Multiplication of hybrid aspen (Populus tremula L. x P. tremuloides Michx.) from cuttings

The primary aim of the present study was to find an efficient and simple method of vegetative propagation for producing large numbers of hybrid aspen (Populus tremuloides L. x P. tremula Michx.) plants for forest plantations. The key objectives were to investigate the main physiological factors that affect the ability of cuttings to regenerate and to determine whether these factors could be manipulated by different growth conditions. In addition, clonal variation in traits related to propagation success was examined. According to our results, with the stem cutting method, depending on the clone, it is possible to obtain only 1−8 plants from one stock plant per year. With the root cutting method the corresponding values for two-year-old stock plants are 81−207 plants. The difference in number of cuttings between one- and two-year-old stock plants is so pronounced that it is economically feasible to grow stock plants for two years. There is no reason to use much older stock plants as a source of cuttings, as it has been observed that rooting ability diminishes as root diameter increases. Clonal variation is the most important individual factor in propagation of hybrid aspen. The fact that the efficiently sprouted clones also rooted best facilitates the selection of clones for large-scale propagation. In practice, root cuttings taken from all parts of the root system of hybrid aspen were capable of producing new shoots and roots. However, for efficient rooting it is important to use roots smaller than one centimeter in diameter. Both rooting and sprouting, as well as sprouting rate, were increased by high soil temperature; in our studies the highest temperature tested (30ºC) was the best. Light accelerated the sprouting of root cuttings, but they rooted best in dark conditions. Rooting is essential because without roots the sprouted cutting cannot survive long. For aspen the criteria for clone selection are primarily fiber qualities and growth rate, but ability to regenerate efficiently is also essential. For large-scale propagation it is very important to find clones from which many cuttings per stock plant can be obtained. In light of production costs, however, it is even more important that the regeneration ability of the produced cuttings be high.

The decline of malaria in Finland – the impact of the vector and social variables

Background: Malaria was prevalent in Finland in the 18th century. It declined slowly without deliberate counter-measures and the last indigenous case was reported in 1954. In the present analysis of indigenous malaria in Finland, an effort was made to construct a data set on annual malaria cases of maximum temporal length to be able to evaluate the significance of different factors assumed to affect malaria trends. Methods: To analyse the long-term trend malaria statistics were collected from 1750–2008. During that time, malaria frequency decreased from about 20,000 – 50,000 per 1,000,000 people to less than 1 per 1,000,000 people. To assess the cause of the decline, a correlation analysis was performed between malaria frequency per million people and temperature data, animal husbandry, consolidation of land by redistribution and household size. Results: Anopheles messeae and Anopheles beklemishevi exist only as larvae in June and most of July. The females seek an overwintering place in August. Those that overwinter together with humans may act as vectors. They have to stay in their overwintering place from September to May because of the cold climate. The temperatures between June and July determine the number of malaria cases during the following transmission season. This did not, however, have an impact on the longterm trend of malaria. The change in animal husbandry and reclamation of wetlands may also be excluded as a possible cause for the decline of malaria. The long-term social changes, such as land consolidation and decreasing household size, showed a strong correlation with the decline of Plasmodium. Conclusion: The indigenous malaria in Finland faded out evenly in the whole country during 200 years with limited or no counter-measures or medication. It appears that malaria in Finland was basically a social disease and that malaria trends were strongly linked to changes in human behaviour. Decreasing household size caused fewer interactions between families and accordingly decreasing recolonization possibilities for Plasmodium. The permanent drop of the household size was the precondition for a permanent eradication of malaria.

