Functional expression and subcellular localization of the Cl- cotransporters KCC2 and NKCC1 in rodent hippocampal and neocortical neurons

The work presented here has focused on the role of cation-chloride cotransporters (CCCs) in (1) the regulation of intracellular chloride concentration within postsynaptic neurons and (2) on the consequent effects on the actions of the neurotransmitter gamma-aminobutyric acid (GABA) mediated by GABAA receptors (GABAARs) during development and in pathophysiological conditions such as epilepsy. In addition, (3) we found that a member of the CCC family, the K-Cl cotransporter isoform 2 (KCC2), has a structural role in the development of dendritic spines during the differentiation of pyramidal neurons. Despite the large number of publications dedicated to regulation of intracellular Cl-, our understanding of the underlying mechanisms is not complete. Experiments on GABA actions under resting steady-state have shown that the effect of GABA shifts from depolarizing to hyperpolarizing during maturation of cortical neurons. However, it remains unclear, whether conclusions from these steady-state measurements can be extrapolated to the highly dynamic situation within an intact and active neuronal network. Indeed, GABAergic signaling in active neuronal networks results in a continuous Cl- load, which must be constantly removed by efficient Cl- extrusion mechanisms. Therefore, it seems plausible to suggest that key parameters are the efficacy and subcellular distribution of Cl- transporters rather than the polarity of steady-state GABA actions. A further related question is: what are the mechanisms of Cl- regulation and homeostasis during pathophysiological conditions such as epilepsy in adults and neonates? Here I present results that were obtained by means of a newly developed method of measurements of the efficacy of a K-Cl cotransport. In Study I, the developmental profile of KCC2 functionality during development was analyzed both in dissociated neuronal cultures and in acute hippocampal slices. A novel method of photolysis of caged GABA in combination with Cl- loading to the somata was used in this study to assess the extrusion efficacy of KCC2. We demonstrated that these two preparations exhibit a different temporal profile of functional KCC2 upregulation. In Study II, we reported an observation of highly distorted dendritic spines in neurons cultured from KCC2-/- embryos. During their development in the culture dish, KCC2-lacking neurons failed to develop mature, mushroom-shaped dendritic spines but instead maintained an immature phenotype of long, branching and extremely motile protrusions. It was shown that the role of KCC2 in spine maturation is not based on its transport activity, but is mediated by interactions with cytoskeletal proteins. Another important player in Cl- regulation, NKCC1 and its role in the induction and maintenance of native Cl- gradients between the axon initial segment (AIS) and soma was the subject of Study III. There we demonstrated that this transporter mediates accumulation of Cl- in the axon initial segment of neocortical and hippocampal principal neurons. The results suggest that the reversal potential of the GABAA response triggered by distinct populations of interneurons show large subcellular variations. Finally, a novel mechanism of fast post-translational upregulation of the membrane-inserted, functionally active KCC2 pool during in-vivo neonatal seizures and epileptiform-like activity in vitro was identified and characterized in Study IV. The seizure-induced KCC2 upregulation may act as an intrinsic antiepileptogenic mechanism.

Valokuvallistettu luonto : I. K. Inhan tuotanto luonnon merkityksellistäjänä

Photographized nature I. K. Inha s work as a signification of nature The dissertation examines, through the work of the Finnish photographer and writer I. K. Inha (formerly Konrad Into Nyström) (1865 1930), the different ways in which the signification of nature is achieved. The principal material consists of Inha s work from 1890 to 1925, from which a number of photographs and texts are considered and upon which the photographization of nature is contemplated. The dissertation addresses the issue of how nature is conceived and how the act of photographizing it can be defined. The methodical context of the study is composed of three thematic baskets that structure the material and which consist of narrations on Finnish national perception, nature conservation and understanding the world. In the first case nature is seen as the natural environment encompassing lakes, seashores, forests, and hills, which at the time were often perceived from a utilitarian viewpoint. By the photographization they generated a pictorial narrative that could be shared. The natural environment was thus turned into landscape by means of photography, following the global pictorial concepts picturesque and sublime, as well as the national canons that had been developed in literature and the visual arts. In the narrations concerning nature conservation, the photographization did not merely occur within the limits of presuppositions, but rather nature was given the opportunity to unfold itself. While the photograph was being established as a basis for supporting nature conservation or to highlight the destruction of nature at the hand of Man, an attempt was made to represent subjects that were difficult to convey in photographs, such as nature s power or the miracle of growth. The thesis suggests, that in the third case the concept of nature broke away from its strict interconnection with the natural environment and led to a contemplation of nature that is perceivable in a person. In this context the photograph and the photographization are interpreted as an attempt to understand a person and his or her very existence in the world, while this same existential wonder is seen as being embodied in Inha s portrait of a rune singer and in his photographs of forest interior and water. Further, the thesis asks whether photographing nature could be interpreted as an action similar to the idea of the phenomenological reduction as a means of bypassing the photographer s prevailing way of being.

