Homogeneous assay technologies in drug screening: Quenching Resonance Energy Transfer (QRET) technique

Information gained from the human genome project and improvements in compound synthesizing have increased the number of both therapeutic targets and potential lead compounds. This has evolved a need for better screening techniques to have a capacity to screen number of compound libraries against increasing amount of targets. Radioactivity based assays have been traditionally used in drug screening but the fluorescence based assays have become more popular in high throughput screening (HTS) as they avoid safety and waste problems confronted with radioactivity. In comparison to conventional fluorescence more sensitive detection is obtained with time-resolved luminescence which has increased the popularity of time-resolved fluorescence resonance energy transfer (TR-FRET) based assays. To simplify the current TR-FRET based assay concept the luminometric homogeneous single-label utilizing assay technique, Quenching Resonance Energy Transfer (QRET), was developed. The technique utilizes soluble quencher to quench non-specifically the signal of unbound fraction of lanthanide labeled ligand. One labeling procedure and fewer manipulation steps in the assay concept are saving resources. The QRET technique is suitable for both biochemical and cell-based assays as indicated in four studies:1) ligand screening study of β2 -adrenergic receptor (cell-based), 2) activation study of Gs-/Gi-protein coupled receptors by measuring intracellular concentration of cyclic adenosine monophosphate (cell-based), 3) activation study of G-protein coupled receptors by observing the binding of guanosine-5’-triphosphate (cell membranes), and 4) activation study of small GTP binding protein Ras (biochemical). Signal-to-background ratios were between 2.4 to 10 and coefficient of variation varied from 0.5 to 17% indicating their suitability to HTS use.

A platform for anti-biofilm assays combining viability, biomass and matrix quantifications in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

Bacteria can exist as planktonic, the lifestyle in which single cells exist in suspension, and as biofilms, which are surface-attached bacterial communities embedded in a selfproduced matrix. Most of the antibiotics and the methods for antimicrobial work have been developed for planktonic bacteria. However, the majority of the bacteria in natural habitats live as biofilms. Biofilms develop dauntingly fast high resistance towards conventional antibacterial treatments and thus, there is a great need to meet the demands of effective anti-biofilm therapy. In this thesis project it was attempted to fill the void of anti-biofilm screening methods by developing a platform of assays that evaluate the effect that screened compounds have on the total biomass, viability and the extracellular polysaccharide (EPS) layer of the biofilms. Additionally, a new method for studying biofilms and their interactions with compounds in a continuous flow system was developed using capillary electrochromatography (CEC). The screening platform was utilized with a screening campaign using a small library of cinchona alkaloids. The assays were optimized to be statistically robust enough for screening. The first assay, based on crystal violet staining, measures total biofilm biomass, and it was automated using a liquid handling workstation to decrease the manual workload and signal variation. The second assay, based on resazurin staining, measures viability of the biofilm, and it was thoroughly optimized for the strain used, but was then a very simple and fast method to be used for primary screening. The fluorescent resazurin probe is not toxic to the biofilms. In fact, it was also shown in this project that staining the biofilms with resazurin prior to staining with crystal violet had no effect on the latter and they can be used in sequence on the same screening plate. This sequential addition step was indeed a major improvement on the use of reagents and consumables and also shortened the work time. As a third assay in the platform a wheat germ agglutinin based assay was added to evaluate the effect a compound has on the EPS layer. Using this assay it was found that even if compounds might have clear effect on both biomass and viability, the EPS layer can be left untouched or even be increased. This is a clear implication of the importance of using several assays to be able to find “true hits” in a screening setting. In the pilot study of screening for antimicrobial and anti-biofilm effects using a cinchona alkaloid library, one compound was found to have antimicrobial effect against planktonic bacteria and prevent biofilm formation at low micromolar concentration. To eradicate biofilms, a higher concentration was needed. It was also shown that the chemical space occupied by the active compound was slightly different than the rest of the cinchona alkaloids as well as the rest of the compounds used for validatory screening during the optimization processes of the separate assays.

Transformation into Platform - How can an organisation move from a single-sided business to platforms? A case study at Lyyti

The research studies the transformation from a single-sided offering to a multi-sided platform. The study aims to define platforms and their benefits, creating a theoretical framework by applying change management models with the platform theory, and by finding critical change points of the transformation. The empirical research was done by utilizing action research. The researcher worked as project manager in the case company, and studied the transformation project by working actively and leading the project team. The result of the project was a study of how the company would be able to manage the transformation. The results clearly showed that the company didn’t have the capabilities to finish the transformation. As a conclusion, the study showed that the critical change points that led to the project failure were, that the project was managed with insufficient change managerial efforts, which later resulted as lack of commitment to re-allocating the resources to complete the transformation. Many of the critical change points were results of combined change managerial and platform-related issues.

