Streptococcus milleri -ryhmä : Tunnistus lajitasolle ja esiintyvyys tietyissä infektioissa

S. milleri -ryhmän bakteerit ovat osa ihmisen suun, nielun, suoliston ja genitaalialueen normaaliflooran bakteeristoa. Kommensaalien lisäksi ryhmään kuuluu myös merkittäviä patogeenejä, jotka esiintyvät varsin runsaina löydöksinä monenlaisissa märkivissä infektioissa. Ryhmään kuuluu kolme lajia: S. anginosus, S. constellatus ja S. intermedius. Lajit ovat varsin samankaltaisia ja raportoidaankin usein vain ryhmänimellä. Lajit ovat kuitenkin erotettavissa, sillä ne eroavat toisistaan tuottamiensa entsyymien suhteen ja esiintyvyydessään kehon eri osissa. Työn tarkoituksena oli tunnistaa erityyppisistä kliinisistä infektioista otetuista näytteistä eristettyjä S. milleri -ryhmään luokiteltuja kantoja lajitasolle ja selvittää niiden esiintymisyleisyyttä näissä infektioissa. Näytteenottopaikat jaettiin viiteen ryhmään: naisten urogenitaalialue (15 kantaa), miesten urogenitaalialue (8 kantaa), oraaliset (28 kantaa), umpisuoli (34 kantaa) ja "muut" (12 kantaa). Lajitunnistuksen lisäksi selvitettiin kantojen hemolyyttisyys ja mahdollinen Lancefield-seroryhmä (A, C, F, G). Lajien erottelu perustuu eroihin bakteerien kyvyissä hajottaa tiettyjä substraatteja (entsyymiprofilointi), hemolyyttisyys määritettiin verimaljalla ja seroryhmitys tehtiin kaupallisella vasta-aine-sakkautumistestillä (Streptex latex Z1- 50). Työssä testattiin käytössä olevia ja kehiteltiin uusia, lähinnä ennalta muodostuneiden entsyymien tunnistamiseen perustuvia erottelumenetelmiä. Vertailtavina oli kolme entsyymiprofiliontimenetelmää, joista yksi on fluorogeeninen (4-Metyyli-umbelliferyyli-subtraatit) ja kaksi kromogeenistä (Weetabs ja RoscoDiagnostic tablets). Kannoilta määritettiin seuraavat aktiivisuudet: ?-fukosidaasi, (?-glukosidaasi, glukosidaasi, ?-galaktosidaaasi, ?-N-asetyyli-galaktosaminidaasi, ?-N-asetyyli-glukosaminidaasi, sialidaasi ja hyaluronidaasi. Työhön sisältyy myös erilaisten kasvatusalustojen sekä pH:n vaikutusten arviointia bakteerienentsyymiaktiivisuuksiin ja testituloksiin. Lisäksi työssä testattiin kromatografisensoluseinärasvahappoanalyysin soveltuvuutta lajien erotteluun. Menetelmiä tarkasteltiin herkkyyden sekäkäytännön suorittamisen ja aiheutuvien kustannusten kannalta. Asetetut tavoitteet saavutettiin. Kaikki käytetyt menetelmät osoittautuivat toimiviksi. Entsyymitestien tuloksetkorreloivat keskenään ja kirjallisuuden kanssa hyvin. Kannat karakterisoitiin, tunnistettiin lajitasolle ja lajiensiintyvyyttä kehon eri osissa voitiin vertailla. Mikään entsyymitesti ei osoittautunut ylivoimaisesti parhaaksi tai huonoimmaksi, vaikkakin yksittäistensubstraattien kohdalla eri testien herkkyydet vaihtelivatkin huomattavasti. Rasvahappoanalyysi ei erotellutkantoja toivotulla tavalla, joten sen käytöstä luovuttiin työn melko varhaisessa vaiheessa. Tutkituista 97 S. milleri -kannasta tunnistettiin 58 S. anginosus-kantaa, 29 S. constellatus-kantaa ja 10 S.ntermedius-kantaa. Eri lajit noudattivat entsyymiprofiiliaan muutamaa poikkeusta lukuunottamatta hyvinkinsäännöllisesti. Lajien sisäinen variaatio hemolyysiominaisuuksissa oli merkittävää ja S. inilleri -ryhmän erilajien sekä hemolyysisltään ja seroryhmältään erilaisten kantojen esiintyvyydessä kehon eri osissa havaittiinselkeitä eroja. Avainsanat: entsyymiprofilointi, fluorogeeniset ja kromogeeniset substraatit, seroryhmä, hemolyysi, esiintyvyys

