Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.

A Model-Based Development and Verification Framework for Distributed System-on-Chip Architecture

The capabilities and thus, design complexity of VLSI-based embedded systems have increased tremendously in recent years, riding the wave of Moore’s law. The time-to-market requirements are also shrinking, imposing challenges to the designers, which in turn, seek to adopt new design methods to increase their productivity. As an answer to these new pressures, modern day systems have moved towards on-chip multiprocessing technologies. New architectures have emerged in on-chip multiprocessing in order to utilize the tremendous advances of fabrication technology. Platform-based design is a possible solution in addressing these challenges. The principle behind the approach is to separate the functionality of an application from the organization and communication architecture of hardware platform at several levels of abstraction. The existing design methodologies pertaining to platform-based design approach don’t provide full automation at every level of the design processes, and sometimes, the co-design of platform-based systems lead to sub-optimal systems. In addition, the design productivity gap in multiprocessor systems remain a key challenge due to existing design methodologies. This thesis addresses the aforementioned challenges and discusses the creation of a development framework for a platform-based system design, in the context of the SegBus platform - a distributed communication architecture. This research aims to provide automated procedures for platform design and application mapping. Structural verification support is also featured thus ensuring correct-by-design platforms. The solution is based on a model-based process. Both the platform and the application are modeled using the Unified Modeling Language. This thesis develops a Domain Specific Language to support platform modeling based on a corresponding UML profile. Object Constraint Language constraints are used to support structurally correct platform construction. An emulator is thus introduced to allow as much as possible accurate performance estimation of the solution, at high abstraction levels. VHDL code is automatically generated, in the form of “snippets” to be employed in the arbiter modules of the platform, as required by the application. The resulting framework is applied in building an actual design solution for an MP3 stereo audio decoder application.

Suunnistussovelluksen suunnittelu Windows Phonelle

Tässä työssä luotiin suunnistussovelluksen demo Windows Phone 8:lle. Työn tavoite oli tehdä sovellus, jonka kanssa suunnistaminen tuntuisi mahdollisimman samalta, kuin oikeakin suunnistus. Työssä kiinnitettiin huomiota lähinnä sovelluksen tekniseen toteutukseen sekä paikannuksen riittävään tarkkuuteen. Työssä vertaillaan eri mobiilialustoja, selainten tietokantaratkaisuja, erilaisia selaimen ja serverin tietokantojen synkronointimenetelmiä sekä olemassaolevia suunnistussovelluksia. Työssä käytettiin indeksitietokantaa, jQuery mobilea ja Cordovaa sekä JSON:ia tietokantojen synkronointiin. Työssä tehty demo on lupaava esitys siitä, miten suunnistustaitojaan voisi harjoittaa tekniikkaa apuna käyttäen. Lopuksi työssä käydään vielä läpi sitä, mitä tällaiseen palveluun voisi toteuttaa työssä toteutetun demon lisäksi.

Upconverting diagnostics: Multiplex assays utilizing upconversion luminescence technology

Conventional diagnostics tests and technologies typically allow only a single analysis and result per test. The aim of this study was to propose robust and multiplex array-inwell test platforms based on oligonucleotide and protein arrays combining the advantages of simple instrumentation and upconverting phosphor (UCP) reporter technology. The UCPs are luminescent lanthanide-doped crystals that have a unique capability to convert infrared radiation into visible light. No autofluorescence is produced from the sample under infrared excitation enabling the development of highly sensitive assays. In this study, an oligonucleotide array-in-well hybridization assay was developed for the detection and genotyping of human adenoviruses. The study provided a verification of the advantages and potential of the UCP-based reporter technology in multiplex assays as well as anti-Stokes photoluminescence detection with a new anti- Stokes photoluminescence imager. The developed assay was technically improved and used to detect and genotype adenovirus types from clinical specimens. Based on the results of the epidemiological study, an outbreak of adenovirus type B03 was observed in the autumn of 2010. A quantitative array-in-well immunoassay was developed for three target analytes (prostate specific antigen, thyroid stimulating hormone, and luteinizing hormone). In this study, quantitative results were obtained for each analyte and the analytical sensitivities in buffer were in clinically relevant range. Another protein-based array-inwell assay was developed for multiplex serodiagnostics. The developed assay was able to detect parvovirus B19 IgG and adenovirus IgG antibodies simultaneously from serum samples according to reference assays. The study demonstrated that the UCPtechnology is a robust detection method for diverse multiplex imaging-based array-inwell assays.

Altmetrics in practice: How are institutional repositories using altmetrics today?

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

How to take advantage of ORCID in institutional repositories

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

Office File Formats – What’s to be Done?

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

Integrating CRIS and repository – an overview of the situation in Finland and in three other Nordic countries

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

Evolver: The Evolution of The Extended EPrints Ecosystem

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

Repository Rant -- Digital Scavenging

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

Ingest into the Digital Preservation Network: Standard Pipelines

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

Preserve your content with LOCKSS-O-Matic

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

Theses and Dissertations Digital Library: Ten years of Open Access and Open Archives in Brazil

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

Digital Preservation Tools, Practices, and Policies in Islandora

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

Promoting Interoperability and Services Through Shared Identifiers

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