Stretching single DNA-molecules with temperature-stabilized optical tweezers

This thesis consists of two parts; in the first part we performed a single-molecule force extension measurement with 10kb long DNA-molecules from phage-λ to validate the calibration and single-molecule capability of our optical tweezers instrument. Fitting the worm-like chain interpolation formula to the data revealed that ca. 71% of the DNA tethers featured a contour length within ±15% of the expected value (3.38 µm). Only 25% of the found DNA had a persistence length between 30 and 60 nm. The correct value should be within 40 to 60 nm. In the second part we designed and built a precise temperature controller to remove thermal fluctuations that cause drifting of the optical trap. The controller uses feed-forward and PID (proportional-integral-derivative) feedback to achieve 1.58 mK precision and 0.3 K absolute accuracy. During a 5 min test run it reduced drifting of the trap from 1.4 nm/min in open-loop to 0.6 nm/min in closed-loop.

Oppimisen kohteen ja oppijan vastavuoroinen kehitys : Teleyrityksen asiakaspalvelun työyhteisöjen oppimiskäytäntöjen uudistaminen osana teknologis-taloudellista kumousta

Reciprocal development of the object and subject of learning. The renewal of the learning practices of front-line communities in a telecommunications company as part of the techno-economical paradigm change. Current changes in production have been seen as an indication of a shift from the techno-economical paradigm of a mass-production era to a new paradigm of the information and communication technological era. The rise of knowledge management in the late 1990s can be seen as one aspect of this paradigm shift, as knowledge creation and customer responsiveness were recognized as the prime factors in business competition. However, paradoxical conceptions concerning learning and agency have been presented in the discussion of knowledge management. One prevalent notion in the literature is that learning is based on individuals’ voluntary actions and this has now become incompatible with the growing interest in knowledge-management systems. Furthermore, commonly held view of learning as a general process that is independent of the object of learning contradicts the observation that the current need for new knowledge and new competences are caused by ongoing techno-economic changes. Even though the current view acknowledges that individuals and communities have key roles in knowledge creation, this conception defies the idea of the individuals’ and communities’ agency in developing the practices through which they learn. This research therefore presents a new theoretical interpretation of learning and agency based on Cultural-Historical Activity Theory. This approach overcomes the paradoxes in knowledge-management theory and offers means for understanding and analyzing changes in the ways of learning within work communities. This research is also an evaluation of the Competence-Laboratory method which was developed as part of the study as a special application of Developmental Work Research methodology. The research data comprises the videotaped competence-laboratory processes of four front-line work communities in a telecommunications company. The findings reported in the five articles included in this thesis are based on the analyses of this data. The new theoretical interpretation offered here is based on the assessment that the findings reported in the articles represent one of the front lines of the ongoing historical transformation of work-related learning since the research site represents one of the key industries of the new “knowledge society”. The research can be characterized as elaboration of a hypothesis concerning the development of work related learning. According to the new theoretical interpretation, the object of activity is also the object of distributed learning in work communities. The historical socialization of production has increased the number of actors involved in an activity, which has also increased the number of mutual interdependencies as well as the need for communication. Learning practices and organizational systems of learning are historically developed forms of distributed learning mediated by specific forms of division of labor, specific tools, and specific rules. However, the learning practices of the mass production era become increasingly inadequate to accommodate the conditions in the new economy. This was manifested in the front-line work communities in the research site as an aggravating contradiction between the new objects of learning and the prevailing learning practices. The constituent element of this new theoretical interpretation is the idea of a work community’s learning as part of its collaborative mastery of the developing business activity. The development of the business activity is at the same time a practical and an epistemic object for the community. This kind of changing object cannot be mastered by using learning practices designed for the stable conditions of mass production, because learning has to change along the changes in business. According to the model introduced in this thesis, the transformation of learning proceeds through specific stages: predefined learning tasks are first transformed into learning through re-conceptualizing the object of the activity and of the joint learning and then, as the new object becomes stabilized, into the creation of new kinds of learning practices to master the re-defined object of the activity. This transformation of the form of learning is realized through a stepwise expansion of the work community’s agency. To summarize, the conceptual model developed in this study sets the tool-mediated co-development of the subject and the object of learning as the theoretical starting point for developing new, second-generation knowledge management methods. Key words: knowledge management, learning practice, organizational system of learning, agency

