Generation of Flat and Curved Membranes by Inverse-BAR Domain Proteins

Biological membranes are tightly linked to the evolution of life, because they provide a way to concentrate molecules into partially closed compartments. The dynamic shaping of cellular membranes is essential for many physiological processes, including cell morphogenesis, motility, cytokinesis, endocytosis, and secretion. It is therefore essential to understand the structure of the membrane and recognize the players that directly sculpt the membrane and enable it to adopt different shapes. The actin cytoskeleton provides the force to push eukaryotic plasma membrane in order to form different protrusions or/and invaginations. It has now became evident that actin directly co-operates with many membrane sculptors, including BAR domain proteins, in these important events. However, the molecular mechanisms behind BAR domain function and the differences between the members of this large protein family remain largely unresolved. In this thesis, the structure and functions of the I-BAR domain family members IRSp53 and MIM were thoroughly analyzed. By using several methods such as electron microscopy and systematic mutagenesis, we showed that these I-BAR domain proteins bind to PI(4,5)P2-rich membranes, generate negative membrane curvature and are involved in the formation of plasma membrane protrusions in cells e.g. filopodia. Importantly, we characterized a novel member of the BAR-domain superfamily which we named Pinkbar. We revealed that Pinkbar is specifically expressed in kidney and epithelial cells, and it localizes to Rab13-positive vesicles in intestinal epithelial cells. Remarkably, we learned that the I-BAR domain of Pinkbar does not generate membrane curvature but instead stabilizes planar membranes. Based on structural, mutagenesis and biochemical work we present a model for the mechanism of the novel membrane deforming activity of Pinkbar. Collectively, this work describes the mechanism by which I-BAR domain proteins deform membranes and provides new information about the biological roles of these proteins. Intriguingly, this work also gives evidence that significant functional plasticity exists within the I-BAR domain family. I-BAR proteins can either generate negative membrane curvature or stabilize planar membrane sheets, depending on the specific structural properties of their I-BAR domains. The results presented in this thesis expand our knowledge on membrane sculpting mechanisms and shows for the first time how flat membranes can be generated in cells.

The effect of structure on the dilute solution properties of branched polysaccharides studied with SEC and AsFlFFF

Cereal arabinoxylans, guar galactomannans, and dextrans produced by lactic acid bacteria(LAB) are a structurally diverse group of branched polysaccharides with nutritional and industrial functions. In this thesis, the effect of the chemical structure on the dilute solution properties of these polysaccharides was investigated using size-exclusion chromatography(SEC) and asymmetric flow field-flow fractionation (AsFlFFF) with multiple-detection. The chemical structures of arabinoxylans were determined, whereas galactomannan and dextran structures were studied in previous investigations. Characterization of arabinoxylans revealed differences in the chemical structures of cereal arabinoxylans. Although arabinoxylans from wheat, rye, and barley fiber contained similar amounts of arabinose side units, the substitution pattern of arabinoxylans from different cereals varied. Arabinoxylans from barley husks and commercial low-viscosity wheat arabinoxylan contained a lower number of arabinose side units. Structurally different dextrans were obtained from different LAB. The structural effects on the solution properties could be studied in detail by modifying pure wheat and rye arabinoxylans and guar galactomannan with specific enzymes. The solution characterization of arabinoxylans, enzymatically modified galactomannans, and dextrans revealed the presence of aggregates in aqueous polysaccharide solutions. In the case of arabinoxylans and dextrans, the comparison of molar mass data from aqueous and organic SEC analyses was essential in confirming aggregation, which could not be observed only from the peak or molar mass distribution shapes obtained with aqueous SEC. The AsFlFFF analyses gave further evidence of aggregation. Comparison of molar mass and intrinsic viscosity data of unmodified and partially debranched guar galactomannan, on the other hand, revealed the aggregation of native galactomannan. The arabinoxylan and galactomannan samples with low or enzymatically extensively decreased side unit content behaved similarly in aqueous solution: lower molar mass samples stayed in solution but formed large aggregates, whereas the water solubility of the higher-molar-mass samples decreased significantly. Due to the restricted solubility of galactomannans in organic solvents, only aqueous galactomannan solutions were studied. The SEC and AsFlFFF results differed for the wheat arabinoxylan and dextran samples. Column matrix effects and possible differences in the separation parameters are discussed, and a problem related to the non-established relationship between the separation parameters of the two separation techniques is highlighted. This thesis shows that complementary approaches in the solution characterization of chemically heterogeneous polysaccharides are needed to comprehensively investigate macromolecular behavior in solution. These results may also be valuable when characterizing other branched polysaccharides.

