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.

GIS assessing traditional and modern agricultural land use/land cover change. A case study 1959-2005: Rekijoki, Somero, SW-Finland

Finland’s rural landscape has gone through remarkable changes from the 1950’s, due to agricultural developments. Changed farming practices have influenced especially traditional landscape management, and modifications in the arable land structure and grasslands transitions are notable. The review of the previous studies reveal the importance of the rural landscape composition and structure to species and landscape diversity, whereas including the relevance in presence of the open ditches, size of the field and meadow patches, topology of the natural and agricultural landscape. This land-change study includes applying remote sensed data from two time series and empirical geospatial analysis in Geographic Information Systems (GIS). The aims of this retrospective research is to detect agricultural landscape use and land cover change (LULCC) dynamics and discuss the consequences of agricultural intensification to landscape structure covering from the aspects of landscape ecology. Measurements of LULC are derived directly from pre-processed aerial images by a variety of analytical procedures, including statistical methods and image interpretation. The methodological challenges are confronted in the process of landscape classification and combining change detection approaches with landscape indices. Particular importance is paid on detecting agricultural landscape features at a small scale, demanding comprehensive understanding of such agroecosystems. Topological properties of the classified arable land and valley are determined in order to provide insight and emphasize the aspect the field edges in the agricultural landscape as important habitat. Change detection dynamics are presented with change matrix and additional calculations of gain, loss, swap, net change, change rate and tendencies are made. Transition’s possibility is computed following Markov’s probability model and presented with matrix, as well. Thesis’s spatial aspect is revealed with illustrative maps providing knowledge of location of the classified landscape categories and location of the dynamics of the changes occurred. It was assured that in Rekijoki valley’s landscape, remarkable changes in landscape has occurred. Landscape diversity has been strongly influenced by modern agricultural landscape change, as NP of open ditches has decreased and the MPS of the arable plot has decreased. Overall change in the diversity of the landscape is determined with the decrease of SHDI. Valley landscape considered as traditional land use area has experienced major transitional changes, as meadows class has lost almost one third of the area due to afforestation. Also, remarkable transitions have occurred from forest to meadow and arable land to built area. Boundaries measurement between modern and traditional landscape has indicated noticeable proportional increase in arable land-forest edge type and decrease in arable land-meadow edge type. Probability calculations predict higher future changes for traditional landscape, but also for arable land turning into built area.

Rakennuksen tietomallien hyödyntämisen edellytykset rakentamisen valmistelu- ja rakennusvaiheessa

Työn tavoitteena oli tutkia, kuinka rakennuksen tietomallia voidaan hyödyntää rakennusliikkeen tuotannonsuunnittelu- ja rakennusvaiheessa sekä mitä sen hyödyntäminen edellyttää rakennuksen tietomallin informaatiosisällöltä. Tavoitteena oli myös tunnistaa tuotannonohjauksen ”pullonkauloja”, joissa kohdin toimintaa voitaisiin tietomallien avulla tehostaa. Työn teoreettisena taustana on aineettoman pääoman merkitys yrityksen kilpailuedun luojana, tietämyksen hallinta ja teknologian hyödyntäminen tietämyksen hallinnassa. Työssä tutkittiin rakennustuotannon johtamista ja tietomallintamisen hyödyntämistä rakentamisessa sekä tietomallien hyödynnettävyyden varmistamista yleisellä tasolla. Työssä tutustuttiin kohdeyritykseen tuotannonohjaukseen ja rakennusvaiheen tietomallien hyödyntämisen nykytilaan. Tuloksina voidaan todeta, että tietomallien tuotannonsuunnittelu- ja rakennusvaiheen tietomallien hyödyntämisen perusedellytys on tietomallien oikeellisuus sekä tietomallien ja perinteisten suunnitteludokumenttien yhdenmukainen tietosisältö. Tämän lisäksi tarvitaan suunnitellut toimintatavat ja toimivat tiedonjakelukanavat sekä kyky hyödyntää tieto- ja viestintäteknologiaa. Tietomallit eivät tämän tutkimuksen perusteella näytä luoneen tarvetta uudenlaisille tuotanto-organisaation roolituksille. Tietomalleilla uskotaan olevan positiivisia vaikutuksia rakennusvaiheen muutostenhallinnassa. Tuotantoorganisaation henkilöillä oli positiivisia odotuksia tietomallien hyödyntämisestä tuotannonohjauksessa. Tietomallien odotetaan tukevan erillisten suunnitelmien muodostamien kokonaisuuksien hahmottamista, rakennusvaiheen osapuolten yhteistyötä ja töiden yhteensovitusta sekä logistiikan suunnittelua ja vaikutusten havainnointia.

