Levosimendaanin vaikutusmekanismi ja nykyinen merkitys sydämen vajaatoiminnan hoidossa

Sydämen vajaatoiminta on erilaisista sydän- ja verisuonisairauksista aiheutuva monimuotoinen oireyhtymä, johon sairastuneiden ja kuolleiden potilaiden määrä on yhä suuri. Sen patofysiologiaan voi kuulua muun muassa sympaattisen hermoston ja reniini-angiotensiini-aldosteroni–järjestelmän aktiivisuutta, huonosti supistuva vasen kammio, sydämen uudelleenmuokkautumista, muutoksia [Ca2+]i:n säätelyssä, kardiomyosyyttien apoptoosia sekä systeeminen tulehdustila. Johonkin osaan sairauden patofysiologiasta eivät nykyiset lääkehoidot riittävästi vaikuta. Klassiset inotroopit lisäävät sydämen supistusvireyttä kasvattamalla solunsisäistä Ca2+-pitoisuutta, mutta ne lisäävät rytmihäiriöriskiä, sydämen hapenkulutusta sekä heikentävät ennustetta. Levosimendaani, kalsiumherkistäjä, lisää sydämen supistusvoimaa [Ca2+]i:ta kohottamatta herkistämällä sydänlihaksen kalsiumin vaikutuksille. Lisäksi levosimendaani avaa sarkolemmaalisia ja mitokondriaalisia K+-kanavia, jotka välittävät vasodilataatiota ja kardioprotektiota. Suurilla annoksilla levosimendaani on selektiivinen PDE3-estäjä. Levosimendaania suositellaan äkillisesti pahentuneen sydämen vajaatoiminnan hoitoon, mutta muitakin lupaavia indikaatioita sille on keksitty. Esimerkiksi kroonisesti annosteltu oraalinen levosimendaani on suojannut kardiovaskulaarijärjestelmää ja parantanut selviytymistä in vivo. Erikoistyössä selvitettiin kroonisesti annostellun oraalisen levosimendaanin, valsartaanin ja näiden kombinaatioterapian vaikutuksia selviytymiseen, verenpaineeseen sekä sydämen hypertrofioitumiseen Dahlin suolaherkillä (Dahl/Rapp) rotilla. Levosimendaanin suojavaikutus ilmeni vähäisempänä kuolleisuutena, mutta ero ei ollut tilastollisesti merkitsevä kontrolliryhmään nähden. Kombinaatioterapia suojasi rottia kardiovaskulaarikuolleisuudelta ja vähensi todennäköisesti verenpaineesta riippuvaisesti sydämen hypertofioitumista niin sydän/kehonpaino–suhteen kuin ultraäänitutkimuksenkin perusteella arvioituna paremmin kuin kumpikaan lääke monoterapiana. Lääkekombinaatio alensi additiivisesti hypertensiota kaikissa mittauspisteissä. Sydämen systolista toimintaa levosimendaani kohensi vain vähäisesti. Dahl/Rapp-rotille kehittyikin pääosin hypertension indusoimaa diastolista sydämen vajaatoimintaa kohonneen IVRT-arvon perusteella. Levosimendaani sekä monoterapiana että kombinaatioterapiana valsartaanin kanssa vähensi sydämen diastolista vajaatoimintaa.

Search for the rare decays B+→μ+μ-K+, B0→μ+μ-K*(892)0, and Bs0→μ+μ-ϕ at CDF

We search for b to s\mu^+\mu^- transitions in B meson (B^+, B^0, or B^0_s) decays with 924pb^{-1} of p pbar collisions at sqrt(s)=1.96 TeV collected with the CDF II detector at the Fermilab Tevatron. We find excesses with significances of 4.5, 2.9, and 2.4 standard deviations in the B^+ to \mu^+\mu^-K^+, B^0 to \mu^+\mu^-K^*(892)^0, and B_s^0 to \mu^+\mu^-\phi decay modes, respectively. Using B to J/psi h (h = K^+, K^*(892)^0, phi) decays as normalization channels, we report branching fractions for the previously observed B^+ and B^0 decays, BR(B^+ to \mu^+\mu^-K^+)=(0.59\pm0.15\pm0.04) x 10^{-6}, and BR(B^0 to \mu^+\mu^-K^*(892)^0)=(0.81\pm0.30\pm0.10) x 10^{-6}, where the first uncertainty is statistical, and the second is systematic. These measurements are consistent with the world average results, and are competitive with the best available measurements. We set an upper limit on the relative branching fraction BR(B_s^0 to \mu^+\mu^-\phi)/BR(B_s^0 to J/\psi\phi)