Biochemical and structural properties of Potato virus A VPg

The potato virus A (PVA) genome linked protein (VPg) is a multifunctional protein that takes part in vital infection cycle events such as replication and movement of the virus from cell to cell. VPg is attached to the 5´ end of the genome and is carried in the tip structure of the filamentous virus particle. VPg is also the last protein to be cleaved from the polyprotein. VPg interacts with several viral and host proteins and is phosphorylated at several positions. These features indicate a central role in virus epidemiology and a requirement for an efficient but flexible mechanism for switching between different functions. -- This study examines some of the key VPg functions in more detail. Mutations in the positively charged region from Ala38 to Lys44 affected the NTP binding, uridylylation, and in vitro translation inhibition activities of VPg, whereas in vivo translation inhibition was not affected. Some of the data generated in this study implicated the structural flexibility of the protein in functional activities. VPg lacks a rigid structure, which could allow it to adapt conformationally to different functions as needed. A major finding of this study is that PVA VPg belongs to the class of ´intrinsically disordered proteins´ (IDPs). IDPs are a novel protein class that has helped to explain the observed lack of structure. The existence of IDPs clearly shows that proteins can be functional and adapt a native fold without a rigid structure. Evidence for the intrinsic disorder of VPg was provided by CD spectroscopy, NMR, fluorescence spectroscopy, bioinformatic analysis, and limited proteolytic digestion. The structure of VPg resembles that of a molten globule-type protein and has a hydrophobic core domain. Approximately 50% of the protein is disordered and an α-helical stabilization of these regions has been hypothesized. Surprisingly, VPg structure was stabilized in the presence of anionic lipid vesicles. The stabilization was accompanied by a change in VPg structure and major morphological modifications of the vesicles, including a pronounced increase in the size and appearance of pore or plaque like formations on the vesicle surface. The most likely scenario seems to be an α-helical stabilization of VPg which induces formation of a pore or channel-like structure on the vesicle surface. The size increase is probably due to fusion or swelling of the vesicles. The latter hypothesis is supported by the evident disruption of the vesicles after prolonged incubation with VPg. A model describing the results is presented and discussed in relation to other known properties of the protein.

Computational studies on new chemical species in the gas-phase and the solid-state.

There is intense activity in the area of theoretical chemistry of gold. It is now possible to predict new molecular species, and more recently, solids by combining relativistic methodology with isoelectronic thinking. In this thesis we predict a series of solid sheet-type crystals for Group-11 cyanides, MCN (M=Cu, Ag, Au), and Group-2 and 12 carbides MC2 (M=Be-Ba, Zn-Hg). The idea of sheets is then extended to nanostrips which can be bent to nanorings. The bending energies and deformation frequencies can be systematized by treating these molecules as an elastic bodies. In these species Au atoms act as an 'intermolecular glue'. Further suggested molecular species are the new uncongested aurocarbons, and the neutral Au_nHg_m clusters. Many of the suggested species are expected to be stabilized by aurophilic interactions. We also estimate the MP2 basis-set limit of the aurophilicity for the model compounds [ClAuPH_3]_2 and [P(AuPH_3)_4]^+. Beside investigating the size of the basis-set applied, our research confirms that the 19-VE TZVP+2f level, used a decade ago, already produced 74 % of the present aurophilic attraction energy for the [ClAuPH_3]_2 dimer. Likewise we verify the preferred C4v structure for the [P(AuPH_3)_4]^+ cation at the MP2 level. We also perform the first calculation on model aurophilic systems using the SCS-MP2 method and compare the results to high-accuracy CCSD(T) ones. The recently obtained high-resolution microwave spectra on MCN molecules (M=Cu, Ag, Au) provide an excellent testing ground for quantum chemistry. MP2 or CCSD(T) calculations, correlating all 19 valence electrons of Au and including BSSE and SO corrections, are able to give bond lengths to 0.6 pm, or better. Our calculated vibrational frequencies are expected to be better than the currently available experimental estimates. Qualitative evidence for multiple Au-C bonding in triatomic AuCN is also found.

