Short-term effects of variable retention on epigaeic spiders and carabid beetles in Finland

Forestry has influenced forest dwelling organisms for centuries in Fennoscandia. For example, in Finland ca. 30% of the threatened species are threatened because of forestry. Nowadays forest management recommendations include practices aimed at maintaining biodiversity in harvesting, such as green-tree retention. However, the effects of these practices have been little studied. In variable retention, different numbers of trees are retained, varying from green-tree retention (at least a few live standing trees in clear-cuts) to thinning (only individual trees removed). I examined the responses of ground-dwelling spiders and carabid beetles to green-tree retention (with small and large tree groups), gap felling and thinning aimed at an uneven age structure of trees. The impacts of these harvesting methods were compared to those of clear-cutting and uncut controls. I aimed to test the hypothesis that retaining more trees positively affects populations of those species of spiders and carabids that were present before harvesting. The data come from two studies. First, spiders were collected with pitfall traps in south-central Finland in 1995 (pre-treatment) and 1998 (after-treatment) in order to examine the effects of clear-cutting, green-tree retention (with 0.01-0.02-ha sized tree groups), gap felling (with three 0.16-ha sized openings in a 1-ha stand), thinning aiming at an uneven age structure of trees and uncut control. Second, spiders and carabids were caught with pitfall traps in eastern Finland in 1998-2001 (pre-treatment and three post-treatment years) in eleven 0.09-0.55-ha sized retention-tree groups and clear-cuts adjacent to them. Original spider and carabid assemblages were better maintained after harvests that retained more trees. Thinning maintained forest spiders well. However, gap felling and large retention-tree groups maintained some forest spider and carabid species in the short-term, but negatively affected some species over time. However, use of small retention-tree groups was associated with negative effects on forest spider populations. Studies are needed on the long-term effects of variable retention on terrestrial invertebrates; especially those directed at defining appropriate retention patch size and on the importance of structural diversity provided by variable retention for invertebrate populations. However, the aims of variable retention should be specified first. For example, are retention-tree groups planned to constitute life-boats , stepping-stones or to create structural diversity? Does it suffice that some species are maintained, or do we want to preserve the most sensitive ones, and how are these best defined? Moreover, the ecological benefits and economic costs of modified logging methods should be compared to other approaches aimed at maintaining biodiversity.

Effects of recreational use and fragmentation on the understorey vegetation and soil microbial communities of urban forests in southern Finland

The impacts of fragmentation and recreational use on the hemiboreal urban forest understorey vegetation and the microbial community of the humus layer (the phospholipid fatty acid (PLFA) pattern, microbial biomass and microbial activity, measured as basal respiration) were examined in the greater Helsinki area, southern Finland. Trampling tolerance of 1) herb-rich OMT, 2) mesic MT, and 3) sub-xeric VT forests (in decreasing order of fertility) was studied by comparing relative understorey vegetation cover (urban/untrampled reference ratio) of the three forest types. The trampling tolerance of forest vegetation increased with the productivity of the site (sub-xeric < mesic < herb-rich). Wear of understorey vegetation correlated positively with the number of residents (i.e., recreational pressure) around the forest patch. An increase of 15000 residents within a radius of 1 km around a forest patch was associated with ca. 30% decrease in the relative understorey vegetation cover. The cover of dwarf shrub Vaccinium myrtillus in particular decreased with increasing levels of wear. The cover of mosses in urban forests was less than half of that in untrampled reference areas. Cover of tree saplings, mainly Sorbus aucuparia, and some resilient herbs was higher than in the reference areas. In small urban forest fragments, broad-leaved trees, grasses and herbs were more abundant and mosses were scarcer than in larger urban forest areas. Thus, due to trampling and edge effects, resilient herb and grass species are replacing sensitive dwarf shrubs, mosses and lichens in urban forests. Differences in the soil microbial community structure were found between paths and untrampled areas and the effects of paths extended more than one meter from the paths. Paths supported approximately 25-30% higher microbial biomass with a transition zone of at least 1 m from the path edge. However, microbial activity per unit of biomass was lower on paths than in untrampled areas. Furthermore, microbial biomass and activity were 30-45% lower at the first 20 m into the forest fragments, due to low moisture content of humus near the edge. The decreased microbial activity detected at forest edges and paths implies decreased litter decomposition rates, and thus, a change in nutrient cycling. Changes in the decomposition and nutrient supply may in turn affect the diversity and function of plant communities in urban forests. Keywords: boreal forest vegetation, edge effects, phospholipid fatty acids, trampling, urban woodlands, wear