Naudanlihan jalostusketjun hiilijalanjälki Suomessa

Kirjallisuustutkimuksen tavoitteena oli perehtyä kasvihuoneilmiön taustoihin ja kartoittaa aiempia tutkimuksia naudan- ja muiden lihatuotteiden kasvihuonekaasupäästöistä. Lisäksi kirjallisuustutkimuksessa perehdyttiin aiemmissa tutkimuksissa elintarvikkeiden hiilijalanjäljen laskemisessa sovellettuun elinkaarianalyysiin ISO 14040-standardin mukaisesti. Kokeellisen osion tavoitteena oli määrittää naudanlihan hiilijalanjälki Suomessa maatilan portilta kuluttajan ruokapöytään. Tavoitteena oli myös ymmärtää jalostusketjun päästöjen merkitys verrattuna koko naudanlihan tuotantoketjuun ja määrittää jalostusketjun vaiheiden merkitys ketjussa. Työn toiminnallisena yksikkönä toimi kilo naudanlihaa. Työ toteutettiin perehtymällä yksityiskohtaisesti yhteen naudanlihan jalostusketjuun Suomessa. Päästöt laskettiin todellisten yhteistyöyritykseltä saatujen prosessitietojen perusteella. Tiedot kerättiin tiedonkeruulomakkeella vierailemalla yhteistyöyrityksen kahdessa tuotantolaitoksessa ja täydentämällä tietoja haastatteluilla. Naudanlihan jalostusketjun päästöt olivat 1240 g CO2-ekv/lihakilo. Eniten päästöjä tuottivat jalostusvaihe (310 g CO2-ekv/lihakilo), teurastus (280 g CO2-ekv/lihakilo) ja lihatuotteiden kuljetus kuluttajalle (210 g CO2-ekv/lihakilo). Koko naudanlihan tuotantoketjusta jalostusketjun päästöt muodostivat alle 4 %, sillä syntymästä maatilan portille syntyviksi päästöiksi laskettiin kirjallisuuden perusteella yli 30 000 g CO2-ekv/lihakilo. Jatkossa naudanlihan hiilijalanjälkeä voitaisiin pääasiassa pienentää kehittämällä prosessia maatilan portille asti. Tämän työn tulokset olivat hyvin samansuuruiset verrattuna aiempaan tutkimukseen broilerin jalostusketjun päästöistä Suomessa (Katajajuuri ym. 2008). Tämä vastasi ennakko-odotuksia, sillä jalostusketjujen vaiheissa ei ollut merkittäviä eroja. Aiempia tutkimuksia naudanlihan jalostusketjun päästöistä ei ollut saatavilla.

Sedimentary zooplankton remains as indicators of lake ecological quality and trophic structure

To protect and restore lake ecosystems under threats posed by the increasing human population, information on their ecological quality is needed. Lake sediments provide a data rich archive that allows identification of various biological components present prior to anthropogenic alterations as well as a constant record of changes. By providing a longer dimension of time than any ongoing monitoring programme, palaeolimnological methods can help in understanding natural variability and long-term ecological changes in lakes. As zooplankton have a central role in the lake food web, their remains can potentially provide versatile information on past trophic structure. However, various taphonomic processes operating in the lakes still raise questions concerning how subfossil assemblages reflect living communities. This thesis work aimed at improving the use of sedimentary zooplankton remains in the reconstruction of past zooplankton communities and the trophic structure in lakes. To quantify interspecific differences in the accumulation of remains, the subfossils of nine pelagic zooplankton taxa in annually laminated sediments were compared with monitoring results for live zooplankton in Lake Vesijärvi. This lake has a known history of eutrophication and recovery, which resulted from reduced external loading and effective fishing of plankti-benthivorous fish. The response of zooplankton assemblages to these known changes was resolved using annually laminated sediments. The generality of the responses observed in Lake Vesijärvi were further tested with a set of 31 lakes in Southern Finland, relating subfossils in surface sediments to contemporary water quality and fish density, as well as to lake morphometry. The results demonstrated differential preservation and retention of cladoceran species in the sediment. Daphnia, Diaphanosoma and Ceriodaphnia were clearly underrepresented in the sediment samples in comparison to well-preserved Bosmina species, Chydorus, Limnosida and Leptodora. For well-preserved species, the annual net accumulation rate was similar to or above the expected values, reflecting effective sediment focusing and accumulation in the deepest part of the lake. The decreased fish density and improved water quality led to subtle changes in zooplankton community composition. The abundance of Diaphanosoma and Limnosida increased after the reduction in fish density, while Ceriodaphnia and rotifers decreased. The most sensitive indicator of fish density was the mean size of Daphnia ephippia and Bosmina (E.) crassicornis ephippia and carapaces. The concentration of plant-associated species increased, reflecting expanding littoral vegetation along with increasing transparency. Several of the patterns observed in Lake Vesijärvi could also be found within the set of 31 lakes. According to this thesis work, the most useful cladoceran-based indices for nutrient status and planktivorous fish density in Finnish lakes were the relative abundances of certain pelagic taxa, and the mean size of Bosmina spp. carapaces, especially those of Bosmina (E.) cf. coregoni. The abundance of plant-associated species reflected the potential area for aquatic plants. Lake morphometry and sediment organic content, however, explained a relatively high proportion of the variance in the species data, and more studies are needed to quantify lake-specific differences in the accumulation and preservation of remains. Commonly occurring multicollinearity between environmental variables obstructs the cladoceran-based reconstruction of single environmental variables. As taphonomic factors and several direct and indirect structuring forces in lake ecosystems simultaneously affect zooplankton, the subfossil assemblages should be studied in a holistic way before making final conclusions about the trophic structure and the change in lake ecological quality.

