Switchable lanthanide fluorescence probes in homogenous molecular diagnostics

The number of molecular diagnostic assays has increased tremendously in recent years.Nucleic acid diagnostic assays have been developed, especially for the detection of human pathogenic microbes and genetic markers predisposing to certain diseases. Closed-tube methods are preferred because they are usually faster and easier to perform than heterogenous methods and in addition, target nucleic acids are commonly amplified leading to risk of contamination of the following reactions by the amplification product if the reactions are opened. The present study introduces a new closed-tube switchable complementation probes based PCR assay concept where two non-fluorescent probes form a fluorescent lanthanide chelate complex in the presence of the target DNA. In this dual-probe PCR assay method one oligonucleotide probe carries a non-fluorescent lanthanide chelate and another probe a light absorbing antenna ligand. The fluorescent lanthanide chelate complex is formed only when the non-fluorescent probes are hybridized to adjacent positions into the target DNA bringing the reporter moieties in close proximity. The complex is formed by self-assembled lanthanide chelate complementation where the antenna ligand is coordinated to the lanthanide ion captured in the chelate. The complementation probes based assays with time-resolved fluorescence measurement showed low background signal level and hence, relatively high nucleic acid detection sensitivity (low picomolar target concentration). Different lanthanide chelate structures were explored and a new cyclic seven dentate lanthanide chelate was found suitable for complementation probe method. It was also found to resist relatively high PCR reaction temperatures, which was essential for the PCR assay applications. A seven-dentate chelate with two unoccupied coordination sites must be used instead of a more stable eight- or nine-dentate chelate because the antenna ligand needs to be coordinated to the free coordination sites of the lanthanide ion. The previously used linear seven-dentate lanthanide chelate was found to be unstable in PCR conditions and hence, the new cyclic chelate was needed. The complementation probe PCR assay method showed high signal-to-background ratio up to 300 due to a low background fluorescence level and the results (threshold cycles) in real-time PCR were reached approximately 6 amplification cycles earlier compared to the commonly used FRET-based closed-tube PCR method. The suitability of the complementation probe method for different nucleic acid assay applications was studied. 1) A duplex complementation probe C. trachomatis PCR assay with a simple 10-minute urine sample preparation was developed to study suitability of the method for clinical diagnostics. The performance of the C. trachomatis assay was equal to the commercial C. trachomatis nucleic acid amplification assay containing more complex sample preparation based on DNA extraction. 2) A PCR assay for the detection of HLA-DQA1*05 allele, that is used to predict the risk of type 1 diabetes, was developed to study the performance of the method in genotyping. A simple blood sample preparation was used where the nucleic acids were released from dried blood sample punches using high temperature and alkaline reaction conditions. The complementation probe HLA-DQA1*05 PCR assay showed good genotyping performance correlating 100% with the routinely used heterogenous reference assay. 3) To study the suitability of the complementation probe method for direct measurement of the target organism, e.g., in the culture media, the complementation probes were applied to amplificationfree closed-tube bacteriophage quantification by measuring M13 bacteriophage ssDNA. A low picomolar bacteriophage concentration was detected in a rapid 20- minute assay. The assay provides a quick and reliable alternative to the commonly used and relatively unreliable UV-photometry and time-consuming culture based bacteriophage detection methods and indicates that the method could also be used for direct measurement of other micro-organisms. The complementation probe PCR method has a low background signal level leading to a high signal-to-background ratio and relatively sensitive nucleic acid detection. The method is compatible with simple sample preparation and it was shown to tolerate residues of urine, blood, bacteria and bacterial culture media. The common trend in nucleic acid diagnostics is to create easy-to-use assays suitable for rapid near patient analysis. The complementation probe PCR assays with a brief sample preparation should be relatively easy to automate and hence, would allow the development of highperformance nucleic acid amplification assays with a short overall assay time.

Metsän teollisen käytön jykevä kokonaispaketti

Kirjallisuusarvostelu

"Järkevästi ujostelemattomat" naiset ja kätilökoulutuksen historia

Kirjallisuusarvostelu

Arkistoluettelo 100: Emil Kauppi

Luettelo Kansalliskirjastossa olevan Emil Kaupin arkiston sisällöstä

Pelillistäminen menetelmänä julkisen liikenteen käytön lisäämisessä

Joukkoliikenteen suosio hiipuu vähitellen, käyttö aste laskee. Tätä ongelmaa lähdimme ratkaisemaan sovelluksen ja pelillistämis menetelmän avulla. Menetelmää hyödyntäen tavoitteena oli luoda sovellus joka koukuttaa käyttäjän sovelluksen käyttöön ja sitä kautta joukkoliikenteen käyttöön. Pyrimme pysäyttämään joukkoliikenteen käyttöasteen laskun tai jopa kääntämään sen kasvuun. Varsinaista tulosta ei tällä työllä saatu, sillä aika ei vain riittänyt sovelluksen testaamiseen käytännössä. Pelillistämis menetelmiä kun tutkii ja vertaa sovellustamme niihin, niin sovelluksellamme on hyvä mahdollisuudet toimia joukkoliikenteen käytön lisäämisen työkaluna.

