From Unspecific Quenching to Specific Signaling: Functional GTPase Assays Utilizing Quenching Resonance Energy Transfer (QRET) Technology

The aim of the work presented in this study was to demonstrate the wide applicability of a single-label quenching resonance energy transfer (QRET) assay based on time-resolved lanthanide luminescence. QRET technology is proximity dependent method utilizing weak and unspecific interaction between soluble quencher molecule and lanthanide chelate. The interaction between quencher and chelate is lost when the ligand binds to its target molecule. The properties of QRET technology are especially useful in high throughput screening (HTS) assays. At the beginning of this study, only end-point type QRET technology was available. To enable efficient study of enzymatic reactions, the QRET technology was further developed to enable measurement of reaction kinetics. This was performed using proteindeoxyribonuclei acid (DNA) interaction as a first tool to monitor reaction kinetics. Later, the QRET was used to study nucleotide exchange reaction kinetics and mutation induced effects to the small GTPase activity. Small GTPases act as a molecular switch shifting between active GTP bound and inactive GDP bound conformation. The possibility of monitoring reaction kinetics using the QRET technology was evaluated using two homogeneous assays: a direct growth factor detection assay and a nucleotide exchange monitoring assay with small GTPases. To complete the list, a heterogeneous assay for monitoring GTP hydrolysis using small GTPases, was developed. All these small GTPase assays could be performed using nanomolar protein concentrations without GTPase pretreatment. The results from these studies demonstrated that QRET technology can be used to monitor reaction kinetics and further enable the possibility to use the same method for screening.

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

The house that the Court built. The retained powers case law of the European Court of Justice and the constitutional structure of the Union

Euroopan unionin perustamissopimusten katsotaan muodostavan EU:n valtiosäännön, jonka rajat ovat kuitenkin perustamissopimusten määräysten väljän muotoilun sekä Euroopan unionin tuomioistuimen tulkintakäytännön valossa epätarkat. Etenkin kysymys unionin ja sen jäsenvaltioiden välisestä toimivallanjaosta on EU-oikeudellisen tutkimuksen klassikoita. Tarkastelen pro gradu -tutkielmassani unionin valtiosääntörakennetta ja unionioikeuden kokonaisvaikutusta jäsenvaltioiden toimivaltojen käyttöön erityisesti EU-tuomioistuimen ratkaisukäytännössään kehittämän niin kutsutun retained powers -doktriinin valossa. Kyseisen opin mukaan EU-oikeus asettaa vaatimuksia jäsenvaltioiden toimivaltojen käytölle myös niillä aloilla, joilla sääntelytoimivalta on jäänyt jäsenvaltioille eikä sitä ole jaettu unionin kanssa. Aiheen teoreettisen tarkastelun pohjalta analysoin Euroopaun unionin tuomioistuimen ratkaisukäytäntöä erityisesti yhtä säilytetyn toimivallan alaa, koulutusta, koskevissa tapauksissa pyrkien havaitsemaan typologioita tuomioistuimen ratkaisutoiminnassa. Tutkimus noudattaa EU-valtiosääntöoikeuden metodologiaa. Keskeisenä lähdemateriaalina on siten käytetty unionituomioistuimen ratkaisukäytäntöä, joka heijastaa perustamissopimuksia tarkemmin unionin valtiosääntörakennetta. Oikeuskäytännön analyysi ja tulkinta on suoritettu peilaten sitä vasten unionin kehitystä markkinaorientoituneesta organisaatiosta yleismaailmalliseksi poliittiseksi unioniksi. Tutkielmani loppupäätelmä on, että jäsenvaltiot ovat tietyissä rajoissa hyväksyneet unionituomioistuimen kehittämän doktriinin, ja unionituomioistuin on siten saanut aikaan tosiasiallisen muutoksen EU:n valtiosääntörakenteessa. Retained powers -doktriini on omiaan syventämään eurooppalaista integraatiota ja nostaa kysymyksiä toimivallanjaon merkityksestä unionioikeudessa, perustamissopimusten kyvystä heijastaa unionin valtiosääntörakennetta sekä unionituomioistuimen toiminnan poliittisesta luonteesta.

Effects of Fin Parameters on Gas-Side Heat Transfer Performance of H-Type Finned Tube Bundle in a Waste Heat Recovery Boiler

Alfa Laval Aalborg Oy designs and manufactures waste heat recovery systems utilizing extended surfaces. The waste heat recovery boiler considered in this thesis is a water-tube boiler where exhaust gas is used as the convective heat transfer medium and water or steam flowing inside the tubes is subject to cross-flow. This thesis aims to contribute to the design of waste heat recovery boiler unit by developing a numerical model of the H-type finned tube bundle currently used by Alfa Laval Aalborg Oy to evaluate the gas-side heat transfer performance. The main objective is to identify weaknesses and potential areas of development in the current H-type finned tube design. In addition, numerical simulations for a total of 15 cases with varying geometric parameters are conducted to investigate the heat transfer and pressure drop performance dependent on H-type fin geometry. The investigated geometric parameters include fin width and height, fin spacing, and fin thickness. Comparison between single and double tube type configuration is also conducted. Based on the simulation results, the local heat transfer and flow behaviour of the H-type finned tube is presented including boundary layer development between the fins, the formation of recirculation zone behind the tubes, and the local variations of flow velocity and temperature within the tube bundle and on the fin surface. Moreover, an evaluation of the effects of various fin parameters on heat transfer and pressure drop performance of H-type finned tube bundle has been provided. It was concluded that from the studied parameters fin spacing and fin width had the most significant effect on tube bundle performance and the effect of fin thickness was the least important. Furthermore, the results suggested that the heat transfer performance would increase due to enhanced turbulence if the current double tube configuration is replaced with single tube configuration, but further investigation and experimental measurements are required in order to validate the results.