Search for the rare decays B+→μ+μ-K+, B0→μ+μ-K*(892)0, and Bs0→μ+μ-ϕ at CDF

We search for b→sμ+μ- transitions in B meson (B+, B0, or Bs0) decays with 924  pb-1 of pp̅ collisions at √s=1.96  TeV collected with the CDF II detector at the Fermilab Tevatron. We find excesses with significances of 4.5, 2.9, and 2.4 standard deviations in the B+→μ+μ-K+, B0→μ+μ-K*(892)0, and Bs0→μ+μ-ϕ decay modes, respectively. Using B→J/ψh (h=K+, K*(892)0, ϕ) decays as normalization channels, we report branching fractions for the previously observed B+ and B0 decays, B(B+→μ+μ-K+)=(0.59±0.15±0.04)×10-6, and B(B0→μ+μ-K*(892)0)=(0.81±0.30±0.10)×10-6, where the first uncertainty is statistical, and the second is systematic. We set an upper limit on the relative branching fraction B(Bs0→μ+μ-ϕ)/B(Bs0→J/ψϕ)<2.6(2.3)×10-3 at the 95(90)% confidence level, which is the most stringent to date.

First simultaneous measurement of the top quark mass in the lepton+jets and dilepton channels at CDF

We present a measurement of the mass of the top quark using data corresponding to an integrated luminosity of 1.9fb^-1 of ppbar collisions collected at sqrt{s}=1.96 TeV with the CDF II detector at Fermilab's Tevatron. This is the first measurement of the top quark mass using top-antitop pair candidate events in the lepton + jets and dilepton decay channels simultaneously. We reconstruct two observables in each channel and use a non-parametric kernel density estimation technique to derive two-dimensional probability density functions from simulated signal and background samples. The observables are the top quark mass and the invariant mass of two jets from the W decay in the lepton + jets channel, and the top quark mass and the scalar sum of transverse energy of the event in the dilepton channel. We perform a simultaneous fit for the top quark mass and the jet energy scale, which is constrained in situ by the hadronic W boson mass. Using 332 lepton + jets candidate events and 144 dilepton candidate events, we measure the top quark mass to be mtop=171.9 +/- 1.7 (stat. + JES) +/- 1.1 (syst.) GeV/c^2 = 171.9 +/- 2.0 GeV/c^2.