Kotimaisen tuorekalan tuoreuden arviointi laatuindeksimenetelmällä QIM®

Kalateollisuus ja kalakauppa tarvitsisivat menetelmän, jolla kalan säilyvyyttä voitaisiin arvioida reaaliaikaisesti ja luotettavasti. Laatuindeksimenetelmä QIM® (engl. Quality Index Method) on käytössä jo useassa Euroopan maassa useille eri kalalajeille. QIM® pyrkii lajikohtaisesti ennustamaan aistinvaraisten ominaisuuksien muutoksien perusteella jäljellä olevaa säilyvyysaikaa. Työn tavoitteena oli luoda QIM® meressä kasvatetulle siialle. Tämä on ensimmäinen suomalaiselle kalalle tehtävä QIM®, ja tavoitteena on saada meressä kasvatetun siian QIM® myös viralliseen QIM®-käsikirjaan. Tutkimus tehtiin Elintarviketurvallisuusvirasto Eviralle. Tutkittavat kalat Evira osti Kalatukku E. Eriksson Oy:ltä. QIM®-tuloksen tueksi tutkittiin yhden erän pH ja indikoitiin pilaantuminen myös mikrobiologisesti. Luotiin myös kasvatetun siian profiili (arvioijia 13). Itse tutkimusosassa kaksi profiiliraatia (n = 9) ja QIM®-raati (n = 5) arvioivat raa’an ja kypsän kalan. Tulos varmistettiin myös aistinvaraisella kalan laadunarviointimenetelmällä (Evira 8001). QIM®-raati loi QIM®-luonnoksen ja luonnoksen toimivuutta testattiin. Kiinteänä osana työn toteutusta oli myös eri vaiheiden muutosten valokuvaus. Tämän tutkimuksen mukaan luotu QIM®-luonnos on toimiva pohja validoitaessaa QIM®-menetelmää siialle. Voidaan myös todeta, että QIM® soveltuu meressä kasvatetulle siialle. Kypsän kalan aistinvaraisella laadunarviointimenetelmällä (Evira 8001) analysoitiin säilytysajankohta, jolloin kypsästä kalasta voitiin todeta kalan kauppakelvottomuus – tätä pidettiin ajanhetkenä, jolloin raa’an kalan tutkiminen voitiin lopettaa. Tutkimuksessa käytetty mikrobiologinen menetelmä ”Mikrobien lukumäärän määrittäminen” (Evira 3420/1) korreloi QIM®-tuloksen kanssa; kalanäyte oli tässä tutkimuksessa niin mikrobiologisesti arvioituna kuin laatuindeksinkin mukaan käyttökelvotonta viidentenätoista päivänä. Tutkittujen kalojen pH-arvoja ei voitu verrata laatuindeksiin, sillä tässä tutkimuksessa mitattujen pH-arvojen tuloksista ei voitu päätellä pilaantumisen etenemistä. Kun QIM® meressä kasvatetulle siialle on validoitu (Evira), valmista meressä kasvatetun siian QI-menetelmää voidaan hyödyntää jatkossa Suomen kalateollisuudessa ja -markkinoilla. Olisi hyvä, jos QIM® luotaisiin myös muille Suomen yleisimmille kauppakaloille, jotta pakkauksiin merkityt viimeiset käyttöpäivät perustuisivat yhteen yhteiseen menetelmään ja näin viimeisellä käyttöpäivällä olisi tieteellinen pohja.

Molecular profiling of indoor microbial communities in moisture damaged and non-damaged buildings