Conservation of Atlantic salmon by supplementary stocking of juvenile fish

Wild salmon stocks in the northern Baltic rivers became endangered in the second half of the 20th century, mainly due to recruitment overfishing. As a result, supplementary stocking was widely practised, and supplementation of the Tornionjoki salmon stock took place over a 25 year period until 2002. The stock has been closely monitored by electrofishing, smolt trapping, mark-recapture studies, catch samples and catch surveys. Background information on hatchery-reared stocked juveniles was also collected for this study. Bayesian statistics was applied to the data as this method offers the possibility of bringing prior information into the analysis and an advanced ability for incorporating uncertainty, and also provides probabilities for a multitude of hypotheses. Substantial divergences between reared and wild Tornionjoki salmon were identified in both demographic and phenological characteristics. The divergences tended to be larger the longer the duration spent in hatchery and the more favourable the hatchery conditions were for fast growth. Differences in environment likely induced most of the divergences, but selection of brood fish might have resulted in genotypic divergence in maturation age of reared salmon. Survival of stocked 1-year old juveniles to smolt varied from about 10% to about 25%. Stocking on the lower reach of the river seemed to decrease survival, and the negative effect of stocking volume on survival raises the concern of possible similar effects on the extant wild population. Post-smolt survival of wild Tornionjoki smolts was on average two times higher than that of smolts stocked as parr and 2.5 times higher than that of stocked smolts. Smolts of different groups showed synchronous variation and similar long-term survival trends. Both groups of reared salmon were more vulnerable to offshore driftnet and coastal trapnet fishing than wild salmon. Average survival from smolt to spawners of wild salmon was 2.8 times higher than that of salmon stocked as parr and 3.3 times higher than that of salmon stocked as smolts. Wild salmon and salmon stocked as parr were found to have similar lifetime survival rates, while stocked smolts have a lifetime survival rate over 4 times higher than the two other groups. If eggs are collected from the wild brood fish, stocking parr would therefore not be a sensible option. Stocking smolts instead would create a net benefit in terms of the number of spawners, but this strategy has serious drawbacks and risks associated with the larger phenotypic and demographic divergences from wild salmon. Supplementation was shown not to be the key factor behind the recovery of the Tornionjoki and other northern Baltic salmon stocks. Instead, a combination of restrictions in the sea fishery and simultaneous occurrence of favourable natural conditions for survival were the main reasons for the revival in the 1990 s. This study questions the effectiveness of supplementation as a conservation management tool. The benefits of supplementation seem at best limited. Relatively high occurrences of reared fish in catches may generate false optimism concerning the effects of supplementation. Supplementation may lead to genetic risks due to problems in brood fish collection and artificial rearing with relaxed natural selection and domestication. Appropriate management of fisheries is the main alternative to supplementation, without which all other efforts for long-term maintenance of a healthy fish resource fail.

Structural basis of cytoskeletal regulation by twinfilin

The actin cytoskeleton is required, in all eukaryotic organisms, for several key cellular functions such as cell motility, cytokinesis, and endocytosis. In cells, actin exists either in a monomeric state (G-actin) or in a filamentous form (F-actin). F-actin is the functional form, which can assemble into various structures and produce direct pushing forces that are required for different motile processes. The assembly of actin monomers into complicated three-dimensional structures is tightly regulated by a large number of actin regulating proteins. One central actin regulating protein is twinfilin. Twinfilin consists of two actin depolymerizing-factor homology (ADF-H) domains, which are capable of binding actin, and is conserved from yeast to mammals. Previously it has been shown that twinfilin binds to and sequesters G-actin, and interacts with the heterodimeric capping protein. More recently it has been found that twinfilin also binds to the fast growing actin filament ends and prevents their growth. However, the cellular role of twinfilin and the molecular mechanisms of these interactions have remained unclear. In this study we characterized the molecular mechanisms behind the functions of twinfilin. We demonstrated that twinfilin forms a high-affinity complex with ADP-bound actin monomers (ADP-G-actin). Both ADF-H domains are capable of binding G-actin, but the C-terminal domain contains the high-affinity binding site. Our biochemical analyses identified twinfilin s C-terminal tail region as the interaction site for capping protein. Contrary to G-actin binding, both ADF-H domains of twinfilin are required for the actin filament barbed end capping activity. The C-terminal domain is structurally homologous to ADF/cofilin and binds to filament sides in a similar manner, providing the main affinity for F-actin during barbed end capping. The structure of the N-terminal domain is more distant from ADF/cofilin, and thus it can only associate with G-actin or the terminal actin monomer at the filament barbed end, where it regulates twinfilin s affinity for barbed ends. These data suggest that the mechanism of barbed end capping is similar for twinfilin and gelsolin family proteins. Taken together, these studies revealed how twinfilin interacts with G-actin, filament barbed ends, and capping protein, and also provide a model for how these activities evolved through a duplication of an ancient ADF/cofilin-like domain.