Product Platform Development from the Product Lines' Perspective: Case of Switching Platform

In the era of fast product development and customized product requirements, the concept of product platform has proven its power in practice. The product platform approach has enabledcompanies to increase the speed of product introductions while simultaneously benefit from efficiency and effectiveness in the development and production activities. The product platforms are technological bases, which can be used to develop several derivative products, and hence, the differentiation can be pushed closer to the product introduction. The product platform development has some specific features, which differ somewhat from the product development of single products. The time horizon is longer, since the product platform¿slife cycle is longer than individual product's. The long time-horizon also proposes higher market risks and the use of new technologies increases the technological risks involved. The end-customer interface might be far away, but there is not a lack of needs aimed at the product platforms ¿ in fact, the product platform development is very much balancing between the varying needs set to it by thederivative products. This dissertation concentrated on product platform development from the internal product lines' perspective of a singlecase. Altogether six product platform development factors were identified: 'Strategic and business fit of product platform', 'Project communication and deliverables', 'Cooperation with product platform development', 'Innovativeness of product platform architecture and features', 'Reliability and quality of product platform', and 'Promised schedules and final product platform meeting the needs'. From the six factors, three were found to influence quite strongly the overall satisfaction, namely 'Strategic and business fit of product platform', 'Reliability and quality of product platform', and 'Promised schedules and final product platform meeting the needs'. Hence, these three factors might be the ones a new product platform development unit should concentrate first in order to satisfy their closest customers, the product lines. The 'Project communication and deliverables' and 'Innovativeness of product platform architecture and features' were weaker contributors to the overall satisfaction. Overall, the factors explained quite well the satisfaction of the product lines with product platform development. Along the research, several interesting aspects about the very basic nature of the product platform development were found. The long time horizon of the product platform development caused challenges in the area of strategic fIT - a conflict between the short-term requirements and long term needs. The fact that a product platform was used as basis of several derivative products resulted into varying needs, and hence the match with the needs and the strategies. The opinions, that the releases of the larger product lines were given higher priorities, give an interesting contribution to the strategy theory of powerand politics. The varying needs of the product lines, the strengths of them as well as large number of concurrent releases set requirements to prioritization. Hence, the research showed the complicated nature of the product platform development in the case unIT - the very basic nature of the product platform development might be its strength (gaining efficiency and effectiveness in product development and product launches) but also the biggest challenge (developing products to meet several needs). As a single case study, the results of this research are not directly generalizable to all the product platform development activities. Instead, the research serves best as a starting point for additional research as well as gives some insights about the factors and challengesof one product development unit.

Lanthanide Chelates as Donors in Fluorescence Resonance Energy Transfer: Exciting Prospects for Bioaffinity Assay Detection

Fluorescence resonance energy transfer (FRET) is a non-radiative energy transfer from a fluorescent donor molecule to an appropriate acceptor molecule and a commonly used technique to develop homogeneous assays. If the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, FRET might occur. As a consequence, the emission of the donor is decreased and the emission of the acceptor (if fluorescent) increased. Furthermore, the distance between the donor and the acceptor needs to be short enough, commonly 10-100 Å. Typically, the close proximity between the donor and the acceptor is achieved via bioaffinity interactions e.g. antibody binding antigen. Large variety of donors and acceptors exist. The selection of the donor/acceptor pair should be done not only based on the requirements of FRET but also the performance expectancies and the objectives of the application should be considered. In this study, the exceptional fluorescence properties of the lanthanide chelates were employed to develop two novel homogeneous immunoassays: a non-competitive hapten (estradiol) assay based on a single binder and a dual-parametric total and free PSA assay. In addition, the quenching efficiencies and energy transfer properties of various donor/acceptor pairs were studied. The applied donors were either europium(III) or terbium(III) chelates; whereas several organic dyes (both fluorescent and quenchers) acted as acceptors. First, it was shown that if the interaction between the donor/acceptor complexes is of high quality (e.g. biotin-streptavidin) the fluorescence of the europium(III) chelate could be quenched rather efficiently. Furthermore, the quenching based homogeneous non-competitive assay for estradiol had significantly better sensitivity (~67 times) than a corresponding homogeneous competitive assay using the same assay components. Second, if the acceptors were chosen to emit at the emission minima of the terbium(III) chelate, several acceptor emissions could be measured simultaneously without significant cross-talk from other acceptors. Based on these results, the appropriate acceptors were chosen for the dual-parameter assay. The developed homogeneous dual-parameter assay was able to measure both total and free PSA simultaneously using a simple mix and measure protocol. Correlation of this assay to a heterogeneous single parameter assay was excellent (above 0.99 for both) when spiked human plasma samples were used. However, due to the interference of the sample material, the obtained concentrations were slightly lower with the homogeneous than the heterogeneous assay, especially for the free PSA. To conclude, in this work two novel immunoassay principles were developed, which both are adaptable to other analytes. However, the hapten assay requires a rather good antibody with low dissociation rate and high affinity; whereas the dual-parameter assay principle is applicable whenever two immunometric complexes can form simultaneously, provided that the requirements of FRET are fulfilled.