Epidemiological studies have shown an elevation in the incidence of asthma, allergic symptoms and respiratory infections among people living or working in buildings with moisture and mould problems. Microbial growth is suspected to have a key role, since the severity of microbial contamination and symptoms show a positive correlation, while the removal of contaminated materials relieves the symptoms. However, the cause-and-effect relationship has not been well established and knowledge of the causative agents is incomplete. The present consensus of indoor microbes relies on culture-based methods. Microbial cultivation and identification is known to provide qualitatively and quantitatively biased results, which is suspected to be one of the reasons behind the often inconsistent findings between objectively measured microbiological attributes and health. In the present study the indoor microbial communities were assessed using culture-independent, DNA based methods. Fungal and bacterial diversity was determined by amplifying and sequencing the nucITS- and16S-gene regions, correspondingly. In addition, the cell equivalent numbers of 69 mould species or groups were determined by quantitative PCR (qPCR). The results from molecular analyses were compared with results obtained using traditional plate cultivation for fungi. Using DNA-based tools, the indoor microbial diversity was found to be consistently higher and taxonomically wider than viable diversity. The dominant sequence types of fungi, and also of bacteria were mainly affiliated with well-known microbial species. However, in each building they were accompanied by various rare, uncultivable and unknown species. In both moisture-damaged and undamaged buildings the dominant fungal sequence phylotypes were affiliated with the classes Dothideomycetes (mould-like filamentous ascomycetes); Agaricomycetes (mushroom- and polypore-like filamentous basidiomycetes); Urediniomycetes (rust-like basidiomycetes); Tremellomycetes and the family Malasseziales (both yeast-like basidiomycetes). The most probable source for the majority of fungal types was the outdoor environment. In contrast, the dominant bacterial phylotypes in both damaged and undamaged buildings were affiliated with human-associated members within the phyla Actinobacteria and Firmicutes. Indications of elevated fungal diversity within potentially moisture-damage-associated fungal groups were recorded in two of the damaged buildings, while one of the buildings was characterized by an abundance of members of the Penicillium chrysogenum and P. commune species complexes. However, due to the small sample number and strong normal variation firm conclusions concerning the effect of moisture damage on the species diversity could not be made. The fungal communities in dust samples showed seasonal variation, which reflected the seasonal fluctuation of outdoor fungi. Seasonal variation of bacterial communities was less clear but to some extent attributable to the outdoor sources as well. The comparison of methods showed that clone library sequencing was a feasible method for describing the total microbial diversity, indicated a moderate quantitative correlation between sequencing and qPCR results and confirmed that culture based methods give both a qualitative and quantitative underestimate of microbial diversity in the indoor environment. However, certain important indoor fungi such as Penicillium spp. were clearly underrepresented in the sequence material, probably due to their physiological and genetic properties. Species specific qPCR was a more efficient and sensitive method for detecting and quantitating individual species than sequencing, but in order to exploit the full advantage of the method in building investigations more information is needed about the microbial species growing on damaged materials. In the present study, a new method was also developed for enhanced screening of the marker gene clone libraries. The suitability of the screening method to different kinds of microbial environments including biowaste compost material and indoor settled dusts was evaluated. The usability was found to be restricted to environments that support the growth and subsequent dominance of a small number microbial species, such as compost material.

Tandemmassaspektrometrinen menetelmä neutraalien isomeeristen oligosakkaridien rakenteen määrittämisessä

Tutkimuksen tavoitteena oli ottaa käyttöön tandemmassaspektrometrinen (MS/MS) menetelmä, jolla voidaan analysoida polysakkarideista purkautuneiden oligosakkaridien rakenteita. Tavoitteena oli, että menetelmällä voidaan määrittää glykosidisten sidosten eri asemat monosakkaridirakenteiltaan samanlaisista neutraaleista lineaarisista oligosakkarideista. Kirjallisuustutkimuksessa tarkasteltiin oligosakkaridien rakenteiden määrittämiseen käytettyjä MS/MS-menetelmiä ja oligosakkaridien pilkkoutumisreaktioita MS/MS-analyysissa. Kirjallisuuden perusteella MS/MS-analyysissa oligosakkaridien pilkkoutuminen voi tapahtua joko glykosidisen sidoksen katkeamisella tai monosakkaridirenkaan halkeamisella. Monosakkaridirenkaan pilkkoutumisesta muodostuvia tuoteioneja voidaan käyttää glykosidisen sidoksen aseman määrittämiseen. Kokeellisessa tutkimuksessa selvitettiin aluksi monosakkaridirakenteiltaan isomeerisilla disakkaridimalliaineilla glykosidisen sidoksen sijainnin vaikutus disakkaridin pilkkoutumiseen MS/MS-analyysissa. Tämän jälkeen pyrittiin löytämään tunnetuista tri- ja tetrasakkaridimalliaineista näitä eri sidoksille tyypillisiä tuoteionien jakaumia. Tunnettujen tri- ja tetrasakkaridien pilkkoutuminen yhdenmukaisesti disakkaridien pilkkoutumisen kanssa antaisi mahdollisuuden pitkäketjuisempien oligosakkaridien glykosidisten sidosten tunnistamiseen sovelletulla MS/MS-menetelmällä. MS/MS-analyysit tehtiin ioniloukkumassadetektorilaitteistolla käyttäen sähkösumutusionisaatiota (ESI). Oligosakkaridit määritettiin positiivisella ionisaatiolla litium- ja natriumaddukti-ioneina ja negatiivisella ionisaatiolla kloridiaddukti-ioneina. Vertaamalla tri- ja tetrasakkarideista MS/MS-analyyseissa muodostuneita tuoteioneja disakkarideista muodostuneisiin tuoteioneihin, voitiin sekä positiivisella että negatiivisella ionisaatiolla määrittää oligosakkaridin pelkistävän pään sidoksen asema. Negatiivisella ionisaatiolla tri- ja tetrasakkarideista muodostuneista tuoteioneista voitiin määrittää myös muiden kuin pelkistävän pään sidosten asemia. Positiivisella ionisaatiolla muiden sidosten määrittäminen ei ollut mahdollista, koska rengasfragmentti-ioneja muodostui pääosin oligosakkaridin pelkistävästä päästä. Glykosidisen sidoksen katkeamisesta muodostuneet tuoteionit analysoitiin edelleen MS3-analyysilla. MS3-analyysissa muodostuneista tuoteioneista ei voitu tulkita sidosten asemia, koska lähtöionit koostuivat sekä terminaalisen että pelkistävän pään isomeerisista ioneista.