Mechanism of RNA Translocation by a Viral Packaging Motor

Molecular motors are proteins that convert chemical energy into mechanical work. The viral packaging ATPase P4 is a hexameric molecular motor that translocates RNA into preformed viral capsids. P4 belongs to the ubiquitous class of hexameric helicases. Although its structure is known, the mechanism of RNA translocation remains elusive. Here we present a detailed kinetic study of nucleotide binding, hydrolysis, and product release by P4. We propose a stochastic-sequential cooperative model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive. Further, we use hydrogen/deuterium exchange detected by high resolution mass spectrometry to visualize P4 conformational dynamics during the catalytic cycle. Concerted changes of exchange kinetics reveal a cooperative unit that dynamically links ATP binding sites and the central RNA binding channel. The cooperative unit is compatible with the structure-based model in which translocation is effected by conformational changes of a limited protein region. Deuterium labeling also discloses the transition state associated with RNA loading which proceeds via opening of the hexameric ring. Hydrogen/deuterium exchange is further used to delineate the interactions of the P4 hexamer with the viral procapsid. P4 associates with the procapsid via its C-terminal face. The interactions stabilize subunit interfaces within the hexamer. The conformation of the virus-bound hexamer is more stable than the hexamer in solution, which is prone to spontaneous ring openings. We propose that the stabilization within the viral capsid increases the packaging processivity and confers selectivity during RNA loading. Finally, we use single molecule techniques to characterize P4 translocation along RNA. While the P4 hexamer encloses RNA topologically within the central channel, it diffuses randomly along the RNA. In the presence of ATP, unidirectional net movement is discernible in addition to the stochastic motion. The diffusion is hindered by activation energy barriers that depend on the nucleotide binding state. The results suggest that P4 employs an electrostatic clutch instead of cycling through stable, discrete, RNA binding states during translocation. Conformational changes coupled to ATP hydrolysis modify the electrostatic potential inside the central channel, which in turn biases RNA motion in one direction. Implications of the P4 model for other hexameric molecular motors are discussed.

Characterization of inhibition of platelet function by paracetamol and its interaction with diclofenac and parecoxib

Aim: To characterize the inhibition of platelet function by paracetamol in vivo and in vitro, and to evaluate the possible interaction of paracetamol and diclofenac or valdecoxib in vivo. To assess the analgesic effect of the drugs in an experimental pain model. Methods: Healthy volunteers received increasing doses of intravenous paracetamol (15, 22.5 and 30 mg/kg), or the combination of paracetamol 1 g and diclofenac 1.1 mg/kg or valdecoxib 40 mg (as the pro-drug parecoxib). Inhibition of platelet function was assessed with photometric aggregometry, the platelet function analyzer (PFA-100), and release of thromboxane B2. Analgesia was assessed with the cold pressor test. The inhibition coefficient of platelet aggregation by paracetamol was determined as well as the nature of interaction between paracetamol and diclofenac by an isobolographic analysis in vitro. Results: Paracetamol inhibited platelet aggregation and TxB2-release dose-dependently in volunteers and concentration-dependently in vitro. The inhibition coefficient was 15.2 mg/L (95% CI 11.8 - 18.6). Paracetamol augmented the platelet inhibition by diclofenac in vivo, and the isobole showed that this interaction is synergistic. Paracetamol showed no interaction with valdecoxib. PFA-100 appeared insensitive in detecting platelet dysfunction by paracetamol, and the cold-pressor test showed no analgesia. Conclusions: Paracetamol inhibits platelet function in vivo and shows synergism when combined with diclofenac. This effect may increase the risk of bleeding in surgical patients with an impaired haemostatic system. The combination of paracetamol and valdecoxib may be useful in patients with low risk for thromboembolism. The PFA-100 seems unsuitable for detection of platelet dysfunction and the cold-pressor test seems unsuitable for detection of analgesia by paracetamol.