Molecular diagnosis of developmental disorders

ABSTRACT Idiopathic developmental disorders (DDs) affect ~1% of the population worldwide. This being a considerable amount, efforts are being made to elucidate the disease mechanisms. One or several genetic factors cause 30-40% of DDs, and only 10% are caused by environmental factors. The remaining 50% of DD patients go undiagnosed, mostly due to a lack of diagnostic techniques. The cause in most undiagnosed cases is though to be a genetic factor or a combination of genetic and environmental factors. Despite the surge of new technologies entering the market, their implementation into diagnostic laboratories is hampered by costs, lack of information about the expected diagnostic yield, and the wide range of selection. This study evaluates new microarray methods in diagnosing idiopathic DDs, providing information about their added diagnostic value. Study I analysed 150 patients by array comparative genomic hybridization (array CGH, 44K and 244K), with a subsequent 18% diagnostic yield. These results are supported by other studies, indicating an enourmous added diagnostic value of array CGH, compared with conventional cytogenetic analysis. Nevertheless, 80% of the patients remained undiagnosed in Study I. In an effort to diagnose more patients, in Study IV the resolution was increased from 8.9 Kb of the 244K CGH array to 0.7 Kb, by using a single-nucleotide polymorphism (SNP) array. However, no additional pathogenic changes were detected in the 35 patients assessed, and thus, for diagnostic purposes, an array platform with ca 9 Kb resolution appears adequate. The recent vast increase in reports of detected aberrations and associated phenotypes has enabled characterization of several new syndromes first based on a common aberration and thereafter by delineation of common clinical characteristics. In Study II, a familial deletion at 9q22.2q22.32 with variable penetrance was described. Despite several reports of aberrations in the adjacent area at 9q associated with Gorlin syndrome, the patients in this family had a unique phenotype and did not present with the syndrome. In Study III, a familial duplication of chromosome 6p22.2 was described. The duplication caused increased expression of an important enzyme of the γ-aminobutyric acid (GABA) degradation pathway, causing oxidative stress of the brain, and thus, very likely, the mild mental retardation of these patients. These two case studies attempted to pinpoint candidate genes and to resolve the pathogenic mechanism causing the clinical characteristics of the patients. Presenting rare genetic and clinical findings to the international science and medical community enables interpretation of similar findings in other patients. The added value of molecular karyotyping in patients with idiopathic DD is evident. As a first line of testing, arrays with a median resolution of at least 9 Kb should be considered and further characterization of detected aberrations undertaken when possible. Diagnostic whole-exome sequencing may be the best option for patients who remain undiagnosed after high-resolution array analysis.

Changing Paradigms in Biblical Criticism : 2 Kings 22:1 23:30 in the Flux of Discourses

The subject and methodology of biblical scholarship has expanded immense-ly during the last few decades. The traditional text-, literary-, source- and form-critical approaches, labeled historical-critical scholarship , have faced the challenge of social sciences. Various new literary, synchronic readings, sometimes characterized with the vague term postmodernism, have in turn challenged historicalcritical, and social-scientific approaches. Widened limits and diverging methodologies have caused a sense of crisis in biblical criticism. This metatheoretical thesis attempts to bridge the gap between philosophical discussion about the basis of biblical criticism and practical academic biblical scholarship. The study attempts to trace those epistemological changes that have produced the wealth of methods and results within biblical criticism. The account of the cult reform of King Josiah of Judah as reported in 2 Kings 22:1 23:30 serves as the case study because of its importance for critical study of the Hebrew Bible. Various scholarly approaches embracing 2 Kings 22:1 23:30 are experimentally arranged around four methodological positions: text, author, reader, and context. The heuristic model is a tentative application of Oliver Jahraus s model of four paradigms in literary theory. The study argues for six theses: 1) Our knowledge of the world is con-structed, fallible and theory-laden. 2) Methodological plurality is the neces-sary result of changes in epistemology and culture in general. 3) Oliver Jahraus s four methodological positions in regard to literature are also an applicable model within biblical criticism to comprehend the methodological plurality embracing the study of the Hebrew Bible. 4) Underlying the methodological discourse embracing biblical criticism is the epistemological ten-sion between the natural sciences and the humanities. 5) Biblical scholars should reconsider and analyze in detail concepts such as author and editor to overcome the dichotomy between the Göttingen and Cross schools. 6) To say something about the historicity of 2 Kings 22:1 23:30 one must bring together disparate elements from various disciplines and, finally, admit that though it may be possible to draw some permanent results, our conclusions often remain provisional.

La contextualisation du discours radiophonique par des moyens prosodiques. L’exemple de cinq grands philosophes français du XXe siècle