Stabilization of Phospholipid Coatings in Capillary Electrophoresis

The development of a simple method of coating a semi-permanent phospholipid layer onto a capillary for electrochromatography use was the focus of this study. The work involved finding good coating conditions, stabilizing the phospholipid coating, and examining the effect of adding divalent cations, cetyltrimethylammonium bromide, and polyethylene glycol (PEG)-lipids on the stability of the coating. Since a further purpose was to move toward more biological membrane coatings, the capillaries were also coated with cholesterol-containing liposomes and liposomes of red blood cell ghost lipids. Liposomes were prepared by extrusion, and large unilamellar vesicles with a diameter of about 100 nm were obtained. Zwitterionic phosphatidylcholine (PC) was used as a basic component, mainly 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) but also eggPC and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Different amounts of sphingomyelin, bovine brain phosphatidylserine, and cholesterol were added to the PC. The stability of the coating in 40 mM N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) solution at pH 7.4 was studied by measuring the electroosmotic flow and by separating neutral steroids, basic proteins, and low-molar-mass drugs. The presence of PC in the coating solution was found to be essential to achieving a coating. The stability of the coating was improved by the addition of negative phosphatidylserine, cholesterol, divalent cations, or PEGylated lipids, and by working in the gel-state region of the phospholipid. Study of the effect on the PC coating of divalent metal ions calcium, magnesium, and zinc showed a molar ratio of 1:3 PC/Ca2+ or PC/Mg2+ to give increased rigidity to the membrane and the best coating stability. The PEGylated lipids used in the study were sterically stabilized commercial lipids with covalently attached PEG chains. The vesicle size generally decreased when PEGylated lipids of higher molar mass were present in the vesicle. The predominance of discoidal micelles over liposomes increased PEG chain length and the average size of the vesicles thus decreased. In the capillary electrophoresis (CE) measurements a highly stable electroosmotic flow was achieved with 20% PEGylated lipid in the POPC coating dispersion, the best results being obtained for disteroyl PEG (3000) conjugates. The results suggest that smaller particles (discoidal micelles) result in tighter packing and better shielding of silanol groups on the silica wall. The effect of temperature on the coating stability was investigated by using DPPC liposomes at temperatures above (45 C) and below (25 C) the main phase transition temperature. Better results were obtained with DPPC in the more rigid gel state than in the fluid state: the electroosmotic flow was heavily suppressed and the PC coating was stabilized. Also dispersions of DPPC with 0−30 mol% of cholesterol and sphingomyelin in different ratios, which more closely resemble natural membranes, resulted in stable coatings. Finally, the CE measurements revealed that a stable coating is formed when capillaries are coated with liposomes of red blood cell ghost lipids.