Konstrukcija mnimosti : K poètike "Voskovoj persony" Ju. Tynjanova

The subject of this work is the poetics of «The Wax Effigy», a short novel or novella by Jurii Tynianov, Russian writer, literary critic, historian of literature and prominent literary theoretician. The plot structure of the novel is based upon a real event, the creation by Bartolomeo Carlo Rastrelli in 1725 of a wax sculpture of the first Russian emperor, Peter the Great. «Construction of the Sham» consists of three chapters, an introduction and a conclusion. Due to the fact that Tynianov was at the same time a prose writer and theoretician of literature it seemed important to consider the reception of his prose and his works on literary theory in relationship to each other. The introduction is devoted to this task. The first chapter is about the history of the creation of the novel and its reception. Tynianov stopped writing one short story in order to write the novel; these two works have some common traits. It seems almost obvious that his work on the first text was a real step toward the creation of the second. In the first story there is an opposition of dead/alive which is semantic prefiguring of a central motif in «The Wax Effigy». An analysis of the reception of the novel demonstrated that almost every critic writing about the novel has described it as nonsense. Critics considered Tynianov's work in terms of «devices» and «content» and could not understand how devices are related to the content of the novel: the novel was thought as a signifier without any signified. Implicitly, critics thought the signified of the novel as a traditional one of the historical novel, as the historiosophical «idea», embodied in the system of literary devices. In this case literature becomes something instrumental, a kind of expression of extraliterary content. In contradistinction to that Tynianov considered literary semantics as an effect of the literary structure. From his point of view the literary sense is immanent to the process of signification accomplished inside the literary text. The second chapter is devoted to a rhetorical analysis of the opposition dead/alive. Tynianov systematically compares both terms of the opposition. As a result of this strategy the wax effigy of the dead emperor becomes «as if» alive and the world of living people «as if» dead. The qualifier «as if» refers to the fact that Tynianov creates an ambiguous semantic system. This rhetoric is related to European Romanticism and his «fantastic literature» (Merimé, Hoffmann, Maupassant etc.). But Tynianov demonstrates a linguistic origin of the strange fantoms created by romantics; he demystifies these idols by parodying the fantastic literature, that is, showing «how it was done». At the same time, the opposition mentioned above refers to his idea of «incongruity» which plays a prominent role in Tynianov s theory but has never been conceptualised. The incongruity is a inner collision of the literary text; from Tynianov's point of view the meaning of the work of literature is always a dynamic collision of semantically heterogeneous elements struggling with each other. In «The Wax Effigy» Tynianov creates a metalevel of the work demonstrating the process of creation of the literary sense. The third chapter is a reconstruction of Tynianov's conception of the historical prose, specifically of the mechanisms by which historical facts are transformed into literary events. Tynianov thought that the task of the historical novelist is to depict his hero as an actor, to demonstrate that as a wearer of many masks he is a creator of appearances, ambiguities. Here, in the «figure of fiction» (Andrei Belyi), the very idea of the historical prose and rhetoric employed in «The Wax Effigy», history and literature meet each other. In his last theoretical work, «On parody» Tynianov writes about the so-called sham structure of parody. In his opinion every parody is a text about other texts and «serious» work which could be read at the same time as a text about «reality». This twofold structure of parody is that of «The Wax Effigy»: that text speaks about ambiguities of the history and about ambiguities of the literary sense, about social reality of the past and - about the working of the literature itself. «The Wax Effigy» is written as a autoreflective text, as an experiment in literary semantics, as a system of literary ambiguities - of hero, rhetoric and the text itself. The meaning of the novel is created not by the embodiment extraliterary idea, but by the process of signification accomplished inside the work of literature. In this sense Tynianov's novel is parody, a break with the tradition of the historical novel preceding «The Wax Effigy».

Olen maa johon tahdot : Timo K. Mukan maailmankuvan poetiikkaa

The dissertation deals with the prose texts of the Finnish writer Timo K. Mukka, renowned for his depictions of his native Lapland. This research concerns the creation of world view in Mukka s prose, which is approached by studying what Mikhail Bakhtin calls generic change. Such genre change is the most characteristic feature of Mukka s prose. His prose is permeated with two genres in particular and changes between them: the ballad-like, archaistic and romantic prose-poem style and naturalistic, even grotesque expressions. In addition, these genres are associated with sublime and grotesque styles so that generic change tends to involve also stylistic changes in Mukka s prose. This study probes the tension-filled interrelationships between the ballad and naturalistic prose by examining the discourse of Mukka s characters. It is shown that these characters invariably find themselves in what Bakhtin calls the chronotope of the threshold; that is, the plots of Mukka s novels and short stories depict situations in which the characters are faced with decisions and deeds that will profoundly impact their lives. The discourse of the threshold affects the characters speech by filling it with dialogical dimensions. This makes their communication ethically loaded and polyphonic. This study is based on Mikhail Bakhtin s theory of the novel and international Bakhtin s studies. I also take into consideration the theoretical developments of Bakhtin s work; for example, the concept of ressentiment, adapted from the Bakhtin scholar Michel André Bernstein, plays an important role. In order to explicate on the psychology of Mukka s characters such as melancholy, abjection, sadism, and taboo I use the concepts familiar from Freudian psychoanalysis. The corpus of my research consists of the following texts: the long prose texts Maa on syntinen laulu. Balladi (1964), Tabu (1965), Täältä jostakin. Romaani (1965), Laulu Sipirjan lapsista. Romaani (1966), Ja kesän heinä kuolee. Kertomus sairaudesta (1968) ja Kyyhky ja unikko (1970) and the short story collections Koiran kuolema (1967) ja Lumen pelko (1970), and with Tabu published short story Sankarihymni , the short story Katkelma laajemmasta laulelmasta from the collection Rakastaa: Kaksitoista novellia rakkaudesta (1965) and also the short stories which were published in various Finnish journals: Yöt (1965), Liisa (1967), Tyttö (1967) ja Näin hetki sitten ketun (1970). I pay particular attention to the novel Maa on syntinen laulu, because it expresses the generic change characteristic of Mukka s world view in a specifically clear and lively way. The dissertation is in Finnish. Key words: Timo K. Mukka, world view, genre change, ballad, naturalism, grotesque realism, Mikhail Bakhtin, dialogism, polyphony, chronotope, sublime, grotesque, ressentiment, Sigmund Freud, melancholy, taboo, abject, sadism, reduced laughter, modern parody.