"Radiodiskurssin kontekstualisointi prosodisin keinoin. Esimerkkinä viisi suurta ranskalaista 1900-luvun filosofia" Väitöskirja käsittelee puheen kontekstualisointia prosodisin keinoin. Toisin sanottuna työssä käsitellään sitä, miten puheen prosodiset piirteet (kuten sävelkulku, intensiteetti, tauot, kesto ja rytmi) ohjaavat puheen tulkintaa vanhastaan enemmän tutkittujen sana- ja lausemerkitysten ohella. Työssä keskitytään seitsemään prosodisesti merkittyyn kuvioon, jotka koostuvat yhden tai usean parametrin silmiinpistävistä muutoksista. Ilmiöitä käsitellään sekä niiden akustisten muotojen että tyypillisten esiintymisyhteyksien ja diskursiivisten tehtävien näkökulmasta. Aineisto koostuu radio-ohjelmista, joissa puhuu viisi suurta ranskalaista 1900-luvun filosofia: Gaston Bachelard, Albert Camus, Michel Foucault, Maurice Merleau-Ponty ja Jean-Paul Sartre. Ohjelmat on lähetetty eri radiokanavilla Ranskassa vuosina 1948–1973. Väitöskirjan tulokset osoittavat, että prosodisesti merkityt kuviot ovat moniulotteisia puheen ilmiöitä, joilla on keskeinen rooli sanotun kontekstualisoinnissa: ne voivat esimerkiksi nostaa tai laskea sanotun informaatioarvoa, ilmaista puhujan voimakasta tai heikkoa sitoutumista sanomaansa, ilmaista rakenteellisen kokonaisuuden jatkumista tai päättymistä, jne. Väitöskirja sisältää myös kontrastiivisia osia, joissa ilmiöitä verrataan erääseen klassisessa pianomusiikissa esiintyvään melodiseen kuvioon sekä erääseen suomen kielen prosodiseen ilmiöön. Tulokset viittaavat siihen, että tietynlaista melodista kuviota käytetään samankaltaisena jäsentämiskeinona sekä puheessa että klassisessa musiikissa. Lisäksi tulokset antavat viitteitä siitä, että tiettyjä melodisia muotoja käytetään samankaltaisten implikaatioiden luomiseen kahdessa niinkin erilaisessa kielessä kuin suomessa ja ranskassa. Yksi väitöskirjan osa käsittelee pisteen ja pilkun prosodista merkitsemistä puheessa. Tulosten mukaan pisteellä ja pilkulla on kummallakin oma suullinen prototyyppinsä: piste merkitään tyypillisesti sävelkulun laskulla ja tauolla, ja pilkku puolestaan sävelkulun nousulla ja tauolla. Merkittävimmät tulokset koskevat kuitenkin tapauksia, joissa välimerkki tulkitaan prosodisesti epätyypillisellä tavalla: sekä pisteellä että pilkulla vaikuttaisi olevan useita eri suullisia vastaavuuksia, ja välimerkkien tehtävät voivat muotoutua hyvin erilaisiksi niiden prosodisesta tulkinnasta riippuen.