The Biocontrol Agent Phlebiopsis gigantea: Efficacy and Impacts on the Stump Bacterial Biota and Conifer tree Defences

Phlebiopsis gigantea has been for a long time known as a strong competitor against Heterobasidion annosum and intensively applied as a biological control agent on stump surfaces of Picea abies in Fennoscandia. However, the mechanism underlying its antagonistic activity is still unknown. A primary concern is the possible impact of P. gigantea treatment on resident non-target microbial biota of conifer stumps. Additional risk factor is the potential of P. gigantea to acquire a necrotrophic habit through adaptation to living wood tissues. This study focused on the differential screening of several P. gigantea isolates from diverse geographical sources as well as the use of breeding approach to enhance the biocontrol efficacy against H. annosum infection. The results showed a significant positive correlation between growth rate in wood and high biocontrol efficacy. Furthermore, with aid of breeding approach, several progeny strains were obtained that had better growth rate and control efficacy than parental isolates. To address the issue of the potential of P. gigantea to acquire necrotrophic capability, a combination of histochemical, molecular and transcript profiling (454 sequencing) were used to investigate the interactions between these two fungi and ten year old P. sylvestris seedlings. The results revealed that both P. gigantea and H. annosum provoked strong necrotic lesions, but after prolonged incubation, P. gigantea lesions shrank and ceased to expand further. Tree seedlings pre-treated with P. gigantea further restricted H. annosum-induced necrosis and had elevated transcript levels of genes important for lignification, cell death regulation and jasmonic acid signalling. These suggest that induced localized resistance is a contributory factor for the biocontrol efficacy of P.gigantea, and it has a comparatively limited necrotrophic capability than H. annosum. Finally, to investigate the potential impact of P. gigantea on the stump bacterial biota, 16S rDNA isolated from tissue samples from stumps of P. abies after 1-, 6- and 13-year post treatment was sequenced using bar-coded 454 Titanium pyrosequencing. Proteobacteria were found to be the most abundant at the initial stages of stump decay but were selectively replaced by Acidobacteria at advanced stages of the decay. Moreover, P. gigantea treatment significantly decreased the bacterial richness at initial decay stage in the stumps. Over time, the bacterial community in the stumps gradually recovered and the negative effects of P. gigantea was attenuated.

Tree mortality, fire scar formation and regeneration 8 years after low-intensity fire in managed Pinus sylvestris stands

Fire is an important driver of the boreal forest ecosystem, and a useful tool for the restoration of degraded forests. However, we lack knowledge on the ecological processes initiated by prescribed fires, and whether they bring about the desired restoration effects. The purpose of this study was to investigate the impacts of low-intensity experimental prescribed fires on four ecological processes in young commercial Scots pine (Pinus sylvestris) stands eight years after the burning. The processes of interest were tree mortality, dead wood creation, regeneration and fire scar formation. These were inventoried in twelve study plots, which were 30 m x 30 m in size. The plots belonged to two different stand age classes: 30-35 years or 45 years old at the time of burning. The study was partly a follow-up of study plots researched by Sidoroff et al. (2007) one year after burning in 2003. Tree mortality increased from 183 stems ha-1 in 2003 to 259 stems ha-1 in 2010, corresponding to 15 % and 21 % of stem number respectively. Most mortality was experienced in the stands of the younger age class, in smaller diameter classes and among species other than Scots pine. By 2010, the average mortality of Scots pine per plot was 18%, but varied greatly ranging from 0% to 63% of stem number. Delayed mortality, i.e. mortality that occurred between 2 and 8 years after fire, seemed to become more important with increasing diameter. The input of dead wood also varied greatly between plots, from none to 72 m3 ha-1, averaging at 12 m3 ha-1. The amount of fire scarred trees per plot ranged from none to 20 %. Four out of twelve plots (43 %) did not have any fire scars. Scars were on average small: 95% of scars were less than 4 cm in width, and 75% less than 40 cm in length. Owing to the light nature of the fire, the remaining overstorey and thick organic layer, regeneration was poor overall. The abundance of pine and other seedlings indicated a viable seed source existed, but the seedlings failed to establish under dense canopy. The number of saplings ranged from 0 to 12 333 stems ha-1. The results of this study indicate that a low intensity fire does not necessarily initiate the ecological processes of tree mortality, dead wood creation and regeneration in the desired scale. Fire scars, which form the basis of fire dating in fire history studies, did not form in all cases.