Litteraturundervisning och ämnessyn i modersmål och litteratur: En kvalitativ undersökning bland 13 modersmålslärare inom den grundläggande utbildningen (åk 7–9)

I denna avhandling undersöker jag hur modersmålslärare inom den grundläggande utbildningen (åk 7–9) undervisar i litteratur. De frågor jag ställer mig är: Hur förverkligar lärarna sin litteraturundervisning i praktiken, hur förhåller de sig till den nya läroplanen i ämnet, Grunderna för läroplanen för den grundläggande utbildningen 2004, samt med vilken ämnes-, litteratur- och kunskapssyn utgår de från i sin undervisning? Undersökningsresultatet analyseras utifrån läroplan och utifrån en läsarorienterad, sociokulturell och funktionell litteratursyn och litteraturpedagogik. Undersökningen är kvalitativ och riktar sig i form av ett frågeformulär till alla modersmålslärare i högstadierna i huvudstadsregionen. Tretton lärare deltar slutligen och åtta av dessa svar uppföljs genom en tematisk intervju. Undersökningen sker våren 2005. Forskningsresultatet visar på att flera av lärarna till stor del bygger upp sin undervisning utifrån färdiga uppgifter och koncept och att undervisningen styrs av metoden, av frågan hur. Dessa lärares undervisning sker således inte utgående från en enhetlig ämnes-, litteratur- och kunskapssyn, utan ämnessynen varierar beroende på uppgift. Recensionen är den arbetsuppgift som är vanligast bland lärarna, även om flera lärare ställer sig tveksamma till den. Övriga uppgifter som förekommer bland flera lärare är uppgiftskoncept kring klassiker och litterära samtal inför klass. Tematisk litteraturundervisning används även av flera lärare, liksom Netlibris, som innebär att två klasser från olika skolor över nätet diskuterar en skönlitterär text. Både Netlibris och tematisk litteraturundervisning är metoder som lärarna förhåller sig positiva till och ser pedagogiska fördelar med. Den tematiska undervisningen sker emellertid ofta i form av koncept som återkommer i samma form år efter år. Två lärare strävar dock efter att arbeta utifrån en enhetlig ämnessyn – den erfarenhetspedagogiska. Dessa lärare använder sig av färre koncept i sin undervisning. De bygger snarare upp sin undervisning utifrån frågan varför än hur, vilket innebär att de i högre grad har möjlighet att beakta elevernas intressen och behov i utformandet av undervisningen. Detta syns bland annat i valet av böcker och teman. En av dessa lärare använder sig även av portfölj- och loggboksmetodik och låter, i enlighet med läroplanen, eleverna själva vara med och utvärdera undervisningen och den egna utvecklingen. Lärarna verkar överlag förhålla sig mera positiva till de uppgifter som bygger på dialog och som tar fasta på läsaren i läsprocessen. Trots att lärarna betonar dialogens betydelse i litteraturundervisningen, bygger undervisningen ändå ofta, och i flera fall helt och hållet, på uppgifter som saknar dialog. En av orsakerna till detta kan vara att gamla koncept, trots nya uppgifter, lever kvar i undervisningen. Att lärarna framhåller de uppgifter som utgår från läsning av gemensamma texter och som tar fasta på en läsarorienterad och sociokulturell litteratursyn, visar dock på att de nya uppgifterna håller på att omkullkasta de gamla; lärarnas litteraturundervisning befinner sig således i förändring. Samtliga lärare förhåller sig positiva till litteraturundervisning och framhåller att litteraturen sedan deras egen skoltid fått en mera framträdande roll i undervisningen. De påpekar också att modersmålsundervisningen idag är mindre färdighetsinriktad och går mot en ökad dialog.