Inhibitory effects of wood extracts on the spoilage flora of marinated poultry meat

Polyphenolic compounds occurring naturally in knotwood of plants are known to have antimicrobial effects. The knots (i.e. the branch bases inside tree stems) and outer branches in pine trees contain a remarkably high concentration of phenolic stilbenes, while lignans are the major phenolic constituents of spruce knots. Large amount of these phenolic compounds can be extracted from wood knots at pulp and paper mills where their presence is undesirable. In Finland, marinating of broiler meat is done not only to increase or add value to the meat, but also to enhance the safety and shelf-life. These products are usually packed under a modified atmosphere for further protection against spoilage microorganisms. However, studies have revealed that addition of marinades to poultry products do not have an inhibitory effect on either some psychrotrophic anaerobic bacteria, such as Brochothrix thermosphacta or lactic acid bacteria associated with spoilage. Also, the activity of pathogenic Campylobacter jejuni is not affected by marinating. The objective of this study was to investigate the inhibitory and lethal activities of extracts from spruce (Picea spp.) and pine (Pinus spp.) knotwood and outer branches that are dissolved in ethanol against the spoilage microorganisms in modified atmosphere packaged marinated broiler products. Modified atmosphere packaged broiler products were separately inoculated with ‘normal’ marinades, marinades with 70% ethanol, marinades with a mixture of spruce and pine extracts dissolved in 70% ethanol or mixture of spruce and pine extracts in powder form. The bacterial colony forming units per gram obtained from each of the samples were analysed on de Man Rogosa and Sharpe agar at days 1, 6, 12 and 15. The results showed that there were significant differences in bacterial colony forming units per gram (P <0.05) between packages with ‘normal’ marinades and packages with extracts added to their marinades on the 12th and 15th day. It can be concluded that the addition of extracts from spruce and pine knotwood to marinades significantly retarded growth of spoilage microorganisms during the 15 day test period. However further research is warranted to characterise and establish the safety and suitability of the compound(s) in spruce and pine knotwood extracts that are responsible for inhibitory or lethal activity against the microbes that may be present in marinated poultry meat.

The roles of nitrification and nitrate reduction pathways in nitrogen cycling of Baltic Sea

The Baltic Sea is one of the largest brackish water bodies in the world. Primary production in the Baltic Sea is limited by nitrogen (N) availability with the exception of river outlets and the northernmost phosphorus limited basin. The excess human induced N load from the drainage basin has caused severe eutrophication of the sea. The excess N loads can be mitigated by microbe mediated natural N removal processes that are found in the oxic-anoxic interfaces in sediments and water column redoxclines. Such interfaces allow the close coupling between the oxic nitrification process, and anoxic denitrification and anaerobic ammonium oxidation (anammox) processes that lead to the formation of molecular nitrogen gas. These processes are governed by various environmental parameters. The effects of these parameters on N processes were investigated in the northern Baltic Sea sediments. During summer months when the sediment organic content is at its highest, nitrification and denitrification reach their maximum rates. However, nitrification had no excess potential, which was probably because of high competition for molecular oxygen (O2) between heterotrophic and nitrification microbes. Subsequently, the limited nitrate (NO3-) availability inhibited denitrification. In fall, winter and spring, nitrification was limited by ammonium availability and denitrification limited by the availability of organic carbon and occasionally by NO3-. Anaerobic ammonium oxidation (anammox) was not an important N removal process in the northern Baltic Sea. Modeling studies suggest that when hypoxia expands in the Baltic Sea, N removal intensifies. However, the results of this study suggest the opposite because bottom water hypoxia (O2< 2 ml l-1) decreased the denitrification rates in sediments. Moreover, N was recycled by the dissimilatory nitrate reduction to ammonium (DNRA) process instead of being removed from the water ecosystem. High N removal potentials were found in the anoxic water column in the deep basins of the Baltic Proper. However, the N removal in the water column appeared to be limited by low substrate availability, because the water at the depths at which the substrate producing nitrification process occurred, rarely mix with the water at the depths at which N removal processes were found. Overall, the natural N removal capacity of the northern Baltic Sea decreased compared to values measured in mid 1990s and early 2000. The reason for this appears to be increasing hypoxia.