Intermolecular Interactions of Noble-Gas-Containing Species

The importance of intermolecular interactions to chemistry, physics, and biology is difficult to overestimate. Without intermolecular forces, condensed phase matter could not form. The simplest way to categorize different types of intermolecular interactions is to describe them using van der Waals and hydrogen bonded (H-bonded) interactions. In the H-bond, the intermolecular interaction appears between a positively charged hydrogen atom and electronegative fragments and it originates from strong electrostatic interactions. H-bonding is important when considering the properties of condensed phase water and in many biological systems including the structure of DNA and proteins. Vibrational spectroscopy is a useful tool for studying complexes and the solvation of molecules. Vibrational frequency shift has been used to characterize complex formation. In an H-bonded system A∙∙∙H-X (A and X are acceptor and donor species, respectively), the vibrational frequency of the H-X stretching vibration usually decreases from its value in free H-X (red-shift). This frequency shift has been used as evidence for H-bond formation and the magnitude of the shift has been used as an indicator of the H-bonding strength. In contrast to this normal behavior are the blue-shifting H-bonds, in which the H-X vibrational frequency increases upon complex formation. In the last decade, there has been active discussion regarding these blue-shifting H-bonds. Noble-gases have been considered inert due to their limited reactivity with other elements. In the early 1930 s, Pauling predicted the stable noble-gas compounds XeF6 and KrF6. It was not until three decades later Neil Bartlett synthesized the first noble-gas compound, XePtF6, in 1962. A renaissance of noble-gas chemistry began in 1995 with the discovery of noble-gas hydride molecules at the University of Helsinki. The first hydrides were HXeCl, HXeBr, HXeI, HKrCl, and HXeH. These molecules have the general formula of HNgY, where H is a hydrogen atom, Ng is a noble-gas atom (Ar, Kr, or Xe), and Y is an electronegative fragment. At present, this class of molecules comprises 23 members including both inorganic and organic compounds. The first and only argon-containing neutral chemical compound HArF was synthesized in 2000 and its properties have since been investigated in a number of studies. A helium-containing chemical compound, HHeF, was predicted computationally, but its lifetime has been predicted to be severely limited by hydrogen tunneling. Helium and neon are the only elements in the periodic table that do not form neutral, ground state molecules. A noble-gas matrix is a useful medium in which to study unstable and reactive species including ions. A solvated proton forms a centrosymmetric NgHNg+ (Ng = Ar, Kr, and Xe) structure in a noble-gas matrix and this is probably the simplest example of a solvated proton. Interestingly, the hypothetical NeHNe+ cation is isoelectronic with the water-solvated proton H5O2+ (Zundel-ion). In addition to the NgHNg+ cations, the isoelectronic YHY- (Y = halogen atom or pseudohalogen fragment) anions have been studied with the matrix-isolation technique. These species have been known to exist in alkali metal salts (YHY)-M+ (M = alkali metal e.g. K or Na) for more than 80 years. Hydrated HF forms the FHF- structure in aqueous solutions, and these ions participate in several important chemical processes. In this thesis, studies of the intermolecular interactions of HNgY molecules and centrosymmetric ions with various species are presented. The HNgY complexes show unusual spectral features, e.g. large blue-shifts of the H-Ng stretching vibration upon complexation. It is suggested that the blue-shift is a normal effect for these molecules, and that originates from the enhanced (HNg)+Y- ion-pair character upon complexation. It is also found that the HNgY molecules are energetically stabilized in the complexed form, and this effect is computationally demonstrated for the HHeF molecule. The NgHNg+ and YHY- ions also show blue-shifts in their asymmetric stretching vibration upon complexation with nitrogen. Additionally, the matrix site structure and hindered rotation (libration) of the HNgY molecules were studied. The librational motion is a much-discussed solid state phenomenon, and the HNgY molecules embedded in noble-gas matrices are good model systems to study this effect. The formation mechanisms of the HNgY molecules and the decay mechanism of NgHNg+ cations are discussed. A new electron tunneling model for the decay of NgHNg+ absorptions in noble-gas matrices is proposed. Studies of the NgHNg+∙∙∙N2 complexes support this electron tunneling mechanism.

Novel CDNF/MANF protein family : Molecular structure, expression and neurotrophic activity

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.

Mechanisms and molecular regulation of mammalian tooth replacement

In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.

Effects of large-scale human land use on Capercaillie (Tetrao urogallus L.) populations in Finland

The Capercaillie (Tetrao urogallus L.) is often used as a focal species for landscape ecological studies: the minimum size for its lekking area is 300 ha, and the annual home range for an individual may cover 30 80 km2. In Finland, Capercaillie populations have decreased by approximately 40 85%, with the declines likely to have started in the 1940s. Although the declines have partly stabilized from the 1990s onwards, it is obvious that the negative population trend was at least partly caused by changes in human land use. The aim of this thesis was to study the connections between human land use and Capercaillie populations in Finland, using several spatial and temporal scales. First, the effect of forest age structure on Capercaillie population trends was studied in 18 forestry board districts in Finland, during 1965 1988. Second, the abundances of Capercaillie and Moose (Alces alces L.) were compared in terms of several land-use variables on a scale of 50 × 50 km grids and in five regions in Finland. Third, the effects of forest cover and fine-grain forest fragmentation on Capercaillie lekking area persistence were studied in three study locations in Finland, on 1000 and 3000 m spatial scales surrounding the leks. The analyses considering lekking areas were performed with two definitions for forest: > 60 and > 152 m3ha 1 of timber volume. The results show that patterns and processes at large spatial scales strongly influence Capercaillie in Finland. In particular, in southwestern and eastern Finland, high forest cover and low human impact were found to be beneficial for this species. Forest cover (> 60 m3ha 1 of timber) surrounding the lekking sites positively affected lekking area persistence only at the larger landscape scale (3000 m radius). The effects of older forest classes were hard to assess due to scarcity of older forests in several study areas. Young and middle-aged forest classes were common in the vicinity of areas with high Capercaillie abundances especially in northern Finland. The increase in the amount of younger forest classes did not provide a good explanation for Capercaillie population decline in 1965 1988. In addition, there was no significant connection between mature forests (> 152 m3ha 1 of timber) and lekking area persistence in Finland. It seems that in present-day Finnish landscapes, area covered with old forest is either too scarce to efficiently explain the abundance of Capercaillie and the persistence of the lekking areas, or the effect of forest age is only important when considering smaller spatial scales than the ones studied in this thesis. In conclusion, larger spatial scales should be considered for assessing the future Capercaillie management. According to the proposed multi-level planning, the first priority should be to secure the large, regional-scale forest cover, and the second priority should be to maintain fine-grained, heterogeneous structure within the separate forest patches. A management unit covering hundreds of hectares, or even tens or hundreds of square kilometers, should be covered, which requires regional-level land-use planning and co-operation between forest owners.