Introducing Continuous Integration for C and C++ Software Development Projects on Linux Platform

Software integration is a stage in a software development process to assemble separate components to produce a single product. It is important to manage the risks involved and being able to integrate smoothly, because software cannot be released without integrating it first. Furthermore, it has been shown that the integration and testing phase can make up 40 % of the overall project costs. These issues can be mitigated by using a software engineering practice called continuous integration. This thesis work presents how continuous integration is introduced to the author's employer organisation. This includes studying how the continuous integration process works and creating the technical basis to start using the process on future projects. The implemented system supports software written in C and C++ programming languages on Linux platform, but the general concepts can be applied to any programming language and platform by selecting the appropriate tools. The results demonstrate in detail what issues need to be solved when the process is acquired in a corporate environment. Additionally, they provide an implementation and process description suitable to the organisation. The results show that continuous integration can reduce the risks involved in a software process and increase the quality of the product as well.

Luminescence-based assay methods in drug discovery

The drug discovery process is facing new challenges in the evaluation process of the lead compounds as the number of new compounds synthesized is increasing. The potentiality of test compounds is most frequently assayed through the binding of the test compound to the target molecule or receptor, or measuring functional secondary effects caused by the test compound in the target model cells, tissues or organism. Modern homogeneous high-throughput-screening (HTS) assays for purified estrogen receptors (ER) utilize various luminescence based detection methods. Fluorescence polarization (FP) is a standard method for ER ligand binding assay. It was used to demonstrate the performance of two-photon excitation of fluorescence (TPFE) vs. the conventional one-photon excitation method. As result, the TPFE method showed improved dynamics and was found to be comparable with the conventional method. It also held potential for efficient miniaturization. Other luminescence based ER assays utilize energy transfer from a long-lifetime luminescent label e.g. lanthanide chelates (Eu, Tb) to a prompt luminescent label, the signal being read in a time-resolved mode. As an alternative to this method, a new single-label (Eu) time-resolved detection method was developed, based on the quenching of the label by a soluble quencher molecule when displaced from the receptor to the solution phase by an unlabeled competing ligand. The new method was paralleled with the standard FP method. It was shown to yield comparable results with the FP method and found to hold a significantly higher signal-tobackground ratio than FP. Cell-based functional assays for determining the extent of cell surface adhesion molecule (CAM) expression combined with microscopy analysis of the target molecules would provide improved information content, compared to an expression level assay alone. In this work, immune response was simulated by exposing endothelial cells to cytokine stimulation and the resulting increase in the level of adhesion molecule expression was analyzed on fixed cells by means of immunocytochemistry utilizing specific long-lifetime luminophore labeled antibodies against chosen adhesion molecules. Results showed that the method was capable of use in amulti-parametric assay for protein expression levels of several CAMs simultaneously, combined with analysis of the cellular localization of the chosen adhesion molecules through time-resolved luminescence microscopy inspection.

The Islandora Digital Humanities Platform

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

Development of agnostic mobile applications with cross-platform cloud computing platforms

Smart phones became part and parcel of our life, where mobility provides a freedom of not being bounded by time and space. In addition, number of smartphones produced each year is skyrocketing. However, this also created discrepancies or fragmentation among devices and OSes, which in turn made an exceeding hard for developers to deliver hundreds of similar featured applications with various versions for the market consumption. This thesis is an attempt to investigate whether cloud based mobile development platforms can mitigate and eventually eliminate fragmentation challenges. During this research, we have selected and analyzed the most popular cloud based development platforms and tested integrated cloud features. This research showed that cloud based mobile development platforms may able to reduce mobile fragmentation and enable to utilize single codebase to deliver a mobile application for different platforms.

Internationalization of IT company: Case study of a Finnish MOOC platform

The interest to small and media size enterprises’ (SMEs) internationalization process is increasing with a growth of SMEs’ contribution to GDP. Internet gives an opportunity to provide variety of services online and reach market niche worldwide. The overlapping of SMEs’ internationalization and online services is the main issue of the research. The most SMEs internationalize according to intuitive decisions of CEO of the company and lose limited resources to worthless attempts. The purpose of this research is to define effective approaches to online service internationalization and selection of the first international market. The research represents single holistic case study of local massive open online courses (MOOCs) platform going global. It considers internationalization costs and internationalization theories applicable to online services. The research includes preliminary screening of the markets and in-depth analysis based on macro parameters of the market and specific characteristics of the customers and expert evaluation of the results. The specific issues as GILT (Globalization, Internationalization, Localization and Translation) approach and Internet-enabled internationalization are considered. The research results include recommendations on international market selection methodology for online services and for effective internationalization strategy development.