The Functions and Regulation of Ornithine Decarboxylase

Ornithine decarboxylase (ODC) regulates the synthesis of polyamines which are involved in many cellular functions e.g. proliferation and differentiation. Due to its critical role, ODC is a tightly regulated enzyme by antizymes and antizyme inhibitors. If the regulation fails, the activity of ODC increases and may lead to malignant transformation of a cell. Increased ODC activity is found in many common cancers, including colon, prostate, and breast cancer. In a transformed cell, dynamics of the actin cytoskeleton is disturbed. A small G-protein, RhoA regulates organization of the cytoskeleton, and its overactivity increases malignant potential of the cell. The present results indicate that covalent attachment of polyamines by transglutaminase is a physiological means of regulating the activity of RhoA. The translocation of RhoA to the plasma membrane, where it exerts its activity is dependent on the presence of catalytically active ODC. As the overactivity of ODC and RhoA are implicated in cell transformation, the results provide a mechanistic explanation of the interrelationship between the polyamine metabolism and the reorganization of the actin cytoskeleton occurring in cancer cells. ODC and polyamines have also an important role in the function of central nervous system. They participate in the regulation of brain morphogenesis in embryos. In adult nervous tissue, polyamines regulate K+ and glutamate channels. K+ inward rectifying channels control membrane potentials and NMDA-type glutamate receptors (NMDAR) regulate synaptic plasticity. High ODC activity and polyamine levels are considered important in the development of ischemic brain damage and they are implicated in the pathogenesis of Alzheimer s disease (AD). A homolog of ODC was cloned from a human brain cDNA library, and several alternatively spliced variants were detected in human brain and testis. The novel protein was nevertheless devoid of ODC catalytic activity. It was subsequently found to be a novel inductor of ODC activity and polyamine synthesis, called antizyme inhibitor 2 (AZIN2). The accumulation of AZIN2 in vesicle-like formations along the axons and beneath the plasma membrane of neurons as well as in steroid hormone producing Leydig cells and luteal cells of the gonads implies that AZIN2 plays a role in secretion and vesicle trafficking. An accumulation of AZIN2 was detected also in specimens of AD brains. This increased expression of AZIN2 was specific for AD and was not found in brains with other neurodegenerative diseases including CADASIL or dementia with Lewy bodies.

Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Yksilöllisyys ja yhteisöllisyys K. E. Nipkowin uskonnonpedagogisen teorian kehityksessä 1960-1990