Quantum Chemical Studies on Tropospheric Nucleation Mechanisms Involving Sulfuric Acid

Nucleation is the first step of the process by which gas molecules in the atmosphere condense to form liquid or solid particles. Despite the importance of atmospheric new-particle formation for both climate and health-related issues, little information exists on its precise molecular-level mechanisms. In this thesis, potential nucleation mechanisms involving sulfuric acid together with either water and ammonia or reactive biogenic molecules are studied using quantum chemical methods. Quantum chemistry calculations are based on the numerical solution of Schrödinger's equation for a system of atoms and electrons subject to various sets of approximations, the precise details of which give rise to a large number of model chemistries. A comparison of several different model chemistries indicates that the computational method must be chosen with care if accurate results for sulfuric acid - water - ammonia clusters are desired. Specifically, binding energies are incorrectly predicted by some popular density functionals, and vibrational anharmonicity must be accounted for if quantitatively reliable formation free energies are desired. The calculations reported in this thesis show that a combination of different high-level energy corrections and advanced thermochemical analysis can quantitatively replicate experimental results concerning the hydration of sulfuric acid. The role of ammonia in sulfuric acid - water nucleation was revealed by a series of calculations on molecular clusters of increasing size with respect to all three co-ordinates; sulfuric acid, water and ammonia. As indicated by experimental measurements, ammonia significantly assists the growth of clusters in the sulfuric acid - co-ordinate. The calculations presented in this thesis predict that in atmospheric conditions, this effect becomes important as the number of acid molecules increases from two to three. On the other hand, small molecular clusters are unlikely to contain more than one ammonia molecule per sulfuric acid. This implies that the average NH3:H2SO4 mole ratio of small molecular clusters in atmospheric conditions is likely to be between 1:3 and 1:1. Calculations on charged clusters confirm the experimental result that the HSO4- ion is much more strongly hydrated than neutral sulfuric acid. Preliminary calculations on HSO4- NH3 clusters indicate that ammonia is likely to play at most a minor role in ion-induced nucleation in the sulfuric acid - water system. Calculations of thermodynamic and kinetic parameters for the reaction of stabilized Criegee Intermediates with sulfuric acid demonstrate that quantum chemistry is a powerful tool for investigating chemically complicated nucleation mechanisms. The calculations indicate that if the biogenic Criegee Intermediates have sufficiently long lifetimes in atmospheric conditions, the studied reaction may be an important source of nucleation precursors.

Asiakassuunnitelma asiakaslähtöistä auttamista tavoitteellistamassa : Kohteen rakentumisen moniääninen menetelmä