Isotopic fingerprints in surficial waters : Stable isotope methods applied in hydrogeological studies

The driving force behind this study has been the need to develop and apply methods for investigating the hydrogeochemical processes of significance to water management and artificial groundwater recharge. Isotope partitioning of elements in the course of physicochemical processes produces isotopic variations to their natural reservoirs. Tracer property of the stable isotope abundances of oxygen, hydrogen and carbon has been applied to investigate hydrogeological processes in Finland. The work described here has initiated the use of stable isotope methods to achieve a better understanding of these processes in the shallow glacigenic formations of Finland. In addition, the regional precipitation and groundwater records will supplement the data of global precipitation, but as importantly, provide primary background data for hydrological studies. The isotopic composition of oxygen and hydrogen in Finnish groundwaters and atmospheric precipitation was determined in water samples collected during 1995 2005. Prior to this study, no detailed records existed on the spatial or annual variability of the isotopic composition of precipitation or groundwaters in Finland. Groundwaters and precipitation in Finland display a distinct spatial distribution of the isotopic ratios of oxygen and hydrogen. The depletion of the heavier isotopes as a function of increasing latitude is closely related to the local mean surface temperature. No significant differences were observed between the mean annual isotope ratios of oxygen and hydrogen in precipitation and those in local groundwaters. These results suggest that the link between the spatial variability in the isotopic composition of precipitation and local temperature is preserved in groundwaters. Artificial groundwater recharge to glaciogenic sedimentary formations offers many possibilities to apply the isotopic ratios of oxygen, hydrogen and carbon as natural isotopic tracers. In this study the systematics of dissolved carbon have been investigated in two geochemically different glacigenic groundwater formations: a typical esker aquifer at Tuusula, in southern Finland and a carbonate-bearing aquifer with a complex internal structure at Virttaankangas, in southwest Finland. Reducing the concentration of dissolved organic carbon (DOC) in water is a primary challenge in the process of artificial groundwater recharge. The carbon isotope method was used to as a tool to trace the role of redox processes in the decomposition of DOC. At the Tuusula site, artificial recharge leads to a significant decrease in the organic matter content of the infiltrated water. In total, 81% of the initial DOC present in the infiltrated water was removed in three successive stages of subsurface processes. Three distinct processes in the reduction of the DOC content were traced: The decomposition of dissolved organic carbon in the first stage of subsurface flow appeared to be the most significant part in DOC removal, whereas further decrease in DOC has been attributed to adsorption and finally to dilution with local groundwater. Here, isotope methods were used for the first time to quantify the processes of DOC removal in an artificial groundwater recharge. Groundwaters in the Virttaankangas aquifer are characterized by high pH values exceeding 9, which are exceptional for shallow aquifers on glaciated crystalline bedrock. The Virttaankangas sediments were discovered to contain trace amounts of fine grained, dispersed calcite, which has a high tendency to increase the pH of local groundwaters. Understanding the origin of the unusual geochemistry of the Virttaankangas groundwaters is an important issue for constraining the operation of the future artificial groundwater plant. The isotope ratios of oxygen and carbon in sedimentary carbonate minerals have been successfully applied to constrain the origin of the dispersed calcite in the Virttaankangas sediments. The isotopic and chemical characteristics of the groundwater in the distinct units of aquifer were observed to vary depending on the aquifer mineralogy, groundwater residence time and the openness of the system to soil CO2. The high pH values of > 9 have been related to dissolution of calcite into groundwater under closed or nearly closed system conditions relative to soil CO2, at a low partial pressure of CO2.