Behavioural responses to anthropogenic disturbances

When a habitat undergoes change, the first response of an individual is often behavioural adjustment. This immediate response can determine whether the population will survive or not, as behavioural flexibility gives time for genetic changes to arise later on. Habitat changes that alter reproductive behaviours can have long-lasting effects on populations. If the selective regime has changed under the new conditions, mate choice cues may no longer reliably reflect an individual s quality. Thus, animals have to be able to adjust their reproductive behaviours to the local conditions. The aim of my thesis was to discuss if and how animals are able to respond to rapid anthropogenic environmental change, and to study the mechanisms of the responses and the evolutionary consequences. The main focus was on the effects of human-induced eutrophication on the reproductive behaviour of fishes. Eutrophication is the result of increased nutrient input and can cause dense underwater vegetation and algal blooms. I used fishes from two very different ecosystems as model species, the Baltic Sea threespine stickleback (Gasterosteus aculeatus) and the desert goby (Chlamydogobius eremius), an endemic species of the Lake Eyre region in Central Australia. I investigated the effects of increased habitat complexity on courtship behaviour and the possibility of local differentiation in courtship and nest building behaviour depending on the level eutrophication in the habitat of origin. Furthermore, I observed the effect of turbidity on stickleback nest building behaviour. The results show that threespine stickleback males, which were born in areas that have been eutrophied for decades, court females at a higher intensity than males from clear water areas. Similarly, male desert gobies increased their courtship effort in dense vegetation. Intense courtship could be an adjustment to reduced visibility and lowered predation risk in the densely vegetated sites. However, there were no clear differences in nest building between males from clear and eutrophied areas under standardized conditions. This was expected as Baltic Sea sticklebacks prefer to nest under vegetation cover and are fairly rigid in adjusting their nest characteristics. Nest building was affected by increased turbidity: males built smaller nests with a larger nest entrance in turbid water. The large variation in the magnitude of phytoplankton blooms may require a rapid adjustment of the optimal nest structure to the current conditions. This thesis highlights the complex interactions that are set- off by human-induced changes in habitats and are followed by the immediate behavioural responses. It also encourages more research to tease apart the phenotypic and genetic components of the observed behavioural differences.

The potential of microbial ecological indicators to guide ecosophisticated management of hydrocarbon-contaminated soils

Soil is an unrenewable natural resource under increasing anthropogenic pressure. One of the main threats to soils, compromising their ability to provide us with the goods and ecosystem services we expect, is pollution. Oil hydrocarbons are the most common soil contaminants, and they disturb not just the biota but also the physicochemical properties of soils. Indigenous soil micro-organisms respond rapidly to changes in the soil ecosystem, and are chronically in direct contact with the hydrophobic pollutants on the soil surfaces. Soil microbial variables could thus serve as an intrinsically relevant indicator of soil quality, to be used in the ecological risk assessment of contaminated and remediated soils. Two contrasting studies were designed to investigate soil microbial ecological responses to hydrocarbons, together with parallel changes in soil physicochemical and ecotoxicological properties. The aim was to identify quantitative or qualitative microbiological variables that would be practicable and broadly applicable for the assessment of the quality and restoration of oil-polluted soil. Soil bacteria commonly react on hydrocarbons as a beneficial substrate, which lead to a positive response in the classical microbiological soil quality indicators; negative impacts were accurately reflected only after severe contamination. Hydrocarbon contaminants become less bioavailable due to weathering processes, and their potentially toxic effects decrease faster than the total concentration. Indigenous hydrocarbon degrader bacteria, naturally present in any terrestrial environment, use specific mechanisms to improve access to the hydrocarbon molecules adsorbed on soil surfaces. Thus when contaminants are unavailable even to the specialised degraders, they should pose no hazard to other biota either. Change in the ratio of hydrocarbon degrader numbers to total microbes was detected to predictably indicate pollutant effects and bioavailability. Also bacterial diversity, a qualitative community characteristic, decreased as a response to hydrocarbons. Stabilisation of community evenness, and community structure that reflected clean reference soil, indicated community recovery. If long-term temporal monitoring is difficult and appropriate clean reference soil unavailable, such comparison could possibly be based on DNA-based community analysis, reflecting past+present, and RNA-based community analysis, showing exclusively present conditions. Microbial ecological indicators cannot replace chemical oil analyses, but they are theoretically relevant and operationally practicable additional tools for ecological risk assessment. As such, they can guide ecologically informed and sustainable ecosophisticated management of oil-contaminated lands.