Konstruktioner och valens : Verbfraser med åt i ett jämförande perspektiv

Det har knappast undgått någon som är språkligt medveten att finlandssvenskan och sverigesvenskan skiljer sig åt till vissa delar. Olikheterna återfinns på olika språkliga nivåer. Mest kända och omskrivna är de lexikologiska skillnaderna, dvs. skillnaderna på ordplanet. Betydligt mindre uppmärksamhet har ägnats syntaktiska skillnader, dvs. skillnader i hur satser och meningar byggs upp. För att öka kunskapen om finlandssvensk syntax initierade Språkvetenskapliga nämnden vid Svenska litteratursällskapet i Finland projektet Svenskan i Finland – syntaktiska drag i ett jämförande perspektiv, som pågick åren 2004–2006. Min avhandling har kommit till inom ramen för det projektet. Prepositionerna (t.ex. av, i, på, för, till, åt osv.) är så kallade funktionsord som har till uppgift att binda samman de mer betydelsetunga orden till satser och meningar. Den finlandssvenska prepositionsanvändningen skiljer sig i viss mån från den sverigesvenska, och ”åt” är en av de prepositioner som ofta lyfts fram som exempel. Finlandssvenskarna säger t.ex. ”han gav en bok åt Lena” i stället för ”han gav en bok till Lena” eller ”han gav Lena en bok”. De säger ”berätta något åt någon” (i stället för ”för”) och de säger ”ringa åt någon” i stället för ”ringa någon”. Ett huvudsyfte med min undersökning är att ta reda på hur pass stora skillnaderna är om man ser till samtliga belägg på ”åt” i ett material och inte bara till sådana som man fäster sig vid för att man vet att de avviker i finlandssvenskan. Undersökningen är korpusbaserad. Det betyder att jag letat efter alla belägg på kombinationer av verb och prepositionen ”åt” i rätt stora textmassor som finns tillgängliga i elektronisk form. Materialet ligger i Språkbanken i Finland och omfattar huvudsakligen tidningstext och skönlitteratur. Jag har använt mig av en textmassa på sammanlagt ungefär 40 miljoner löpande ord, drygt 23 miljoner finlandssvenska och drygt 19 miljoner sverigesvenska. Det materialet gav ca 20 000 åt-belägg att studera, och det visade sig något oväntat att ”åt” inte alls är vanligare i finlandssvenskan än i sverigesvenskan när det gäller skriftspråk, åtminstone inte i professionella skribenters språk. Om man kompenserar för att den finlandssvenska och den sverigesvenska korpusen inte är helt lika i fråga om genrefördelning och ålder, kommer man fram till i stort sett samma frekvens för ”åt” i båda korpusarna. För den närmare analysen av vilka mönster åt-beläggen uppvisar har jag först och främst utnyttjat konstruktionsgrammatik men också ramsemantik och valensteori. Konstruktionsgrammatiken är ingen enhetlig teori, men tanken om grammatiska konstruktioner är gemensam. Konstruktioner representerar allt från generella syntaktiska mönster till specifika mönster för språkliga enskildheter. Uppfattningen om vad som ska inbegripas i begreppet varierar, men definitionen av ”konstruktion” som ”par (eller konstellationer) av form och betydelse” är gemensam. ”Konstruktion” avser aldrig konkreta belägg i texter eller yttranden utan alltid det abstrakta mönstret bakom dessa. Och varje yttrande är resultatet av att en stor mängd konstruktioner samverkar. I min analys har jag utgått ifrån att beläggen med ”åt” kan återföras på olika konstruktioner eller mönster utifrån vad som är gemensamt för grupper av belägg. Jag har sett på vad åt-frasen i samverkan med verbet har för funktion i beläggen. En åt-fras är syntaktiskt en prepositionsfras och består av en preposition och en rektion. Exempelvis utgör ordparet ”åt skogen” en prepositionsfras där ”skogen” är rektion. Ur mitt material har jag kunnat abstrahera fram fem övergripande mönster där referenten för rektionen har olika så kallade semantiska roller. Åt-frasen kan i kombination med verbet ange mål eller riktmärke, som i t.ex. svänga åt höger, dra åt helvete, ta sig åt hjärtat, luta åt en seger för IFK. Den kan för det andra ange mottagare (t.ex. ge varsin kaka åt hundarna, bygga en bastu åt sina svärföräldrar, skaffa biljetter åt en kompis). För det tredje kan åt-frasen avse en referent som har nytta (eller skada) av en aktion (t.ex. klippa häcken åt grannen, ställa in digitalboxen åt sin moster). Åt-frasen kan slutligen avse den eller det som är föremål antingen för en kommunikationsaktion (vinka åt sin son, skratta åt eländet) eller en attityd eller känsla (glädja sig åt framgången). Utöver dessa huvudmönster finns det ett antal smärre grupper av belägg som bildar egna mönster, men de utgör sammanlagt under 3 % i bägge korpusarna. Inom grupperna kan undermönster urskiljas. I t.ex. mottagargruppen representerar ”ge varsin kaka åt hundarna” överföringskonstruktion, ”bygga en bastu åt sina svärföräldrar” produktionskonstruktion och ”skaffa biljetter åt en kompis” ombesörjningskonstruktion. Alla typer är gemensamma för bägge materialen, men andelen belägg som representerar de olika typerna skiljer sig betydligt. I det sverigesvenska materialet står t.ex. det mönster där åt-frasen avser mål eller riktmärke för en mycket större andel av beläggen än i finlandssvenskan. Också andelen belägg där åt-frasen avser någon som har nytta (eller skada) av en aktion är mycket högre i det sverigesvenska materialet. I det finlandssvenska materialet står i gengäld mottagarbeläggen för över 50 % av beläggen medan andelen i det sverigesvenska materialet är bara 30 %. Inom gruppen utgör belägg av produktions- och ombesörjningstyp dessutom en mindre andel i det finlandssvenska materialet än i det sverigesvenska. Dessa står till sin funktion nära den typ som avser den som har nytta av aktionen. De konkreta beläggen på överföring (ge varsin kaka åt hundarna) utgör en större andel i det finlandssvenska materialet än i det sverigesvenska (ca 8 % mot 3 %), men typiskt för båda materialen är hög kollokationsgrad (”kollokation” avser par eller grupper av ord som uppträder oftare tillsammans än de statiskt sett skulle göra vid helt slumpmässig förekomst). Största delen av mottagarbeläggen utgörs av fraser av typen ”ge arbete åt någon, ge eftertryck åt något, ge liv åt något; ägna tid åt något, ägna sitt liv åt något, ägna uppmärksamhet åt något”. De här slutsatserna gäller alltså skriftspråk. I talspråk ser fördelningen annorlunda ut. Typiskt för prepositionen ”åt” är överhuvudtaget hög kollokationsgrad. Det förefaller som om språkanvändarna har tydliga, färdiga mallar för var ”åt” kan komma in. Det enda mönster som verkar helt produktivt, i den meningen att elementen är i stort sett fritt kombinerbara, är kombinationer av verb och åt-fras där åt-frasen avser den som har nytta av något. Att någon utför något för någons räkning verkar överlag kunna uttryckas med prepositionen ”åt”: t.ex. ”tvätta bilen åt pappa, ringa efter en taxi åt kunden”. Till och med belägg av typen ”hon drömde åt honom att bli ordinarie adjunkt” förekommer i någon mån. Konstruktionen är produktiv i båda språkvarieteterna men uppenbart är att konstruktion med mottagare har tolkningsföreträde i vissa fall i finlandssvenskan: ”Filip skrev ett brev åt sin syster” tolkas av sverigesvenskar som att Filip skrev brevet för systerns räkning, medan finlandssvenskar överlag uppenbarligen tolkar det som att Filip skrev till sin syster, att systern var mottagare av brevet. Ungefär 20 % av alla belägg i båda materialen representerar fall där ”åt” utgör partikel. Verb och ”åt” är närmare förbundna med varandra än när ”åt” utgör normal preposition. Exempel på partikelbelägg är ”han kom inte åt strömbrytaren, det gick åt mängder med saft, landet får dra åt svångremmen, de roffade åt sig de bästa platserna”. Också partikelmaterialet ser på ett generellt plan väldigt lika ut i båda språkvarieteterna. Den största skillnaden uppvisar den reflexiva typen ”roffa åt sig”. Medan typen är mycket homogen i det sverigesvenska materialet är variationen större i det finlandsvenska. Dels uppträder fler verb i kombinationen (han köpte åt sig ett par jeans), dels vacklar ordföljden (han nappade åt sig ett paraply ~ han nappade ett paraply åt sig). Att ”åt” används mer i vissa funktioner i finlandsvenskan brukar förklaras med påverkan från finskans allativ (ändelsen -lle: hän antoi kirjan Astalle > hon gav en bok åt Asta). Allt tyder dock på att den finlandssvenska åt-användningen delvis är en relikt. I äldre sverigesvenska källor träffar man på ”åt” i sådana kontexter som numera är typiska för finlandsvenskan. Det finlandssvenska språkområdet ligger ute i periferin i relation till det språkliga centrum som förändringar sprider sig från (för svenskans del främst Stockholmstrakten) och typiskt för perifera områden är att de uppvisar ålderdomliga drag också när inga kontaktfenomen spelar in. Allativen kan naturligtvis ha bidragit till att bevara användningen av ”åt” i finlandssvenskan. Att det är just ”åt” som används” beror antagligen på att prepositionen har flest funktioner gemensamt med allativen rent kognitivt om man jämför med de betydligt mer frekventa prepositionerna ”till” och ”för”. Uppenbart är också att åt-användningen därtill lever sitt eget liv i finlandssvenskan. I vissa varieteter av finlandssvenska kan man t.ex. höra yttranden av typ ”alla fiskarna dog åt dom”. Som språklig enskildhet har det ingen finsk förebild med allativ. Yttrandet är ett exempel på töjning av en svensk konstruktion. Modell finns dels i det mönster där åt avser den som har nytta eller skada av något, dels i relationell användning av ”åt”: han är hantlangare åt Eriksson ~ han är Erikssons hantlangare. Vid språkkontakt är det överlag konstruktioner som har förebild i det låntagande språket som lånas in från det långivande språket, medan konstruktioner som saknar förebild är betydligt mindre benägna att vinna insteg.