Population dynamics of blue mussels in a variable environment at the edge of their range

Spatial and temporal variation in the abundance of species can often be ascribed to spatial and temporal variation in the surrounding environment. Knowledge of how biotic and abiotic factors operate over different spatial and temporal scales in determining distribution, abundance, and structure of populations lies at the heart of ecology. The major part of the current ecological theory stems from studies carried out in central parts of the distributional range of species, whereas knowledge of how marginal populations function is inadequate. Understanding how marginal populations, living at the edge of their range, function is however in a key position to advance ecology and evolutionary biology as scientific disciplines. My thesis focuses on the factors affecting dynamics of marginal populations of blue mussels (Mytilus edulis) living close to their tolerance limits with regard to salinity. The thesis aims to highlight the dynamics at the edge of the range and contrast these with dynamics in more central parts of the range in order to understand the potential interplay between the central and the marginal part in the focal system. The objectives of the thesis are approached by studies on: (1) factors affecting regional patterns of the species, (2) long-term temporal dynamics of the focal species spaced along a regional salinity gradient, (3) selective predation by increasing populations of roach (Rutilus rutilus) when feeding on their main food item, the blue mussel, (4) the primary and secondary effects of local wave exposure gradients and (5) the role of small-scale habitat heterogeneity as determinants of large-scale pattern. The thesis shows that populations of blue mussels are largely determined by large scale changes in sea water salinity, affecting mainly recruitment success and longevity of local populations. In opposite to the traditional view, the thesis strongly indicate that vertebrate predators strongly affect abundance and size structure of blue mussel populations, and that the role of these predators increases towards the margin where populations are increasingly top-down controlled. The thesis also indicates that the positive role of biogenic habitat modifiers increases towards the marginal areas, where populations of blue mussels are largely recruitment limited. Finally, the thesis shows that local blue mussel populations are strongly dependent on high water turbulence, and therefore, dense populations are constrained to offshore habitats. Finally, the thesis suggests that ongoing sedimentation of rocky shores is detrimental for the species, affecting recruitment success and post-recruit survival, pushing stable mussel beds towards offshore areas. Ongoing large scale changes in the Baltic Sea, especially dilution processes with attendant effects, are predicted to substantially contract the distributional range of the mussel, but also affect more central populations. The thesis shows that in order to understand the functioning of marginal populations, research should (1) strive for multi-scale approaches in order to link ecosystem patterns with ecosystem processes, and (2) challenge the prevailing tenets that origin from research carried out in central areas that may not be valid at the edge.

Evaluation and modelling of the spatial and temporal variability of particulate matter in urban areas

This thesis contains three subject areas concerning particulate matter in urban area air quality: 1) Analysis of the measured concentrations of particulate matter mass concentrations in the Helsinki Metropolitan Area (HMA) in different locations in relation to traffic sources, and at different times of year and day. 2) The evolution of traffic exhaust originated particulate matter number concentrations and sizes in local street scale are studied by a combination of a dispersion model and an aerosol process model. 3) Some situations of high particulate matter concentrations are analysed with regard to their meteorological origins, especially temperature inversion situations, in the HMA and three other European cities. The prediction of the occurrence of meteorological conditions conducive to elevated particulate matter concentrations in the studied cities is examined. The performance of current numerical weather forecasting models in the case of air pollution episode situations is considered. The study of the ambient measurements revealed clear diurnal variation of the PM10 concentrations in the HMA measurement sites, irrespective of the year and the season of the year. The diurnal variation of local vehicular traffic flows seemed to have no substantial correlation with the PM2.5 concentrations, indicating that the PM10 concentrations were originated mainly from local vehicular traffic (direct emissions and suspension), while the PM2.5 concentrations were mostly of regionally and long-range transported origin. The modelling study of traffic exhaust dispersion and transformation showed that the number concentrations of particles originating from street traffic exhaust undergo a substantial change during the first tens of seconds after being emitted from the vehicle tailpipe. The dilution process was shown to dominate total number concentrations. Minimal effect of both condensation and coagulation was seen in the Aitken mode number concentrations. The included air pollution episodes were chosen on the basis of occurrence in either winter or spring, and having at least partly local origin. In the HMA, air pollution episodes were shown to be linked to predominantly stable atmospheric conditions with high atmospheric pressure and low wind speeds in conjunction with relatively low ambient temperatures. For the other European cities studied, the best meteorological predictors for the elevated concentrations of PM10 were shown to be temporal (hourly) evolutions of temperature inversions, stable atmospheric stability and in some cases, wind speed. Concerning the weather prediction during particulate matter related air pollution episodes, the use of the studied models were found to overpredict pollutant dispersion, leading to underprediction of pollutant concentration levels.