Suolistopatogeenisten Escherichia coli -bakteerien esiintyvyys ripulipotilailla ja raa’oissa lihatuotteissa Burkina Fasossa sekä enterohemorraagisten E. coli -kantojen karakterisointi

Suolistopatogeeniset Escherichia coli -bakteerit eli ripulikolit aiheuttavat ihmisellä suolistoinfektioita. Kuten normaalimikrobiston E. coli -bakteerit, ne esiintyvät ihmisen lisäksi muiden nisäkkäiden, etenkin märehtijöiden, ja lintujen suolistossa. Lisäksi ne voivat esiintyä maaperässä ja vesistöissä. Ihminen voi saada tartunnan eläinperäisten elintarvikkeiden välityksellä tai juomalla eläinten tai ihmisen ulosteilla saastunutta vettä. Ripulikolit voidaan jakaa ainakin viiteen ryhmään perustuen niiden erilaisiin virulenssiominaisuuksiin: enteropatogeeninen E. coli (EPEC), enterotoksigeeninen E. coli (ETEC), enterohemorraaginen E. coli (EHEC), enteroinvasiivinen E. coli (EIEC) ja enteroaggregatiivinen E. coli (EAEC). EPEC aiheuttaa etenkin kehitysmaissa pikkulapsille ripulia. ETEC aiheuttaa turistiripulia ja vastasyntyneiden ripulia kehitysmaissa. EHEC aiheuttaa ripulia tai veriripulia, joka voi varsinkin pienillä lapsilla johtaa hemolyyttis-ureemiseen oireyhtymään (HUS) ja munuaisten vaurioitumiseen. EIEC aiheuttaa Shigellan kaltaista ripulia, joka voi olla veristä. EAEC on yhdistetty lähinnä pitkittyneisiin ripuleihin. Tutkimuksessa selvitettiin suolistopatogeenisten E. coli -bakteerien esiintyvyyttä Burkina Fasossa, josta ei ole saatavilla aikaisempaa tietoa ripulikolien esiintymisestä ihmisissä ja elintarvikkeissa. Ulostenäytteitä otettiin ripulia sairastavilta alle viisivuotiailta lapsilta maaseudulta kahdesta kylästä, Boromosta ja Gourcysta, ja maan pääkaupungista Ouagadougousta (110 näytettä). Lihanäytteitä (kanaa, nautaa, lammasta ja naudan suolta, jota käytetään ihmisravinnoksi) otettiin Ouagadougoun toreilla myytävistä kypsentämättömistä lihoista (120 näytettä). Näytteistä saadut bakteerisekaviljelmät tutkittiin monialukkeisella PCR-menetelmällä, joka tunnistaa viiden ripulikoliryhmän virulenssigeenejä. Lisäksi lihanäytteistä eristettiin 20 EHEC-kantaa shigatoksiinin stx-geenin havaitsemiseen perustuvalla pesäkehybridisaatiolla ja PCR-seulonnalla, ja karakterisoitiin mahdollisten virulenssiominaisuuksien selvittämiseksi. Tutkimus osoitti, että ripulikolien aiheuttamat suolistoinfektiot ovat yleisiä ripulia sairastavilla pikkulapsilla Burkina Fasossa. Ulostenäytteistä 59 % oli positiivisia. Useimmiten lapsilla esiintyi EAEC- (32 %) ETEC- (31 %) ja EPEC-patoryhmiä (20 %). EIEC- (2 %) ja EHEC-patoryhmiä (1 %) esiintyi vähän. Myös useamman patoryhmän sekainfektiot olivat yleisiä (24 %). Eri paikkakuntien välillä oli tilastollisesti merkitseviä eroja ripulikolien esiintymisessä. Gourcyssa ripulikoleja esiintyi useammin kuin Ouagadougoussa ja Boromossa. Tutkimuksessa kävi ilmi, että Ouagadougoun toreilla myytävissä lihoissa on paljon ripulikoleja. Lihanäytteistä 43 % oli positiivisia. Yleisimmin lihoissa esiintyi EHEC (28 %), EPEC (20 %), ETEC (8 %) ja EAEC (5 %). EIEC-ryhmää ei havaittu lihoissa. Myös useamman patoryhmän sekakontaminaatioita löytyi (17 %) lihoista. Ripulikolien esiintyvyydessä eri lihojen välillä ei ollut tilastollisesti merkitseviä eroja, kun tarkasteltiin kaikkia patoryhmiä yhdessä. Eri patoryhmien esiintyvyyttä tarkasteltaessa EHEC-patoryhmää ei esiintynyt ollenkaan kanassa ja ero oli tilastollisesti merkitsevä muihin lihoihin verrattuna. Lihoista eristetyt 20 EHEC-kantaa kuuluivat 14 eri serotyyppiin, joista osa on aikaisemmin eristetty suolistoinfektioihin ja HUSoireyhtymään sairastuneilta ihmisiltä. Kaikki kannat olivat stx1-positiivisia ja puolella oli lisäksi stx2-geeni, jota pidetään shigatoksiinin virulentimpana muotona. Kahdelta EHEC-kannalta löytyi myös ETECpatoryhmän lämpöstabiilin enterotoksiini Ia:n geeni eli kannat olivat kahden patoryhmän välimuotoa ja osoitus geenien siirtymisestä eri patoryhmien välillä. Vaikka nuorimmat näytteen antaneet lapsipotilaat tuskin söivät lihaa, sen voidaan ajatella silti olevan edustava näyte lasten elinympäristöstä, sillä lasten ruoka valmistetaan usein samoissa oloissa, joissa raakaa lihaa käsitellään. Saastunut liha voi siten olla pikkulasten ripulikoli-infektioiden aiheuttaja.

