carbonatite patches, which display mantle-like O- and C-isotope values, as well as trace element characteristics consistent with a magmatic origin, and could thus be among the oldest known carbonatites in the world. Sanukitoid (2.73 2.71 Ga) and quartz diorite suites (2.70 Ga) overlap within error margins and display compositional similarities, but can be differentiated from each other on the basis of higher Ba, K2O and LREE contents of the sanukitoids. The Likamännikkö complex, sanukitoids and quartz diorites are interpreted as originating from the metasomatized mantle and mark the diversification of the granitoid clan after 200 Ma of evolution dominated by the TTG suite. Widespread migmatization and the intrusion of anatectic leucogranitoids as dykes and intrusions of varying size took place at 2.70 2.69 Ga, following collisional thickening of the crust. The leucogranitoids and leucosomes of migmatized TTGs are compositionally alike and characterized by high silica contents and a leucocratic appearance. Due to compositional overlap, definitive discrimination between leucogranitoids and transitional TTGs requires isotope datings and/or knowledge of field relationships. Leucogranitoids represent partial melts of the local TTGs, both the sensu stricto and transitional types, mostly derived under water fluxed conditions, with possible fluid sources being late sanukitoids and quartz diorites as well as dehydrating lower crust. The Paleoproterozoic 2.44 2.39 Ga A-type granitoids of the Kianta Complex emplaced in an extensional environment are linked to the coeval and more widespread mafic intrusions and dykes observed over most of the Archean nucleus of the Fennoscandian shield. The A-type intrusions in the Suomussalmi area are interpreted as partial melts of the Archean lower crust and display differences in composition and magnetite content, which indicate differences in the composition and oxidation state of the source.

Ekotuotteet kuluttajan arjessa – vastuullisuutta, puhtautta ja arjen luksusta

Tutkielmassa tarkastellaan kuluttajien näkemyksiä ekotuotteiden valintaan vaikuttavista tekijöistä. Analysoin ekotuotteiden hankintaa kolmen teeman kautta. Ne ovat vihreys ja vastuullisuus, puhtaus tuotevalinnoissa sekä niin sanottu arjen luksus. Tutkimukseni empiirinen osa koostuu 10 teemahaastattelusta. Tutkimuskohteena on ekokauppa Ruohonjuuressa ostoksiaan tekevät kuluttajat. Haastateltavia etsin ilmoituksella ekokauppa Ruohonjuuresta sekä kaupan Facebook-sivuilta. Lisäksi oma haastattelupäivä Ruohonjuuren myymälässä tuotti haastateltavia mukaan tutkimukseen. Kirjoitin haastatteluista yhteenvedon ja analysoin aineistoa teemoittelun avulla. Nykyiset ympäristöongelmat vaikuttavat siihen, millaisena koemme arjen tulevaisuudessa. Vihreä ja vastuulllinen kuluttaja ottaa huomioon kulutuspäätöksiensä vaikutukset ympäristöön. Vihreät kulutuspäätökset tarkoittavat kestäviä kulutustapoja kuten jätteiden lajittelua, kirpputorikierrättämistä ja ympäristöä säästävien ekotuotteiden valintaa. Aineiston perusteella voi todeta, että ekotuotteiden ympäristömyönteisyyteen liitetään läheisesti luomutuotanto ja luomutuotteet. Ekotuotteet nähdään myös eettisinä ja moraalisina valintoina, joiden avulla halutaan vaikuttaa myös muiden hyvinvointiin. Ekotuotteisiin kohdistuu siten monenlaisia odotuksia, mutta myös epäilyjä. Aineistoni perusteella tuotetta ei välttämättä koettu ekotuotteeksi, jos sen valmistamiseen on käytetty paljon resursseja. Kuluttajat ovat kiinnostuneita ruoan alkuperästä ja sen aitoudesta. Ekotuotteet koetaan muita tuotteita päinvastoin puhtaiksi vaihtoehdoiksi. Puhtaus ekotuotteissa mielletään laadultaan turvallisiksi ja terveellisiksi tuotteiksi, jotka maistuvat hyvältä. Haastatteluaineiston perusteella voi todeta, että ekotuotteet koettiin myös arjen ostosten erikoisuudeksi. Arjen luksus lisää käyttäjälleen mielihyvän elämyksiä. Ekotuotteita ostamalla rakennetaan myös omaa elämäntyyliä ja erottaudutaan muista. Aineiston perusteella ekokauppaan mennään kiertelemään, tekemään heräteostoksia ja etsimään uutuuksia. Shoppailu ekokaupassa voi olla nautinnollista ja miellyttävää toimintaa, vaikka ostamista vain harkitaan. Ekotuote lahjana kertoo lahjan antajasta ja tuo lahjan saajalle palan luksusta vaikkapa luomusuklaan muodossa.