Trust, participation and health among ageing people : A cross-sectional cohort study of men and women born in 1926-30, 1936-40, and 1946-50 in the Lahti region, southern Finland

The purpose of the present study was to explore the associations between good self-rated health and economic and social factors in different regions among ageing people in the Päijät-Häme region in southern Finland. The data of this study were collected in 2002 as part of the research and development project Ikihyvä 2002 2012 (Good Ageing in Lahti region GOAL project). The baseline data set consisted of 2,815 participants born in 1926 30, 1936 40, and 1946 50. The response rate was 66 %. According to the previous studies, trust in other people and social participation as the main aspects of social capital are associated with self-rated health. In addition, socioeconomic position (SEP) and self-rated health are associated, but all SEP indicators do not have identical associations with health. However, there is a lack of knowledge of the health associations and regional differences with these factors, especially among ageing people. Regarding these questions, the present study gives new information. According to the results of this study, self-perceived adequacy of income was significantly associated with good self-rated health, especially in the urban areas. Similar associations were found in the rural areas, though education was also considered an important factor. Adequacy of income was an even stronger predictor of good health than the actual income. Women had better self-rated health than men only in the urban areas. The youngest respondents had quite equally better self-rated health than the others. Social participation and access to help when needed were associated with good self-rated health, especially in the urban area and the sparsely populated rural areas. The result was comparable in the rural population centres. The correlation of trust with self-rated health was significant in the urban area. High social capital was associated with good self-rated health in the urban area. The association was quite similar in the other areas, though it was statistically insignificant. High social capital consisted of co-existent high social participation and high trust. The association of traditionalism (low participation and high trust) with self-rated health was also substantial in the urban area. The associations of self-rated health with low social capital (low participation and low trust) and the miniaturisation of community (high participation and low trust) were less significant. From the forms of single participation, going to art exhibitions, theatre, movies, and concerts among women, and studying and self-development among men were positively related to self-rated health. Unexpectedly, among women, active participation in religious events and voluntary work was negatively associated with self-rated health. This may indicate a coping method with ill-health. As a whole, only minor variations in self-rated health were found between the areas. However, the significance of the factors associated with self-rated health varied according to the areas. Economic factors, especially self-perceived adequacy of income was strongly associated with good self-rated health. Also when adjusting for economic and several other background factors social factors (particularly high social capital, social participation, and access to help when needed) were associated with self-rated health. Thus, economic and social factors have a significant relation with the health of the ageing, and improving these factors may have favourable effects on health among ageing people.

Molecular genetic studies on nemaline myopathy and related disorders

Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.