Individuality and the Community in the Development of K. E. Nipkow's Theory of Religious Education from 1960 to 1990 The purpose of this study was to describe and analyze the development occurred between 1960 and 1990 of the theory of religious education as proposed by K. E. Nipkow, the German Religious Education specialist, from the point of view of individuality and the community. Nipkow's methodological approach of dialectic convergence theory resulted in a dialogue between theological and educational factors, which supported the thirty-year development of Nipkow's models, theoretical foundations, and theory of religious education. Nipkow's doctoral dissertation, published in 1960, deals with individuality in the thinking of Pestalozzi, Humboldt and Schleiermacher. Nipkow regarded individuality as one of the basic concepts of education, which were to be interpreted anew as social and historical situations changed. In the late 1960s Nipkow developed the so-called experiential hermeneutically oriented context model for the needs of religious education. In this model, individuality is expressed in the attention paid to pupils' life situations and the educational reality. The multi-dimensional theoretical framework of religious education in 1975 emphasized supporting identity as a fundamental task of religious education. The concept of individuality was thus given a new form, in accordance with contemporary theories of developmental psychology. Other fundamental tasks, such as the socio-ethical task, the task of critical religious thinking, and that of ecumenical learning, meant a more specific emphasis on the community. It was an outline of a liberating education, which faced the individualistic-existential and social-ecclesiastical challenges of the time with a critical attitude. The further development of the theoretical outline in 1982 continued to uphold the perspectives of both individuality and the community, as Nipkow combined a historical-social dimension with theories of developmental psychology, especially that of life-span research. According to him, the development of the individual and communal life-reality belonged together. The fundamental task of religious education came to be learning to live and believe together. Nipkow transferred the idea of dialogue into inter-generational learning and developed elementarization as a methodology of Religious Education, which takes into account the point of departure of each age group. His theory of educational responsibility in the church (1990) contained the tasks of walking alongside the individual and the renewal of church communities as prerequisites of communicating the Christian faith in an era characterized by multifaceted Christianity. The "geisteswissenschaftliche" school and its concepts (Ger. Individualität; Bildung) were found to be the explanatory factor of the concepts of individuality and the community in the development of Nipkow's theory of religious education. The concept of education employed by Nipkow (Ger. Bildung) implies, on one hand, the individuality, autonomy, freedom and personal responsibility of people of different ages, and on the other hand, the dialogical nature of education in the community facilitated by this concept. Theologically, Nipkow associates himself in his views on individuality and the community with Schleiermacher's understanding of faith, of which openness towards the world was characteristic. The significance of individuality and the community in Nipkow's thinking was, furthermore, deepened by his participation, as a member of working parties, in the educational discussions of the World Council of Churches.

Structural and functional roles of KCC2 in developing cortex

Cation chloride cotransporters (CCCs) are critical for controlling intracellular chloride homeostasis. The CCC family is composed of four isoforms of K-Cl cotransporters (KCC1-4), two isoforms of Na-K-2Cl cotransporters (NKCC1-2), one Na-Cl cotransporter (NCC) and two the structurally related proteins with unknown function, CCC8 also known as cation-chloride cotransporter interaction protein, CIP, and CCC9. KCC2 is a neuron-specific isoform, which plays a prominent role in controlling the intracellular Cl- concentration in neurons and is responsible for producing the negative shift of GABAA responses from depolarizing to hyperpolarizing during neuronal maturation. In the present studies we first used in situ hybridization to examine the developmental expression patterns of the cation-chloride cotransporters KCC1-4 and NKCC1. We found that they display complementary expression patterns during embryonic brain development. Most interestingly, KCC2 expression in the embryonic central nervous system strictly follows neuronal maturation. In vitro data obtained from primary and organotypic neuronal cultures support this finding and revealed a temporal correlation between the expression of KCC2 and synaptogenesis. We found that KCC2 is highly expressed in filopodia and mature spines as well as dendritic shaft and investigated the role of KCC2 in spine formation by analyzing KCC2-/- neurons in vitro. Our studies revealed that KCC2 is a key factor in the maturation of dendritic spines. Interestingly, the effect of KCC2 in spine formation is not due to Cl- transport activity, but mediated through the interaction between KCC2 C-terminal and intracellular protein associated with cytoskeleton. The interacting protein we found is protein 4.1N by immunoprecipitation. Our results indicate a structural role for KCC2 in the development of functional glutamatergic synapses and suggest KCC2 as a synchronizer for the functional development of glutamatergic and GABAergic synapses in neuronal network. Studies on the regulatory mechanisms of KCC2 expression during development and plasticity revealed that synaptic activity of both the glutamatergic and GABAergic system is not required for up-regulation of KCC2 during development, whereas in acute mature hippocampal slices which undergo continuous synchronous activity induced by the absence of Mg2+ solution, KCC2 mRNA and protein expression were down-regulated in CA1 pyramidal neurons subsequently leading to a reduced capacity for neuronal Cl- extrusion. This effect is mediated by endogenous BDNF-TrkB down-stream cascades involving both Shc/FRS-2 and PLCγ-CREB signaling. BDNF mediated changes in KCC2 expression indicate that KCC2 is significantly involved in the complex mechanisms of neuronal plasticity during development and pathophysiological conditions.