Yhteisestä toiminnasta yhteiseen kieleen : keskustelunanalyyttinen tutkimus toisen kielen oppimisesta kielikylpypäiväkodin arkitilanteissa

This dissertation examines how Finnish-speaking children learn Swedish in an immersion kindergarten where the method of Canadian immersion is used. Within the framework of conversation analysis, this study explores how second language learning is situated in interaction and evidenced in the participants´ verbal and non-verbal behavior. The database consists of 40 hours of videotaped data collected in naturally occurring situations in a group of 15 four-year-old children during the first two years of their immersion. Due to the immersion method, all the children share the same L1, in this case Finnish, and the teachers understand Finnish. However, they speak only Swedish to the children in all situations and Swedish is learned in interaction without formal teaching. The aim of the study is to discover how the children´s second language competence gradually increases when they participate in interaction with the Swedish-speaking teachers. The study also sheds light on the methodological question of how second language learning can be analyzed with the method of conversation analysis. The focus is on showing how the second language is learned in interaction, especially on how learning is achieved collaboratively. In this study, the emerging second language competence is explored by investigating how the children show understanding of the teachers´ non-verbal and verbal actions during the first and the second semester of the immersion. The children´s use of Swedish is analyzed by investigating how they recycle lexical items and later even syntactic structures from the teachers´ Swedish turns. The results show that the teachers´ actions are largely understood by the children even at the beginning of the immersion. The analyzes of the children´s responsive turns reveal that they interpret the teachers´ turns on the basis of non-verbal cues at first. Especially at the beginning of the immersion, the participants orient to the progress of interaction and not to problems in understanding. Even in situations where the next actions show that the children do not understand what is said, they tend to display understanding rather than non-understanding. This behavior changes, however, when the children´s competence in their second language increases. At the second semester, the children both show understanding of the teachers´ verbal turns and also display their non-understanding by initiating repair when they do not understand. Understanding of the teachers´ verbal turns, including their syntactic structure, is manifested in the ways the children tie their turns to the teachers´ turns. Recycling, on the other hand, proves to be the way by which the children start to speak the second language. In this study, the children´s common L1 is evidenced to be an important resource in interaction. It allows the children to participate in their individual ways and to share their experiences both with each other and with the teachers. It also enables them to co-construct conversations that lead to collaborative learning. Moreover, the uninhibited use of L1 proves to be an important analytic tool that makes the immersion data especially fruitful for conversation analytic research on second language learning, since the children´s interpretations of the second language are in evidence even when they do not speak the second language.

Effects of gender, and SLCO1B1 and CYP7A1 polymorphisms on plasma bile acids in humans and the pharmacokinetics of therapeutic ursodeoxycholic acid