Potato virus A as a heterologous protein expression tool in plants

Viruksien käyttö tuotekehityksen ja tutkimuksen vaatimien proteiinien tuottamiseen, syötävien rokotteiden kehittämiseen ja geeniterapiaan edustavat kasvavia biotekniikan sovellusalueita. Perunan A-virus (PVA) kuuluu potyviruksiin, joiden proteiinit tuotetaan aluksi yhtenä suurena molekyylinä, joka pilkotaan yksittäisiksi proteiineiksi viruksen itsensä tuottamilla entsyymeillä. Siten virusgenomiin lisätty vieras geeni käännetään proteiiniksi virusproteiinien mukana. Lopputuloksena kaikkia proteiineja tuotetaan kasvisoluissa samansuuruinen määrä. Lisäksi, viruksen proteiinikuoren koontimekanismi sallii perintöaineksen merkittävän lisäyksen ilman että viruksen tartutuskyky merkittävästi heikkenee. Koska virus monistuu ja leviää koko kasviin, jo melko pieni määrä kasveja riittää huomattavan proteiinimäärän tuottamiseen esimerkiksi säännösten mukaisessa kasvihuoneessa. Tämän työn tarkoituksena oli muuntaa PVA:n genomia siten, että virus soveltuisi yhden vieraan proteiinin tai useiden erilaisten proteiinien samanaikaiseen tuottamiseen kasveissa. Aluksi kokeiltiin viruksen replikaasia ja kuoriproteiinia koodaavien genomialueiden välistä kohtaa ja ihmisestä peräisi olevaa geeniä, joka tuotti S-COMT-entsyymiä (katekoli-O-metyylitransferaasi). Sen aktiivisuuden rajoittaminen auttaa Parkinsonintaudin hoidossa. Kasvissa tuotettua S-COMT:ia voitaisiin käyttää lääkekehityksessä estolääkkeiden testaukseen. Kahden viikon kuluttua tartutuksesta tupakan lehdissä oli entsymaattisesti aktiivista S-COMT:ia n. 1 % lehden liukoisista proteiineista. PVA:n P1-proteiinia koodaavalta alueelta oli paikannettu kohta, johon ehkä voitaisiin siirtää vieras geeni. Asia varmistettiin siirtämällä tähän kohtaan meduusan geeni, joka tuottaa UV-valossa vihreänä fluoresoivaa proteiinia (GFP). GFP-geeniä kantava PVA levisi kasvissa ja lisääntyi n. 30-50 %:iin viruksen normaalista pitoisuudesta. Koko kasvi fluoresoi vihreänä UV-valossa. Vieras geeni voidaan sijoittaa myös potyviruksen P1- ja HCpro-proteiineja koodaavien alueiden väliin. Samaan PVA-genomiin siirrettiin kolme geeniä, yksi kuhunkin kolmesta kloonauskohdasta: GFP-geeni P1:n sisälle, merivuokon lusiferaasigeeni P1/HCpro-kohtaan ja bakteerin beta-glukuronidaasigeeni (GUS) replikaasi/kuoriproteiini-kohtaan. Virusgenomin ja itse viruksen pituudet kasvoivat 38 %, mutta virus säilytti tartutuskykynsä. Se levisi kasveissa saavuttaen n. 15 % viruksen normaalista pitoisuudesta. Kaikki kolme vierasta proteiinia esiintyivät lehdissä aktiivisina.

Sweet potato chlorotic stunt virus: Studies on viral synergism and suppression of RNA silencing

The studies presented in this thesis aimed to a better understanding of the molecular biology of Sweet potato chlorotic stunt virus (SPCSV, Crinivirus, Closteroviridae) and its role in the development of synergistic viral diseases. The emphasis was on the severe sweet potato virus disease (SPVD) that results from a synergistic interaction of SPCSV and Sweet potato feathery mottle virus (SPFMV, Potyvirus, Potyviridae). SPVD is the most important disease affecting sweetpotato. It is manifested as a significant increase in symptom severity and SPFMV titres. This is accompanied by a dramatic sweetpotato yield reduction. SPCSV titres remain little affected in the diseased plants. Viral synergistic interactions have been associated with the suppression of an adaptive general defence mechanism discovered in plants and known as RNA silencing. In the studies of this thesis two novel proteins (RNase3 and p22) identified in the genome of a Ugandan SPCSV isolate were shown to be involved in suppression of RNA silencing. RNase3 displayed a dsRNA-specific endonuclease activity that enhanced the RNA-silencing suppression activity of p22. Comparative analyses of criniviral genomes revealed variability in the gene content at the 3´end of the genomic RNA1. Molecular analyses of different isolates of SPCSV indicated a marked intraspecific heterogeneity in this region where the p22 and RNase3 genes are located. Isolates of the East African strain of SPCSV from Tanzania and Peru and an isolate from Israel were missing a 767-nt fragment that included the p22 gene. However, regardless of the absence of p22, all SPCSV isolates acted synergistically with SPFMV in co-infected sweetpotato, enhanced SPFMV titres and caused SPVD. These results showed that p22 is dispensable for development of SPVD. The role of RNase3 in SPVD was then studied by generating transgenic plants expressing the RNase3 protein. These plants had increased titres of SPFMV (ca. 600-fold higher in comparison with nontransgenic plants) 2-3 weeks after graft inoculation and displayed the characteristic SPVD symptoms. RNA silencing suppression (RSS) activity of RNase3 was detected in agroinfiltrated leaves of Nicotiana bethamiana. In vitro studies showed that RNase3 was able to cleave small interferring RNAs (siRNA) to products of ~14-nt. The data thus identified RNase3 as a suppressor of RNA silencing able to cleave siRNAs. RNase3 expression alone was sufficient for breaking down resistance to SPFMV in sweetpotato and for the development of SPVD. Similar RNase III-like genes exist in animal viruses which points out a novel and possibly more general mechanism of RSS by viruses. A reproducible method of sweetpotato transformation was used to target RNA silencing against the SPCSV polymerase region (RdRp) with an intron-spliced hairpin construct. Hence, engineered resistance to SPCSV was obtained. Ten out of 20 transgenic events challenged with SPCSV alone showed significantly reduced virus titres. This was however not sufficient to prevent SPVD upon coinfection with SPFMV. Immunity to SPCSV seems to be required to control SPVD and targeting of different SPCSV regions need to be assessed in further studies. Based on the identified key role of RNase3 in SPVD the possibility to design constructs that target this gene might prove more efficient in future studies.

Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Heterotrophic Bacteria Associated with Cyanobacteria in Recreational and Drinking Water

Cyanobacterial mass occurrences, also known as water blooms, have been associated with adverse health effects of both humans and animals. They can also be a burden to drinking water treatment facilities. Risk assessments of the blooms have generally focused on the cyanobacteria themselves and their toxins. However, heterotrophic bacteria thriving among cyanobacteria may also be responsible for many of the adverse health effects, but their role as the etiological agents of these health problems is poorly known. In addition, studies on the water purification efficiency of operating water treatment plants during cyanobacterial mass occurrences in their water sources are rare. In the present study, over 600 heterotrophic bacterial strains were isolated from natural freshwater, brackish water or from treated drinking water. The sampling sites were selected as having frequent cyanobacterial occurrences in the water bodies or in the water sources of the drinking water treatment plants. In addition, samples were taken from sites where cyanobacterial water blooms were surmised to have caused human health problems. The isolated strains represented bacteria from 57 different genera of the Gamma-, Alpha- or Betaproteobacteria, Actinobacteria, Flavobacteria, Sphingobacteria, Bacilli and Deinococci classes, based on their partial 16S rRNA sequences. Several isolates had no close relatives among previously isolated bacteria or cloned 16S rRNA genes of uncultivated bacteria. The results show that water blooms are associated with a diverse community of cultivable heterotrophic bacteria. Chosen subsets of the isolated strains were analysed for features such as their virulence gene content and possible effect on cyanobacterial growth. Of the putatively pathogenic haemolytic strains isolated in the study, the majority represented the genus Aeromonas. Therefore, the Aeromonas spp. strains isolated from water samples associated with adverse health effects were screened for the virulence gene types encoding for enterotoxins (ast, alt and act/aerA/hlyA), flagellin subunits (flaA/flaB), lipase (lip/pla/lipH3/alp-1) and elastase (ahyB) by PCR. The majority (90%) of the Aeromonas strains included one or more of the six screened Aeromonas virulence gene types. The most common gene type was act, which was present in 77% of the strains. The fla, ahyB and lip genes were present in 30 37% of the strains. The prevalence of the virulence genes implies that the Aeromonas may be a factor in some of the cyanobacterial associated health problems. Of the 183 isolated bacterial strains that were studied for possible effects on cyanobacterial growth, the majority (60%) either enhanced or inhibited growth of cyanobacteria. In most cases, they enhanced the growth, which implies mutualistic interactions. The results indicate that the heterotrophic bacteria have a role in the rise and fall of the cyanobacterial water blooms. The genetic and phenotypic characteristics and the ability to degrade cyanobacterial hepatotoxins of 13 previously isolated Betaproteobacteria strains, were also studied. The strains originated from Finnish lakes with frequent cyanobacterial occurrence. Tested strains degraded microcystins -LR and -YR and nodularin. The strains could not be assigned to any described bacterial genus or species based on their genetic or phenotypic features. On the basis of their characteristics a new genus and species Paucibacter toxinivorans was proposed for them. The water purification efficiency of the drinking water treatment processes during cyanobacterial water bloom in water source was assessed at an operating surface water treatment plant. Large phytoplankton, cyanobacterial hepatotoxins, endotoxins and cultivable heterotrophic bacteria were efficiently reduced to low concentrations, often below the detection limits. In contrast, small planktonic cells, including also possible bacterial cells, regularly passed though the water treatment. The passing cells may contribute to biofilm formation within the water distribution system, and therefore lower the obtained drinking water quality. The bacterial strains of this study offer a rich source of isolated strains for examining interactions between cyanobacteria and the heterotrophic bacteria associated with them. The degraders of cyanobacterial hepatotoxins could perhaps be utilized to assist the removal of the hepatotoxins during water treatment, whereas inhibitors of cyanobacterial growth might be useful in controlling cyanobacterial water blooms. The putative pathogenicity of the strains suggests that the health risk assessment of the cyanobacterial blooms should also cover the heterotrophic bacteria.