Characterizing Physical Properties of Wetting Paper by Air Coupled Ultrasound and Light

This thesis reports investigations into the paper wetting process and its effects on the surface roughness and the out-of-plane (ZD) stiffness of machine-made paper. The aim of this work was to test the feasibility of employing air-borne ultrasound methods to determine surface roughness (by reflection) and ZD stiffness (by through transmission) of paper during penetration of distilled water, isopropanol and their mixtures. Air-borne ultrasound provides a non-contacting way to evaluate sample structure and mechanics during the liquid penetration event. Contrary to liquid immersion techniques, an air-borne measurement allows studying partial wetting of paper. In addition, two optical methods were developed to reveal the liquid location in paper during wetting. The laser light through transmission method was developed to monitor the liquid location in partially wetted paper. The white light reflection method was primarily used to monitor the penetration of the liquid front in the thickness direction. In the latter experiment the paper was fully wetted. The main results of the thesis were: 1) Liquid penetration induced surface roughening was quantified by monitoring the ultrasound reflection from the paper surface. 2) Liquid penetration induced stiffness alteration in the ZD of paper could be followed by measuring the change in the ultrasound ZD resonance in paper. 3) Through transmitted light revealed the liquid location in the partially wetted paper. 4) Liquid movement in the ZD of the paper could be observed by light reflection. The results imply that the presented ultrasonic means can without contact measure the alteration of paper roughness and stiffness during liquid transport. These methods can help avoiding over engineering the paper which reduces raw material and energy consumption in paper manufacturing. The presented optical means can estimate paper specific wetting properties, such as liquid penetration speed, transport mechanisms and liquid location within the paper structure. In process monitoring, these methods allow process tuning and manufacturing of paper with engineered liquid transport characteristics. With such knowledge the paper behaviour during printing can be predicted. These findings provide new methods for paper printing, surface sizing, and paper coating research.

Observations of production and transport of NOx formed by energetic particle precipitation in the polar night atmosphere

This work is focused on the effects of energetic particle precipitation of solar or magnetospheric origin on the polar middle atmosphere. The energetic charged particles have access to the atmosphere in the polar areas, where they are guided by the Earth's magnetic field. The particles penetrate down to 20-100 km altitudes (stratosphere and mesosphere) ionising the ambient air. This ionisation leads to production of odd nitrogen (NOx) and odd hydrogen species, which take part in catalytic ozone destruction. NOx has a very long chemical lifetime during polar night conditions. Therefore NOx produced at high altitudes during polar night can be transported to lower stratospheric altitudes. Particular emphasis in this work is in the use of both space and ground based observations: ozone and NO2 measurements from the GOMOS instrument on board the European Space Agency's Envisat-satellite are used together with subionospheric VLF radio wave observations from ground stations. Combining the two observation techniques enabled detection of NOx enhancements throughout the middle atmosphere, including tracking the descent of NOx enhancements of high altitude origin down to the stratosphere. GOMOS observations of the large Solar Proton Events of October-November 2003 showed the progression of the SPE initiated NOx enhancements through the polar winter. In the upper stratosphere, nighttime NO2 increased by an order of magnitude, and the effect was observed to last for several weeks after the SPEs. Ozone decreases up to 60 % from the pre-SPE values were observed in the upper stratosphere nearly a month after the events. Over several weeks the GOMOS observations showed the gradual descent of the NOx enhancements to lower altitudes. Measurements from years 2002-2006 were used to study polar winter NOx increases and their connection to energetic particle precipitation. NOx enhancements were found to occur in a good correlation with both increased high-energy particle precipitation and increased geomagnetic activity. The average wintertime polar NOx was found to have a nearly linear relationship with the average wintertime geomagnetic activity. The results from this thesis work show how important energetic particle precipitation from outside the atmosphere is as a source of NOx in the middle atmosphere, and thus its importance to the chemical balance of the atmosphere.