Tiedon jakaminen heterogeenisessa projektiverkostossa

Tutkielman tavoitteena on selvittää, mitkä tekijät vaikuttavat tiedon jakamiseen heterogeenisessa projektiverkostossa. Tutkielma sisältää sekä teoreettisen että empiirisen osan. Teoriaosassa käsitellään kahta pääaihetta: tiedon jakamisen verkostomaista kontekstia ja projektiverkostoon sekä sen toimijoihin liittyviä, tiedon jakamiseen vaikuttavia tekijöitä. Empiirinen osuus sisältää kvalitatiivisen haastattelututkimuksen, missä on kerätty case-projektin toimijoiden mielipiteitä niistä seikoista, jotka vaikuttavat tiedon jakamiseen projektiverkostossa. Tutkielman lopuksi kirjallisuuskatsauksen ja haastattelujen tuloksia on verrattu toisiinsa. Tutkielman tulosten perusteella voidaan todeta, että tiedon jakamiseen vaikuttavat projektiverkoston rakenne, projektikumppaneiden motivaatio, osaaminen ja resurssit, projektintarjoama tiedon jakamisen infrastruktuuri, projektin päämäärän selkeys, projektin toimintaa varten laaditut pelisäännöt, sekä projektikoordinaattorin kyky johtaa ja ohjata projektin toimintaa.

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.

Spotlight: A Blacklight Plugin for Showcasing Digital Collections

Presentation at Open Repositories 2014, Helsinki, Finland, June 9-13, 2014

Prognostic Factors In Breast Cancer. With Special Reference to Cyclins A, B1, D1 and E, MMP-1 and Decorin

Breast cancer is a highly heterogenous malignancy, which despite of the similar histological type shows different clinical behaviour and response to therapy. Prognostic factors are used to estimate the risk for recurrence and the likelihood of treatment effectiveness. Because breast cancer is one of the most common causes of cancer death in women worldwide, identification of new prognostic markers are needed to develop more specific and targeted therapies. Cancer is caused by uncontrolled cell proliferation. The cell cycle is controlled by specific proteins, which are known as cyclins. They function at important checkpoints by activating cyclin-dependent kinase enzymes. Overexpression of different cyclins has been linked to several cancer types and altered expression of cyclins A, B1, D1 and E has been associated with poor survival. Little is known about the combined expression of cyclins in relation to the tumour grade, breast cancer subtype and other known prognostic factors. In this study cyclins A, B1 and E were shown to correlate with histological grade, Ki-67 and HER2 expression. Overexpression of cyclin D1 correlated with receptor status and non-basal breast cancer suggesting that cyclin D1 might be a marker of good prognosis. Proteolysis in the surrounding tumour stroma is increased during cancer development. Matrix metalloproteinases (MMPs) are proteolytic enzymes that are capable of degrading extracellular matrix proteins. Increased expression and activation of several MMPs have been found in many cancers and MMPs appear to be important regulators of invasion and metastasis. In this study MMP-1 expression was analysed in breast cancer epithelial cells and in cancer associated stromal cells. MMP-1 expression by breast cancer epithelial cells was found to carry an independent prognostic value as did Ki-67 and bcl-2. The results suggest that in addition to stromal cells MMP-1 expression in tumour cells control breast cancer progression. Decorin is a small proteoglycan and an important component of the extracellular matrix. Decorin has been shown to inhibit growth of tumour cells and reduced decorin expression is associated with a poor prognosis in several cancer types. There has been some suspicion wheather different cancer cells express decorin. In this study decorin expression was shown to localize only in the cells of the original stroma, while breast cancer epithelial cells were negative for decorin expression. However, transduction of decorin in decorin-negative human breast cancer cells markedly modulated the growth pattern of these cells. This study provides evidence that targeted decorin transduction to breast cancer cells could be used as a novel adjuvant therapy in breast malignancies.

Pienen mittakaavan reaktiivisuuskoelaitteen mallinnus

Tässä kandidaatintyössä tavoitteena on selvittää, ymmärretäänkö heterogeenisen reakti-on ominaisuuksia paremmin mallinnuksen avulla. Työssä tarkastellaan heterogeenisia reaktioita ja niiden makrotason ilmiöitä. Työn kirjallisuusosassa käsitellään, mikä on heterogeeninen reaktio ja missä tilanteissa heterogeenisiä reaktioita esiintyy. Hetero-geenisiä reaktioita voidaan tutkia Lappeenrannan teknillisen yliopiston energiatekniikan osaston rakentamalla koelaitteistolla. Tässä kandidaatintyössä esitellään pintapuolisesti tämä laitteisto. Työn pääpaino on mallinnuksessa. Työssä selvitetään, miksi mallinnusta tehdään ja miten malli rakennetaan kuvaamaan koelaitteistolla saatuja mittaustuloksia. Lopuksi tehdään johtopäätökset ja arvioidaan edellä kuvatun prosessin toimivuutta. Tämä kandidaatintyö antaa perusteet sille, miksi ja miten mallinnuksen avulla voidaan ymmärtää reaktioiden ominaisuuksia paremmin ja yksityiskohtaisemmin.