Bile acids are important steroid-derived molecules essential for fat absorption in the small intestine. They are produced in the liver and secreted into the bile. Bile acids are transported by bile flow to the small intestine, where they aid the digestion of lipids. Most bile acids are reabsorbed in the small intestine and return to the liver through the portal vein. The whole recycling process is referred to as the enterohepatic circulation, during which only a small amount of bile acids are removed from the body via faeces. The enterohepatic circulation of bile acids involves the delicate coordination of a number of bile acid transporters expressed in the liver and the small intestine. Organic anion transporting polypeptide 1B1 (OATP1B1), encoded by the solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene, mediates the sodium independent hepatocellular uptake of bile acids. Two common SNPs in the SLCO1B1 gene are well known to affect the transport activity of OATP1B1. Moreover, bile acid synthesis is an important elimination route for cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production. The aim of this thesis was to investigate the effects of SLCO1B1 polymorphism on the fasting plasma levels of individual endogenous bile acids and a bile acid synthesis marker, and the pharmacokinetics of exogenously administered ursodeoxycholic acid (UDCA). Furthermore, the effects of CYP7A1 genetic polymorphism and gender on the fasting plasma concentrations of individual endogenous bile acids and the bile acid synthesis marker were evaluated. Firstly, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of bile acids was developed (Study I). A retrospective study examined the effects of SLCO1B1 genetic polymorphism on the fasting plasma concentrations of individual bile acids and a bile acid synthesis marker in 65 healthy subjects (Study II). In another retrospective study with 143 healthy individuals, the effects of CYP7A1 genetic polymorphism and gender as well as SLCO1B1 polymorphism on the fasting plasma levels of individual bile acids and the bile acid synthesis marker were investigated (Study III). The effects of SLCO1B1 polymorphism on the pharmacokinetics of exogenously administered UDCA were evaluated in a prospective genotype panel study including 27 healthy volunteers (Study IV). A robust, sensitive and simple HPLC-MS/MS method was developed for the simultaneous determination of 16 individual bile acids in human plasma. The method validation parameters for all the analytes met the requirements of the FDA (Food and Drug Administration) bioanalytical guidelines. This HPLC-MS/MS method was applied in Studies II-IV. In Study II, the fasting plasma concentrations of several bile acids and the bile acid synthesis marker seemed to be affected by SLCO1B1 genetic polymorphism, but these findings were not replicated in Study III with a larger sample size. Moreover, SLCO1B1 polymorphism had no effect on the pharmacokinetic parameters of exogenously administered UDCA. Furthermore, no consistent association was observed between CYP7A1 genetic polymorphism and the fasting plasma concentrations of individual bile acids or the bile acid synthesis marker. In contrast, gender had a major effect on the fasting plasma concentrations of several bile acids and also total bile acids. In conclusion, gender, but not SLCO1B1 or CYP7A1 polymorphisms, has a major effect on the fasting plasma concentrations of individual bile acids. Moreover, the common genetic polymorphism of CYP7A1 is unlikely to influence the activity of CYP7A1 under normal physiological conditions. OATP1B1 does not play an important role in the in vivo disposition of exogenously administered UDCA.