The research focuses on client plan in the field of health care and social work on families with children. The purpose of the plan is to create objectives for helping the client and to assist in coordinating the ever-increasing multi-professional work. In general, the plan is understood in terms of assignments and as a contract specifying what to do in client cases. Taking this into consideration, the plan is outsourced into a written document. Instead of understanding the plan as a tool that stabilizes the objectives of action, documents it and facilitates evaluation, the client plan is conceptualized in this study as a practice. This kind of practice mediates client work as being itself also a process of action that focuses on an object whose gradual emergence and definition is the central question in multi-professional collaboration with a client. The plan is examined empirically in a non-stabilized state which leads to the research methodology being based on the dynamics between stabilization and emerging, non-stabilized entities the co-creation and formulation of practice and context. The theoretical approach of the research is the micro analytic approach of activity theory (Engeström R. 1999b). Grounding on this, the research develops a method of qualitative analysis which follows an emerging object with multiple voices. The research data is composed of the videotaped sessions from client meetings with three families, the interviews with the client and the workers as well as client documents that are used to follow up on client processes for at least one year. The research questions are as follows: 1) How is the client plan constructed between the client and different professional agents? 2) How are meanings constructed in a client-centred plan? 3) What are the elements of client-employee relationships that support the co-configuration necessitated by the changes in the client s everyday life? The study shows that the setting of objectives were limited by the palette of institutional services, which caused that the clients interpretations and acts of giving meaning to the kinds of help that was required were left out of the plan. Conceptually, the distinctions between client-centred and client-specific ways of working as well as an action-based working method are addressed. Central to this action-based approach is construing the everyday life of the client, recognizing different meanings and analyzing them together with the client as well as focusing attention on developing the prerequisites for social agency of the clients. The research portrays the elements for creating an action-based client plan. Key words: client plan, user perspective, multi-voiced meaning, multi-professional social work with children and families, agency

Polymeereillä stabiloitu amorfinen formulaatio

Nowadays growing number of new active pharmaceutical ingredients (API) have large molecular weight and are hydrophobic. The energy of their crystal lattice is bigger and polarity has decreased. This leads to weakened solubility and dissolution rate of the drug. These properties can be enhanced for example by amorphization. Amorphous form has the best dissolution rate in the solid state. In the amorphous form drug molecules are randomly arranged, so the energy required to dissolve molecules is lower compared to the crystalline counterpart. The disadvantage of amorphous form is that it is unstable. Amorphous form tends to crystallize. Stability of amorphous form can be enhanced by adding an adjuvant to drug product. Adjuvant is usually a polymer. Polymers prevent crystallization both by forming bonds with API molecules and by steric hindrance. The key thing in stabilizing amorphous form is good miscibility between API and polymer. They have to be mixed in a molecular level so that the polymer is able to prevent crystallization. The aim of this work was to study miscibility of drug and polymer and stability of their dispersion with different analytical methods. Amorphous dispersions were made by rotary evaporator and freeze dryer. Amorphicity was confirmed with X-ray powder diffraction (XRPD) right after preparation. Itraconazole and theophylline were the chosen molecules to be stabilized. Itraconazole was expected to be easier and theophylline more difficult to stabilize. Itraconazole was stabilized with HPMC and theophylline was stabilized with PVP. Miscibility was studied with XRPD and differential scanning calorimetry (DSC). In addition it was studied with polarized light microscope if miscibility was possible to see visually. Dispersions were kept in stressed conditions and the crystallization was analyzed with XRPD. Stability was also examined with isothermal microcalorimetry (IMC). The dispersion of itraconazole and theophylline 40/60 (w/w) was completely miscible. It was proved by linear combination of XRPD results and single glass transition temperature in DSC. Homogenic well mixed film was observed with light microscope. Phase separation was observed with other compositions. Dispersions of theophylline and PVP mixed only partly. Stability of itraconazole dispersions were better than theophylline dispersions which were mixed poorer. So miscibility was important thing considering stability. The results from isothermal microcalorimetry were similar to results from conventional stability studies. Complementary analytical methods should be used when studying miscibility so that the results are more reliable. Light microscope is one method in addition to mostly used XRPD and DSC. Analyzing light microscope photos is quite subjective but it gives an idea of miscibility. Isothermal microcalorimetry can be one option for conventional stability studies. If right conditions can be made where the crystallization is not too fast, it may be possible to predict stability with isothermal microcalorimetry.

