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.

Integrating environmental education in primary school education in Tanzania : teachers’ perceptions and teaching practices

The study focuses on primary school teachers’ perceptions of environmental education, its integration into primary school education and teachers’ teaching practices in Tanzania. The thesis is based on empirical research. The theoretical underpinnings of the study are based on Palmer’s (1998) model of environmental education. According to the model, meaningful environmental education should include education about, in or through and for the environment. The study is supported by national and international literature from research done on environmental education and education for sustainable development and policy statements. The study is qualitative in nature, adopting phenomenography and phenomenology as points of departure. The empirical data was collected from four primary schools in Morogoro region in Tanzania. The study sample consisted of 31 primary school teachers. Data was collected through interviews and lesson observations. According to the results of the study, primary school teachers expressed variations in their perceptions of environmental education and education for sustainable development. Most of the teachers focused on the aspect of knowledge acquisition. According to Tanzanian education and training policy, environmental education has to be integrated into all subjects. Although there is environmental education in the primary school curriculum, it is not integrated on an equal footing in all subjects. Some subjects like science, social studies and geography have more environmental content than other subjects. Teachers claim that the approach used to integrate environmental education into the school curriculum was not favoured because many claimed that what is to be taught as environmental education in the various subjects is not shown clearly. As a result, many teachers suggested that to ensure that it is taught properly it should be included in the curriculum as an independent subject or as specific topics. The study revealed that teachers’ teaching practices in integrating environmental education varied from one subject to another. Although most of the teachers said that they used participatory methods, lesson observations showed that they limited themselves to question and answer and group discussion. However, the teachers faced a number of barriers in the teaching of environmental education, some of which include lack of teaching and learning resources, time and large class size. The role of teachers in the implementation of environmental education in developing an environmentally literate citizenry is of great significance. The responsibility of the government in developing a curriculum with clear goals and content, developing teachers’ capacity in the teaching of environmental education and provision of teaching and learning materials needs to be taken seriously by the government in educational plans and programs.

Limits of Meritocracy. How Fields of Study and Gender Segregation Affect Social Stratification in Finland

This dissertation critically reviews the idea of meritocracy from both a theoretical and an empirical perspective. Based on a discussion of classical texts of social philosophy and sociology, it is argued that meritocracy as a concept for social stratification is best compatible with the sociological tradition of status attainment research: both frame social inequality in primarily individualistic terms, centring on the role of ascribed (e.g., gender, social background) and achieved (e.g., educational qualifications) characteristics for determining individuals’ socioeconomic rewards. This theoretical argument introduces the research problem at the core of this dissertation: to what extent can the individualistic conception of social stratification be maintained empirically? Fields of study and their interaction with educational attainment levels play a prominent role in the analysis of this question. Drawing on sociological versions of segmented labour market theory, it is assumed that fields of study may channel individuals into heterogeneous political-economic contexts on the labour market, which potentially modify the socioeconomic benefit individuals derive from their qualification levels. The focus on fields of study may also highlight economic differentials between men and women that derive from the persisting segregation of men’s and women’s occupational and educational specializations rather than direct gender discrimination on the labour market. The quantitative analyses in this dissertation consist of three research articles, which are based primarily on Finnish data, but occasionally extend the view to other European countries. The data sources include register-based macro- and microdata as well as survey data. Article I examines the extent and the patterns of gender segregation within the Finnish educational system between 1981 and 2005. The results show that differences between men’s and women’s field specializations have for the most part remained stable during this period, with particularly high levels of gender segregation observed at lower educational levels. The focus in Article II rests on the effects of gender-segregated fields of study on higher education graduates’ occupational status. It is shown that fields of study matter for accessing professional jobs and avoiding low-skilled positions in Finland: at the early career stage, particularly polytechnic graduates from female-dominated fields are less likely to work in professional positions. Finnish university graduates from male-dominated fields were more likely than their peers with different specializations to work as professionals, yet they also faced a greater risk of being sorted into lowskilled jobs if they failed to make use of this advantage. Article III proceeded to analyse the joint impact of educational qualification levels and fields of study on young adults’ median earnings in Finland between 1985 and 2005. The results show that qualification levels do not confer a consistent benefit in the process of earnings stratification. Advanced qualifications raise median earnings most clearly among individuals specializing in the same field of study. When comparing individuals with different field specializations, on the other hand, higher-level qualifications do not necessarily lead to higher median earnings. Overall, the findings of this dissertation reveal a heterogeneous effect of education for achieving social positions, which challenges individual-centred, meritocratic accounts of social stratification and underlines the problematic lack of structural and institutional dimensions in the dominant account of social status attainment.

Feasibility Study and Market Review of Small Scale Bioenergy Heating Plants in North-West Russia

The paper is focused on feasibility study and market review of small scale bioenergy heating plants in the Russian North-West region. The main focus is effective and competitive usage of low-grade wood for heating purposes in the region. As example of economical feasibility estimation it was chosen the project of reconstruction of small scale boiler plant in Leningrad region that Brofta Oy is planning to implement the nearest time. It includes calculation the payback time with and without interest, the estimation of probable investments, the evaluation of possible risks and research on the potential of small scale heating plants projects. Calculations show that the profitability of this kind of projects is high, but payback time is not very short, because of high level of initial investments. Though, the development of small scale bioenergy heating plants in the region is considered to be the best way to solve the problems of heat supply in small settlements using own biomass resources.

Towards the Best A++ Rating Productivity of Research and Teaching in Finnish Universities

Tämän tutkimusraportin suomenkielinen versio on osoitteessa: http://urn.fi/URN:ISBN:978-951-29-4509-2