Moonlighting Proteins of Lactobacillus crispatus : Extracellular Localization, Cell Wall Anchoring and Interactions with the Host

Moonlighting functions have been described for several proteins previously thought to localize exclusively in the cytoplasm of bacterial or eukaryotic cells. Moonlighting proteins usually perform conserved functions, e. g. in glycolysis or as chaperonins, and their traditional and moonlighting function(s) usually localize to different cell compartments. The most characterized moonlighting proteins in Grampositive bacteria are the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which function in bacteria-host interactions, e. g. as adhesins or plasminogen receptors. Research on bacterial moonlighting proteins has focused on Gram-positive bacterial pathogens, where many of their functions have been associated with bacterial virulence. In this thesis work I show that also species of the genus Lactobacillus have moonlighting proteins that carry out functions earlier associated with bacterial virulence only. I identified enolase, GAPDH, glutamine synthetase (GS), and glucose-6-phosphate isomerase (GPI) as moonlighting proteins of Lactobacillus crispatus strain ST1 and demonstrated that they are associated with cell surface and easily released from the cell surface into incubation buffer. I also showed that these lactobacillar proteins moonlight either as adhesins with affinity for basement membrane and extracellular matrix proteins or as plasminogen receptors. The mechanisms of surface translocation and anchoring of bacterial moonlighting proteins have remained enigmatic. In this work, the surface localization of enolase, GAPDH, GS and GPI was shown to depend on environmental factors. The members of the genus Lactobacillus are fermentative organisms that lower the ambient pH by producing lactic acid. At acidic pH enolase, GAPDH, GS and GPI were associated with the cell surface, whereas at neutral pH they were released into the buffer. The release did not involve de novo protein synthesis. I showed that purified recombinant His6-enolase, His6-GAPDH, His6-GS and His6-GPI reassociate with cell wall and bind in vitro to lipoteichoic acids at acidic pH. The in-vitro binding of these proteins localizes to cell division septa and cell poles. I also show that the release of moonlighting proteins is enhanced in the presence of cathelicidin LL- 37, which is an antimicrobial peptide and a central part of the innate immunity defence. I found that the LL-37-induced detachment of moonlighting proteins from cell surface is associated with cell wall permeabilization by LL-37. The results in this thesis work are compatible with the hypothesis that the moonlighting proteins of L. crispatus associate to the cell wall via electrostatic or ionic interactions and that they are released into surroundings in stress conditions. Their surface translocation is, at least in part, a result from their release from dead or permeabilized cells and subsequent reassociation onto the cell wall. The results of this thesis show that lactobacillar cells rapidly change their surface architecture in response to environmental factors and that these changes influence bacterial interactions with the host.

The function and regulation of the U11-48K protein in U12-dependent splicing

The removal of noncoding sequences, or introns, from the eukaryotic messenger RNA precursors is catalyzed by a ribonucleoprotein complex known as the spliceosome. In most eukaryotes, two distinct classes of introns exist, each removed by a specific type of spliceosome. The major, U2-type introns account for over 99 % of all introns, and are almost ubiquitous. The minor, U12-type introns are found in most but not all eukaryotes, and reside in conserved locations in a specific set of genes. Due to their slow excision rates, the U12-type introns are expected to be involved in the regulation of the genes containing them by inhibiting the maturation of the messenger RNAs. However, little information is currently available on how the activity of the U12-dependent spliceosome itself is regulated. The levels of many known splicing factors are regulated through unproductive alternative splicing events, which lead to inclusion of premature STOP codons, targeting the transcripts for destruction by the nonsense-mediated decay pathway. These alternative splice sites are typically found in highly conserved sequence elements, which also contain binding sites for factors regulating the activation of the splice sites. Often, the activation is achieved by binding of products of the gene in question, resulting in negative feedback loops. In this study, I show that U11-48K, a protein factor specific to the minor spliceosome, specifically recognizes the U12-type 5' splice site sequence, and is essential for proper function of the minor spliceosome. Furthermore, the expression of U11-48K is regulated through a feedback mechanism, which functions through conserved sequence elements that activate alternative splicing and nonsense-mediated decay. This mechanism is conserved from plants to animals, highlighting both the importance and early origin of this mechanism in regulating splicing factors. I also show that the feedback regulation of U11-48K is counteracted by a component of the major spliceosome, the U1 small nuclear ribonucleoprotein particle, as well as members of the hnRNP F/H protein family. These results thus suggest that the feedback mechanism is finely tuned by multiple factors to achieve precise control of the activity of the U12-dependent spliceosome.