The many ways to delimit species: hairs, genes and surface chemistry

Species identification forms the basis for understanding the diversity of the living world, but it is also a prerequisite for understanding many evolutionary patterns and processes. The most promising approach for correctly delimiting and identifying species is to integrate many types of information in the same study. Our aim was to test how cuticular hydro- carbons, traditional morphometrics, genetic polymorphisms in nuclear markers (allozymes and DNA microsatellites) and DNA barcoding (partial mitochondrial COI gene) perform in delimiting species. As an example, we used two closely related Formica ants, F. fusca and F. lemani, sampled from a sympatric population in the northern part of their distribu- tion. Morphological characters vary and overlap in different parts of their distribution areas, but cuticular hydrocarbons include a strong taxonomic signal and our aim is to test the degree to which morphological and genetic data correspond to the chemical data. In the morphological analysis, species were best separated by the combined number of hairs on pro- notum and mesonotum, but individual workers overlapped in hair numbers, as previously noted by several authors. Nests of the two species were separated but not clustered according to species in a Principal Component Analysis made on nuclear genetic data. However, model-based Bayesian clustering resulted in perfect separation of the species and gave no indication of hybridization. Furthermore, F. lemani and F. fusca did not share any mitochondrial haplotypes, and the species were perfectly separated in a phylogenetic tree. We conclude that F. fusca and F. lemani are valid species that can be separated in our study area relatively well with all methods employed. However, the unusually small genetic differen- tiation in nuclear markers (FST = 0.12) shows that they are closely related, and occasional hybridization between F. fusca and F. lemani cannot be ruled out.

Myymälämarkkinointi osana markkinointiviestintää – Kuluttajien arvostamat tekijät päivittäistavarakaupassa. CASE: K-supermarket Torpparinmäki

Tässä tutkimuksessa tarkastellaan K-supermarket Torpparinmäen asiakkaiden arvostuksia myymälän sisäisiin toimiin liittyen. Myymälän sisäisiin toimiin lasketaan tässä tutkimuksessa kuuluvan laaja kirjo asioita kuten hinnat, tarjoukset, valikoiman laajuus eri tuoteryhmissä, elämyksellisyys, hyllysijoittelu, kaupan selkeys, asiakaspalvelu sekä viihtyisyys. Myös merkityksellisten ja vähemmän merkityksellisten asioiden toteutumisesta kyseisessä myymälässä halutaan vastauksia. Tutkimuksessa nostetaan esille myös erilaiset tiedotuskanavat kaupan markkinoinnissa. Tämän lisäksi sivutaan aihetta kuluttajien päivittäistavarakaupan valintakriteereistä. Tutkimusote on kvantitatiivinen ja kysely suoritettiin survey-tutkimuksena. Tutkimuksessa haluttiin tämän lisäksi kerätä mahdollisimman paljon myös avoimia kommentteja sekä myymälästä että päivittäistavarakaupan valintaan liittyvistä kriteereistä. K-supermarket Torpparinmäen pahin kilpailija alueellaan on S-market Torpparinmäki. Asiakkailta haluttiin kysyä avoimesti, miksi asioivat juuri kyseisessä myymälässä. Kysely suoritettiin lauantaipäivänä K-supermarket Torpparinmässä asioineille 18-89-vuotiaille henkilöille. Kyselyyn vastasi 80 asiakasta, joista 65 % oli naisia ja 35 % miehiä. Tutkimuksen viitekehys jakaa myymälämarkkinoinnin neljään pääkategoriaan: viihtyvyystekijä, palvelutekijä, hintatekijä sekä valikoimatekijä. Näiden tekijöiden merkityksiä asiakkaille haluttiin tutkimuksessa asettaa tärkeysjärjestykseen. Myös näiden tekijöiden toteumaa K-supermarket Torpparinmäessä haluttiin selvittää. Tulosten analysoinnin perusteella edellä mainituista pääkategorioista tärkeimmäksi muodostui valikoimatekijä. Valikoimien monipuolisuutta pidettiin kaikista tärkeimpänä tekijänä myymälässä. Myös luomu- ja lähiruoan valikoimia pidettiin tärkeäpänä kuin valmisruokien. Toiseksi eniten asiakkaat arvostivat asiakaspalvelua ja vasta kolmannelle sijalle päätyi hintatekijä. Neljänneksi tärkeimpänä asiakkaat pitivät viihtyvyystekijää. Mikään tekijöistä ei erottunut suuresti toisistaan, mutta erot olivat kuitenkin selkeitä.