ONKI Light -ontologiaselaimen käytettävyystestaus: toinen testikierros

Kansalliskirjaston ONKI-projektin ylläpitämä Finto-palvelu käyttää projektissa kehitettävää Skosmos-ohjelmaa (entinen ONKI Light). Skosmos on työkalu kontrolloitujen sanastojen, kuten asiasanastojen ja asiasanastotyyppisesti käytettävien ontologioiden julkaisuun. Työkalu tarjoaa selailu- ja hakukäyttöliittymän sanastoille sekä avoimet rajapinnat koneellista käyttöä varten. Käyttöliittymä on monikielinen sisältäen tällä hetkellä suomen-, ruotsin- sekä englanninkieliset käyttöliittymäversiot.

Skosmoksen edeltäjälle ONKI Lightille on tehty ONKI-projektissa jo aikaisempi käytettävyystesti, jonka raportti on luettavissa Doriassa.

Käytettävyystestien perusteella vaikuttaisi siltä, että ontologian selaamiseen asiasanoituksessa vaikuttaisi ainakin käyttäjän käyttämä järjestelmä, asiasanoitukseen käytettävä aika, asiasanoitustottumukset ja -kokemus sekä sanaston tuttuus. Jos aikaa ei ole juurikaan varattu asiasanoitukselle, ei käsitteitä juurikaan selailla, vaikka muuten tuki ontologioiden hyödyntämiselle olisi olemassa. Myöskään sanastoa ei juurikaan selailla, mikäli aihe ja sanasto ovat tuttuja, jolloin asiasanojen merkitykset ovat etukäteen tiedossa.

Reproductive phenology, pollination and seed dispersal syndromes on sandstone outcrop vegetation in the "Chapada Diamantina", northeastern Brazil: population and community analyses

Vegetation on rock outcrops in the "Chapada Diamantina" (soil islands) is often aggregated and surrounded by nude rock surfaces, thus creating natural units with well defined limits. The flowering and fruiting cycles of plants on 58 soil islands at altitudes between 1,100 and 1,140 meters above sea leavel were analyzed at Mãe Inácia Peak (12°27' S and 41°28' W) in the "Chapada Diamantina", Bahia, Brazil. The presence/absence of flowering and fruiting species on each soil island, and their respective cover areas were analyzed at both the population and community levels, and the phenophases of flowering and fruiting were observed during 24 successive months. The analyses of pollination and seed dispersal syndromes indicated that animals are more important in pollination than in seed dispersal (which is predominantly by anemochory and autochory). The flowering and fruiting of plants with animal pollination syndromes were correlated with rainfall and temperature. The flowering season varied during the year according to the pollination syndrome involved: entomophily was predominant from summer through autumn, ornithophily was predominant during winter, and anemophily in the spring. The staggered timing of flowering and fruiting among different species provides a nearly continuous supply of resources for the local fauna.

Neonatal handling induces anovulatory estrous cycles in rats

Since previous work has shown that stimulation early in life decreases sexual receptiveness as measured by the female lordosis quotient, we suggested that neonatal handling could affect the function of the hypothalamus-pituitary-gonadal axis. The effects of neonatal handling on the estrous cycle and ovulation were analyzed in adult rats. Two groups of animals were studied: intact (no manipulation, N = 10) and handled (N = 11). Pups were either handled daily for 1 min during the first 10 days of life or left undisturbed. At the age of 90 days, a vaginal smear was collected daily at 9:00 a.m. and analyzed for 29 days; at 9:00 a.m. on the day of estrus, animals were anesthetized with thiopental (40 mg/kg, ip), the ovaries were removed and the oviduct was dissected and squashed between 2 glass slides. The number of oocytes of both oviductal ampullae was counted under the microscope. The average numbers for each phase of the cycle (diestrus I, diestrus II, proestrus and estrus) during the period analyzed were compared between the two groups. There were no significant differences between intact and handled females during any of the phases. However, the number of handled females that showed anovulatory cycles (8 out of 11) was significantly higher than in the intact group (none out of 10). Neonatal stimulation may affect not only the hypothalamus-pituitary-adrenal axis, as previously demonstrated, but also the hypothalamus-pituitary-gonadal axis in female rats.