Bentsodiatsepiinien ja amfetamiinijohdannaisten seulonta virtsasta mikropilarisähkösumutusmassaspektrometrian avulla

Väärinkäytettyjen aineiden seulontaan käytetyn menetelmän tulee olla herkkä, selektiivinen, yksinkertainen, nopea ja toistettava. Työn tavoitteena oli kehittää yksinkertainen, mutta herkkä, esikäsittelymenetelmä bentsodiatsepiinien ja amfetamiinijohdannaisten kvalitatiiviseen seulomiseen virtsasta mikropilarisähkösumutussirun (μPESI) avulla, mikä tarjoaisi vaihtoehdon seulonnassa käytetyille immunologisille menetelmille, joiden herkkyys ja selektiivisyys ovat puutteellisia. Tavoitteena oli samalla tarkastella mikropilarisähkösumutussirun toimivuutta biologisten näytteiden analyysissa. Esikäsittely optimoitiin erikseen bentsodiatsepiineille ja amfetamiinijohdannaisille. Käytettyjä esikäsittelymenetelmiä olivat neste-nesteuutto, kiinteäfaasiuutto Oasis HLB-patruunalla ja ZipTip®-pipetinkärjellä sekä laimennus ja suodatus ilman uuttoa. Mittausten perusteella keskityttiin optimoimaan ZipTip®-uuttoa. Optimoinnissa tutkittavia yhdisteitä spiikattiin 0-virtsaan niiden ennaltamääritetyn raja-arvon verran, bentsodiatsepiineja 200 ng/ml ja amfetamiinijohdannaisia 300 ng/ml. Bentsodiatsepiinien kohdalla optimoitiin kutakin uuton vaihetta ja optimoinnin tuloksena näytteen pH säädettiin arvoon 5, faasi kunnostettiin asetonitriililla, tasapainotettiin ja pestiin veden (pH 5) ja asetonitriilin (10 % v/v) seoksella ja eluoitiin asetonitriilin, muurahaishapon ja veden (95:1:4 v/v/v) seoksella. Amfetamiinijohdannaisten uutossa optimoitiin näytteen ja liuottimien pH-arvoja ja tuloksena näytteen pH säädettiin arvoon 10, faasi kunnostettiin veden ja ammoniumvetykarbonaatin(pH 10, 1:1 v/v) seoksella, tasapainotettiin ja pestiin asetonitriilin ja veden (1:5 v/v) seoksella ja eluoitiin metanolilla. Optimoituja uuttoja testattiin Yhtyneet Medix Laboratorioista toimitetuilla autenttisilla virtsanäytteillä ja saatuja tuloksia verrattiin kvantitatiivisen GC/MS-analyysin tuloksiin. Bentsodiatsepiininäytteet hydrolysoitiin ennen uuttoa herkkyyden parantamiseksi. Autenttiset näytteet analysoitiin Q-TOF-laitteella Viikissä. Lisäksi hydrolysoidut bentsodiatsepiininäytteet mitattiin Yhtyneet Medix Laboratorioiden TOF-laitteella. Kehitetty menetelmä vaatii tulosten perusteella lisää optimointia toimiakseen. Ongelmana oli etenkin toistoissa ilmennyt tulosten hajonta. Manuaalista näytteensyöttöä tulisi kehittää toistettavammaksi. Autenttisten bentsodiatsepiininäytteiden analyysissa ongelmana olivat virheelliset negatiiviset tulokset ja amfetamiinijohdannaisten analyysissa virheelliset positiiviset tulokset. Virheellisiä negatiivisia tuloksia selittää menetelmän herkkyyden puute ja virheellisiä positiivisia tuloksia mittalaitteen, sirujen tai liuottimien likaantuminen.