Mykorritsa fosforin kierrättäjänä

Suomen maatalousmaihin kertynyttä fosforia hyödynnetään tehottomasti, ja samalla muokkauskerroksen suuri fosforimäärä on alttiina huuhtoutumiselle. Arbuskelimykorritsaa (AM) hyödyntämällä on mahdollista tehostaa viljelykasvin fosforinottoa ja kasvua, ja siten vähentää fosforin huuhtoutumista. Tämän tutkielman tavoitteena oli selvittää mykorritsan vaikutus kasvin kasvuun ja fosforinottoon karjanlantalannoituksella mineraalilannoitukseen verrattuna sekä näiden lannoitusten pitkäaikaisvaikutusta AM-sieniyhteisöihin. Jotta lannoituskäytäntöjen vaikutus mykorritsaan voitiin suhteuttaa muihin maan laatutekijöihin, näiden käytäntöjen vaikutus myös satomääriin sekä muihin maan laatumittareihin arvioitiin. Pitkäaikainen kenttäkoe perustettiin kolmelle paikkakunnalle Pohjois-Ruotsissa vuosina 1965–66. Kuusivuotinen viljelykierto koostui joko viisivuotisesta nurmesta ja ohrasta tai ohramonokulttuurista. Lannoituskäsittelyt 32-vuoden ajan olivat suositusten mukainen (NPK) ja edelliseen nähden kaksinkertainen (2NPK) mineraalilannoitus sekä karjanlantalannoitus (KL), jonka ravinnemäärä vastasi NPK -käsittelyä. Kolmen lannoituskäsittelyn vaikutusta mykorritsan tehokkuuteen kasvin kasvun ja fosforiravitsemuksen näkökulmasta tutkittiin astiakokeissa. Mykorritsasieniyhteisöjen toiminnallisten erojen selvittämiseksi tehtiin takaisin- ja ristiinsiirrostuskoe. (5 v-%) steriloitua maanäytettä NPK- ja KL -käsittelyistä siirrostettiin käsittelemättömiin maanäytteisiin, jotka olivat samoista lannoituskäsittelyistä. Mykorritsan positiivinen vaikutus kasvin kasvuun ja fosforiravitsemukseen oli suurin kun käytettiin karjanlantaa. NPK ja 2NPK -käsittelyiden välillä ei havaittu eroja. Takaisin- ja ristiinsiirrostuskokeessa ei ollut tilastollisesti merkitseviä eroja. Nurmi- ja ohrasadot olivat suurimmat kun mineraalilannoitetta annettiin suosituksiin nähden kaksinkertainen määrä. Satomäärät olivat yhtä suuret tai suuremmat kun käytettiin karjanlantaa NPK –lannoituksen sijaan. Karjanlantakäsittely lisäsi maaperän kokonaishiili- ja kokonaistyppipitoisuutta verrattuna NPK -käsittelyyn, joka sisälsi saman määrän ravinteita. Samalla huuhtoutumiselle altis liukoisen fosforin pitoisuus säilyi alhaisella tasolla. Karjanlanta edisti mykorritsan toimintaedellytyksiä, ja siksi mykorritsasta saatua hyötyä fosforinotossa ja kasvuvaikutuksena mineraalilannoitteisiin verrattuna, mutta se ei vaikuttanut mykorritsasieniyhteisön toiminnallisiin ominaisuuksiin. Karjanlantalannoitus paransi mitattuja maan ominaisuuksia kokonaisuudessaan, eikä se vähentänyt satoja.

Puheen suuntia luokkahuoneessa : Oppilaat osallistujina yläkoulun suomi toisena kielenä -tunnilla

This study describes how students influence their possibilities of participating in whole-class conversation. The main objective is to investigate the verbal and non-verbal resources used by students to modify the participant roles of the ongoing conversation. The resources studied are attention-getting devices such as hand-raising and address terms, recycling and other forms of collaborative talk, means of reference to persons, such as pronouns, as well as gaze and other embodied resources. The theoretical and methodological framework adopted in this study is that of conversation analysis. The data consist of ten videotaped lessons of Finnish as a second language in three secondary schools (grades 7 9) in southern Finland; the number of students per group varies from five to ten. Finnish has a triple role in the data as the medium of teaching, the target language, and the lingua franca used by the participants. The findings show that the multi-party context of the classroom conversation is both a disadvantage and an affordance for student participation. The students possess multiple tools to overcome and deal with the encumbrances posed by the large number of participants. They combine various techniques in order to actively compete for public turns, and they monitor the ongoing conversation carefully to adjust their participation to the activities of other participants. Sometimes the whole-class conversation splits into two separate conversations, but participants usually orient to the overlapping nature of the talk and tend to bring the conversations together rapidly. On the other hand, students skilfully make use of other participants and their talk to increase and diversify their own possibilities to participate. For example, they recycle elements of each other s turns or refer to the currently speaking student in order to gain access to the conversation. Students interact with each other even during the public whole-class conversation. Students orient to one another often even when talking to the teacher, but they also address talk directly to one another, as part of the public conversation. In this way students increase each other s possibilities of participation. The interaction is constantly multi-layered: in addition to the pedagogic agenda, the students orient to social goals, for example, by teasing each other and putting on humorous performances for their peer audience. The student student participation arises spontaneously from a genuine need to communicate and thus represents authentic language use: by talking to each other, often playfully, the students appropriate Finnish vocabulary, grammar, and expressions. In this way the structure of the interaction reflects the particular nature of Finnish as a second language lessons: all talk serves the pedagogic goal of enabling students to communicate in the target language.