Neuronal K-Cl Cotransporter : Transcriptional Mechanisms of KCC2 Gene Regulation

K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.

Ion-regulatory proreins in neuronal development and communication

Brain function is critically dependent on the ionic homeostasis in both the extra- and intracellular compartment. The regulation of brain extracellular ionic composition mainly relies on active transport at blood brain and at blood cerebrospinal fluid interfaces whereas intracellular ion regulation is based on plasmalemmal transporters of neurons and glia. In addition, the latter mechanisms can generate physiologically as well as pathophysiologically significant extracellular ion transients. In this work I have studied molecular mechanisms and development of ion regulation and how these factors alter neuronal excitability and affect synaptic and non-synaptic transmission with a particular emphasis on intracellular pH and chloride (Cl-) regulation. Why is the regulation of acid-base equivalents (H+ and HCO3-) and Cl- of such interest and importance? First of all, GABAA-receptors are permeable to both HCO3- and Cl-. In the adult mammalian central nervous system (CNS) fast postsynaptic inhibition relies on GABAA-receptor mediated transmission. Today, excitatory effects of GABAA-receptors, both in mature neurons and during the early development, have been recognized and the significance of the dual actions of GABA on neuronal communication has become an interesting field of research. The transmembrane gradients of Cl- and HCO3- determine the reversal potential of GABAA-receptor mediated postsynaptic potentials and hence, the function of pH and Cl- regulatory proteins have profound consequences on GABAergic signaling and neuronal excitability. Secondly, perturbations in pH can cause a variety of changes in cellular function, many of them resulting from the interaction of protons with ionizable side chains of proteins. pH-mediated alterations of protein conformation in e.g. ion channels, transporters, and enzymes can powerfully modulate neurotransmission. In the context of pH homeostasis, the enzyme carbonic anhydrase (CA) needs to be taken into account in parallel with ion transporters: for CO2/HCO3- buffering to act in a fast manner, CO2 (de)hydration must be catalyzed by this enzyme. The acid-base equivalents that serve as substrates in the CO2 dehydration-hydration reaction are also engaged in many carrier and channel mediated ion movements. In such processes, CA activity is in key position to modulate transmembrane solute fluxes and their consequences. The bicarbonate transporters (BTs; SLC4) and the electroneutral cation-chloride cotransporters (CCCs; SLC12) belong the to large gene family of solute carriers (SLCs). In my work I have studied the physiological roles of the K+-Cl- cotransporter KCC2 (Slc12a5) and the Na+-driven Cl--HCO3- exchanger NCBE (Slc4a10) and the roles of these two ion transporters in the modualtion of neuronal communication and excitability in the rodent hippocampus. I have also examined the cellular localization and molecular basis of intracellular CA that has been shown to be essential for the generation of prolonged GABAergic excitation in the mature hippocampus. The results in my Thesis provide direct evidence for the view that the postnatal up-regulation of KCC2 accounts for the developmental shift from depolarizing to hyperpolarizing postsynaptic EGABA-A responses in rat hippocampal pyramidal neurons. The results also indicate that after KCC2 expression the developmental onset of excitatory GABAergic transmission upon intense GABAA-receptor stimulation depend on the expression of intrapyramidal CA, identified as the CA isoform VII. Studies on mice with targeted Slc4a10 gene disruption revealed an important role for NCBE in neuronal pH regulation and in pH-dependent modulation of neuronal excitability. Furthermore, this ion transporter is involved in the basolateral Na+ and HCO3- uptake in choroid plexus epithelial cells, and is thus likely to contribute to cerebrospinal fluid production.