Method-MS, final report 2010

Radiometric determination methods, such as alpha spectrometry require long counting times when low activities are to be determined. Mass spectrometric techniques as Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Thermal Ionisation Mass Spectrometry (TIMS) and Accelerator Mass Spectrometry (AMS) have shown several advantages compared to traditional methods when measuring long-lived radionuclides. Mass spectrometric methods for determination of very low concentrations of elemental isotopes, and thereby isotopic ratios, have been developed using a variety of ion sources. Although primarily applied to the determination of the lighter stable element isotopes and radioactive isotopes in geological studies, the techniques can equally well be applied to the measurement of activity concentrations of long-lived low-level radionuclides in various samples using “isotope dilution” methods such as those applied in inductively coupled plasma mass spectrometry (ICP-MS). Due to the low specific activity of long-lived radionuclides, many of these are more conveniently detected using mass spectrometric techniques. Mass spectrometry also enables the individual determination of Pu-239 and Pu-240, which cannot be obtained by alpha spectrometry. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) are rapidly growing techniques for the ultra-trace analytical determination of stable and long-lived isotopes and have a wide potential within environmental science, including ecosystem tracers and radio ecological studies. Such instrumentation, of course needs good radiochemical separation, to give best performance. The objectives of the project is to identify current needs and problems within low-level determination of long-lived radioisotopes by ICP-MS, to perform intercalibration and development and improvement of ICP-MS methods for the measurement of radionuclides and isotope ratios and to develop new methods based on modified separation chemistry applied to new auxiliary equipment.

Bioremediation of diesel oil contaminated soil and water

Diesel spills contaminate aquatic and terrestrial environments. To prevent the environmental and health risks, the remediation needs to be advanced. Bioremediation, i.e., degradation by microbes, is one of the suitable methods for cleaning diesel contamination. In monitored natural attenuation technique are natural processes in situ combined, including bioremediation, volatilization, sorption, dilution and dispersion. Soil bacteria are capable of adapting to degrade environmental pollutants, but in addition, some soil types may have indigenous bacteria that are naturally suitable for degradation. The objectives for this work were (1) to find a feasible and economical technique to remediate oil spilled into Baltic Sea water and (2) to bioremediate soil contaminated by diesel oil. Moreover, the aim was (3) to study the potential for natural attenuation and the indigenous bacteria in soil, and possible adaptation to degrade diesel hydrocarbons. In the aquatic environment, the study concentrated on diesel oil sorption to cotton grass fiber, a natural by-product of peat harvesting. The impact of diesel pollution was followed in bacteria, phytoplankton and mussels. In a terrestrial environment, the focus was to compare the methods of enhanced biodegradation (biostimulation and bioaugmentation), and to study natural attenuation of oil hydrocarbons in different soil types and the effect that a history of previous contamination may have on the bioremediation potential. (1) In the aquatic environment, rapid removal of diesel oil was significant for survival of tested species and thereby diversity maintained. Cotton grass not only absorbed the diesel but also benefited the bacterial growth by providing a large colonizable surface area and hence oil-microbe contact area. Therefore use of this method would enhance bioremediation of diesel spills. (2) Biostimulation enhances bioremediation, and (3) indigenous diesel-degrading bacteria are present in boreal environments, so microbial inocula are not always needed. In the terrestrial environment experiments, the combination of aeration and addition of slowly released nitrogen advanced the oil hydrocarbon degradation. Previous contamination of soil gives the bacterial community the potential for rapid adaptation and efficient degradation of the same type of contaminant. When the freshly contaminated site needs addition of diesel degraders, previously contaminated and remediated soil could be used as a bacterial inoculum. Another choice of inoculum could be conifer forest soil, which provides a plentiful population of degraders, and based on the present results, could be considered as a safe non-polluted inoculum. According to the findings in this thesis, bioremediation (microbial degradation) and monitored natural attenuation (microbial, physical and chemical degradation) are both suitable techniques for remediation of diesel-contaminated sites in Finland.