Working fluid selection and design of small-scale waste heat recovery systems based on organic Rankine cycles

Demand for the use of energy systems, entailing high efficiency as well as availability to harness renewable energy sources, is a key issue in order to tackling the threat of global warming and saving natural resources. Organic Rankine cycle (ORC) technology has been identified as one of the most promising technologies in recovering low-grade heat sources and in harnessing renewable energy sources that cannot be efficiently utilized by means of more conventional power systems. The ORC is based on the working principle of Rankine process, but an organic working fluid is adopted in the cycle instead of steam. This thesis presents numerical and experimental results of the study on the design of small-scale ORCs. Two main applications were selected for the thesis: waste heat re- covery from small-scale diesel engines concentrating on the utilization of the exhaust gas heat and waste heat recovery in large industrial-scale engine power plants considering the utilization of both the high and low temperature heat sources. The main objective of this work was to identify suitable working fluid candidates and to study the process and turbine design methods that can be applied when power plants based on the use of non-conventional working fluids are considered. The computational work included the use of thermodynamic analysis methods and turbine design methods that were based on the use of highly accurate fluid properties. In addition, the design and loss mechanisms in supersonic ORC turbines were studied by means of computational fluid dynamics. The results indicated that the design of ORC is highly influenced by the selection of the working fluid and cycle operational conditions. The results for the turbine designs in- dicated that the working fluid selection should not be based only on the thermodynamic analysis, but requires also considerations on the turbine design. The turbines tend to be fast rotating, entailing small blade heights at the turbine rotor inlet and highly supersonic flow in the turbine flow passages, especially when power systems with low power outputs are designed. The results indicated that the ORC is a potential solution in utilizing waste heat streams both at high and low temperatures and both in micro and larger scale appli- cations.

Cycle modulation of insulin-like growth factor-binding protein 1 in human endometrium

Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1) interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP) have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5%) followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia.

Nopeusrajoituksen 100 km/h alentamisen liikenneturvallisuusvaikutukset Varsinais-Suomen ja Satakunnan alueella, esisuunnitelma

Työn tavoitteena ovat valtakunnalliset liikenneturvallisuustavoitteet vuoteen 2020 mennessä ja niiden saavuttaminen tienpitäjän toimenpitein.Tässä työssä tarkasteltiin maanteitä, joilla on kesäaikana voimassa 100 km/h nopeusrajoitus. Työssä vertailtiin nykyisten 100 km/h väylien tierekisteritietoja uusiin nopeus-rajoitusohjeisiin (LVM:n yleisohje 2009) sekä vertailtiin ko. tiejaksojen onnettomuustiheyksiä ja -asteita valtakunnallisiin lukuihin (Liikennevirasto, 2012). Lisäksi 100 km/h tieosuuksien ympäristön asukastiheyttä tarkasteltiin Rakennus- ja huoneistorekisteritietojen perusteella. Näiden tietojen perusteella muodostuivat mahdolliset nopeusrajoituksen alentamista vaativat tiejaksot. Nopeusrajoituksen alentamista liikenneturvallisuustilanteen parantamiseksi tarkasteltiin seuraavien priorisointiperiaatteiden mukaisesti: 1. 0-vision ja ihmisen kestokyvyn mukainen priorisointi: • Millä 100 km/h tiejaksoilla ei ole keskikaidetta? 2. Vastaavatko 100 km/h tiejaksot nykyisiä nopeusrajoitusohjeita? 3. Esiintyykö 100 km/h tiejaksoilla korkeita onnettomuustiheyksiä ja -asteita? Varsinais-Suomen ELY-keskuksen liikennevastuualueen johtotiimin päätöksen mukaisesti nopeusrajoitusta alennetaan 80 km/h:iin yhteensä 23 kilometrin matkalla. Lisäksi talvinopeusrajoitusta alennetaan yhteensä 9 kilometrin pituisella tiejaksolla. Nopeusrajoitustoimenpiteiden lisäksi yhden liittymän kohdalle päätettiin asettaa ohituskiellot ja yhdelle tiejaksolle esitettiin tehtäväksi puuston raivausta. Toimenpiteillä saavutetaan noin 0,78 vuosittainen vähenemä henkilövahinkoon johtaneissa onnettomuuksissa. Toimenpiteiden yhteiskuntataloudelliset kustannukset ovat yhteensä 600 000 euroa/vuosi.