Effects of dietary phosphorus and calcium-to-phosphorus ratio on calcium and bone metabolism in healthy 20- to 43-year-old Finnish women

Dietary habits have changed during the past decades towards an increasing consumption of processed foods, which has notably increased not only total dietary phosphorus (P) intake, but also intake of P from phosphate additives. While the intake of calcium (Ca) in many Western countries remains below recommended levels (800 mg/d), the usual daily P intake in a typical Western diet exceeds by 2- to 3-fold the dietary guidelines (600 mg/d). The effects of high P intake in healthy humans have been investigated seldom. In this thesis healthy 20- to 43-year-old women were studied. In the first controlled study (n = 14), we examined the effects of P doses, and in a cross-sectional study (n = 147) the associations of habitual P intakes with Ca and bone metabolism. In this same cross-sectional study, we also investigated whether differences exist between dietary P originating from natural P sources and phosphate additives. The second controlled study (n = 12) investigated whether by increasing the Ca intake, the effects of a high P intake could be reduced. The associations of habitual dietary calcium-to-phosphorus ratios (Ca:P ratio) with Ca and bone metabolism were determined in a cross-sectional study design (n = 147). In the controlled study, the oral intake of P doses (495, 745, 1245 and 1995 mg/d) with a low Ca intake (250 mg/d) increased serum parathyroid hormone (S-PTH) concentration in a dose-dependent manner. In addition, the highest P dose decreased serum ionized calcium (S-iCa) concentration and bone formation and increased bone resorption. In the second controlled study with a dietary P intake of 1850 mg/d, by increasing the Ca intake from 480 mg/d to 1080 mg/d and then to 1680 mg/d, the S-PTH concentration decreased, the S-iCa concentration increased and bone resorption decreased dose-dependently. However, not even the highest Ca intake could counteract the effect of high dietary P on bone formation, as indicated by unchanged bone formation activity. In the cross-sectional studies, a higher habitual dietary P intake (>1650 mg/d) was associated with lower S-iCa and higher S-PTH concentrations. The consumption of phosphate additive-containing foods was associated with a higher S-PTH concentration. Moreover, habitual low dietary Ca:P ratios (≤0.50, molar ratio) were associated with higher S-PTH concentrations and 24-h urinary Ca excretions, suggesting that low dietary Ca:P ratios may interfere with homeostasis of Ca metabolism and increase bone resorption. In summary, excessive dietary P intake in healthy Finnish women seems to be detrimental to Ca and bone metabolism, especially when dietary Ca intake is low. The results indicate that by increasing dietary Ca intake to the recommended level, the negative effects of high P intake could be diminished, but not totally prevented. These findings imply that phosphate additives may be more harmful than natural P. Thus, reduction of an excessively high dietary P intake is also beneficial for healthy individuals.

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.

Properties of Ammonium Nitrate based fertilisers

The text is divided into three parts; Properties, Application and Safety of Ammonium Nitrate (AN) based fertilisers. In Properties, the structures and phase transitions of ammonium and potassium nitrate are reviewed. The consequences of phase transitions affect the proper use of fertilisers. Therefore the products must be stabilised against the volume changes and consequent loss of bulk density and hardness, formation of dust and finally caking of fertilisers. The effect of different stabilisers is discussed. Magnesium nitrate, ammonium sulphate and potassium nitrate are presented as a good compromise. In the Application part, the solid solutions in the systems (K+,NH4+)NO3- and (NH4+,K+)(Cl-,NO3-) are presented based on studies made with DSC and XRD. As there are clear limits for solute content in the solvent lattice, a number of disproportionation transitions exist in these process phases, e.g., N3 (solid solution isomorphous to NH4NO3-III) disproportionates to phases K3 (solid solution isomorphous to KNO3-III) and K2 (solid solution isomorphous to KNO3-II). In the crystallisation experiments, the formation of K3 depends upon temperature and the ratio K/(K+NH4). The formation of phases K3, N3, and K2 was modelled as a function of temperature and the mole ratios. In introducing chlorides, two distinct maxima for K3 were found. Confirmed with commercial potash samples, the variables affecting the reaction of potassium chloride with AN are the particle size, time, temperature, moisture content and amount of organic coating. The phase diagrams obtained by crystallisation studies were compared with a number of commercial fertilisers and, with regard to phase composition, the temperature and moisture content are critical when the formation and stability of solid solutions are considered. The temperature where the AN-based fertiliser is solidified affects the amount of compounds crystallised at that point. In addition, the temperature where the final moisture is evaporated affects the amount and type of solid solution formed at this temperature. The amount of remaining moisture affects the stability of the K3 phase. The K3 phase is dissolved by the moisture and recrystallised into the quantities of K3, which is stable at the temperature where the sample is kept. The remaining moisture should not be free; it should be bound as water in the final product. The temperatures during storage also affect the quantity of K3 phase. As presented in the figures, K3 phase is not stable at temperatu¬res below 30 °C. If the temperature is about 40 °C, the K3 phase can be formed due to the remaining moisture. In the Safety part, self-sustaining decomposition (SSD), oxidising and energetic properties of fertilisers are discussed. Based on the consequence analysis of SSD, early detection of decomposition in warehouses and proper temperature control in the manufacturing process is important. SSD and oxidising properties were found in compositions where K3 exists. It is assumed that potassium nitrate forms a solid matrix in which AN can decompose. The oxidising properties can be affected by the form of the product. Granular products are inherently less oxidising. Finally energetic properties are reviewed. The composition of the fertiliser has an importance based on theoretical calculations supported by experimental studies. Materials such as carbonates and sulphates act as diluents. An excess of ammonium ions acts as a fuel although this is debatable. Based on the experimental work, the physical properties have a major importance over the composition. A high bulk density is of key importance for detonation resistance.