Effect of water vapour stabilization of raw materials on the quality of the inhalation product

Generation of raw materials for dry powder inhalers by different size reduction methods can be expected to influence physical and chemical properties of the powders. This can cause differences in particle size, size distribution, shape, crystalline properties, surface texture and energy. These physical properties of powders influence the behaviour of particles before and after inhalation. Materials with an amorphous surface have different surface energy compared to materials with crystalline surface. This can affect the adhesion and cohesion of particles. Changes in the surface nature of the drug particles results in a change in product performance. By stabilization of the raw materials the amorphous surfaces are converted into crystalline surfaces. The primary aim of the study was to investigate the influence of the surface properties of the inhalation particles on the quality of the product. The quality of the inhalation product is evaluated by measuring the fine particle dose (FPD). FDP is the total dose of particles with aerodynamic diameters smaller than 5,0 μm. The secondary aim of this study was to achieve the target level of the FPD and the stability of the FPD. This study was also used to evaluate the importance of the stabilization of the inhalation powders. The study included manufacturing and analysing drug substance 200 μg/dose inhalation powder batches using non-stabilized or stabilized raw materials. The inhaler formulation consisted of micronized drug substance, lactose <100μm and micronized lactose <10μm. The inhaler device was Easyhaler®. Stabilization of the raw materials was done in different relative humidity, temperature and time. Surface properties of the raw materials were studied by dynamic vapour sorption, scanning electron microscopy and three-point nitrogen adsorption technique. Particle size was studied by laser diffraction particle size analyzer. Aerodynamic particle size distribution from inhalers was measured by new generation impactor. Stabilization of all three raw materials was successful. A clear difference between nonstabilized and stabilized raw materials was achieved for drug substance and lactose <10μm. However for lactose <100μm the difference wasn’t as clear as wanted. The surface of the non-stabilized drug substance was more irregular and the particles had more roughness on the surface compared to the stabilized drug substances particles surface. The surface of the stabilized drug particles was more regular and smoother than non-stabilized. Even though a good difference between stabilized and non-stabilized raw materials was achieved, a clear evidence of the effect of the surface properties of the inhalation particles on the quality of the product was not observed. Stabilization of the raw materials didn’t lead to a higher FPD. Possible explanations for the unexpected result might be too rough conditions in the stabilization of the drug substance or smaller than wanted difference in the degree of stabilization of the main component of the product <100μm. Despite positive effects on the quality of the product were not seen there appears to be some evidence that stabilized drug substance results in smaller particle size of dry powder inhalers.

Suomussalmen varhaisproterotsooisten diabaasien mineralogia tektonisessa tulkinnassa

The object of this research is to study the mineralogy of the diabase dykes in Suomussalmi and the relevance of the mineralogy to tectonic events, specifically large block movements in the Archaean crust. Sharp tectonic lines separate two anomalies in the dyke swarms, shown on a geomagnetic map as positive anomalies. In one of these areas, the Toravaara anomaly, the diabases seem to contain pyroxenes as a main component. Outside the Toravaara anomaly hornblende is the main ferromagnesian mineral in diabases. The aim of this paper is to research the differences in the diabases inside and outside the anomalies and interpret the processes that formed the anomalies. The data for this sudy consist of field observations, 120 thin sections, 334 electron microprobe analyses, 19 whole-rock chemical analyses, a U-Pb age analysis and geomagnetic low-altitude aerial survey maps. The methods are interpretation of field observations, chemical analyses, microprobe analyses of single minerals and radiometric age determination, microscopic studies of the thin sections, geothermometers and geobarometers. On the basis of field observations and petrographic studies the diabases in the area are divided into pyroxene diabases, hornblende diabases and the Lohisärkkä porphyritic dyke swarm. Hornblende diabases are found in the entire study area, while the pyroxene diabases concentrate on the area of the Toravaara geomagnetic anomaly. The Lohisärkkä swarm transects the whole area as a thin line from east to west. The diabases are fairly homogenous both chemically and by mineral composition. The few exceptions are part of rarer older swarms or are significantly altered. The Lohisärkkä dyke swarm was dated as 2,21 Ga old, significantly older than the most common 1,98 Ga swarm in the area. The geothermometers applied showed that the diabases on the Toravaara anomaly were stabilized at a much higher temperature than the dykes outside the anomaly. The geobarometers showed the pyroxenes to have crystallized at varying depths. The research showed the Toravaara anomaly to have formed by a vertical block movement, and the fault on its west side to have a total lateral transfer of only a few kilometers. The formation of the second anomaly was also interpreted to be tectonic in nature. In addition, the results of the geothermobarometry uncovered necessary conditions for the study of diabase emplacement depth: the minerals for the study must be chosen by minimum crystallization depth, and a geobarometer capable of determining the magmatic temperature must be used. In addition, it would be more suitable to conduct this kind of study in an area where the dykes are more exposed.