Jazzfestival Balver Höhle 1978 : traditional Jazz aus der Bundesrepublik Deutschland

Konserttitaltiointi Balven luolan jazzfestivaaleilta 1978.

Hydraulisen puomin avoimen piirin koordinaattiohjaus paine-ero-tilavuusvirta-approksimaation avulla

Diplomityön tavoitteena oli kehittää mahdollisimman hyvä koordinaattiohjaus. Sen tuli olla jälkiasennettavissa perinteisen ohjauksen rinnalle työkoneisiin, joissa on käytetty sähköohjattuja proportionaaliventtiileitä. Työssä keskityttiin tutkimaan suuntaventtiilin yli vallitsevasta paine-erosta saatavan tilavuusvirtatiedon hyödyntämistä ohjauksessa. Työn ensimmäisessä vaiheessa koordinaattiohjaus toteutettiin käyttäen 0-peittoisilla karoilla ja karan asematakaisinkytkennällä varustettuja suuntaventtiileitä. Hydrauliseen kuristukseen perustuen saatiin paine-erosta käyttökelpoista tilavuusvirtasignaalia ja koordinaattiohjauksen liikeradan seurannassa oli parhaimmillaan vain 3 cm:n virhe koenosturin työliikkeen pituudella. Toisessa vaiheessa käytettiin työkoneissa yleisesti esiintyvää positiivisin karapeitoin varustettua mobiiliventtiilistöä, jossa oli karakohtaiset painekompensaattorit. Painekompensaattoreiden takia ei paine-eron mittaaminen puhtaasti suuntaventtiilin karan yli ollut mahdollista, jonka takia tyydyttiin koordinaattiohjaus toteuttamaan ilman paineen mittausta luottaen painekompensaattoreiden toimintaan. Käytetyn venttiilistön kavitoinninestotoiminnon huomiointi ohjauksessa jäi ratkaisematta ja se ohitettiin vastusvastaventtiileiden avulla. Koordinaattiohjauksen tarkkuus mobiiliventtiileillä oli vaatimaton ja tulosten toistettavuus heikko. Tulosten perusteella todettiin avoimellakin koordinaattiohjauksella olevan mahdollista saavuttaa lupaava tarkkuus ammattikuljettajiin verrattuna. Mobiiliventtiilistöön liittyvät, työn aikana esiinnousseet epäkohdat olisi ratkaistava ennen käytettyjen menetelmien soveltamista käytännön kohteisiin.

Utilization of steelmaking waste materials for production of calcium carbonate (CaCO3)

The steel industry produces, besides steel, also solid mineral by-products or slags, while it emits large quantities of carbon dioxide (CO2). Slags consist of various silicates and oxides which are formed in chemical reactions between the iron ore and the fluxing agents during the high temperature processing at the steel plant. Currently, these materials are recycled in the ironmaking processes, used as aggregates in construction, or landfilled as waste. The utilization rate of the steel slags can be increased by selectively extracting components from the mineral matrix. As an example, aqueous solutions of ammonium salts such as ammonium acetate, chloride and nitrate extract calcium quite selectively already at ambient temperature and pressure conditions. After the residual solids have been separated from the solution, calcium carbonate can be precipitated by feeding a CO2 flow through the solution. Precipitated calcium carbonate (PCC) is used in different applications as a filler material. Its largest consumer is the papermaking industry, which utilizes PCC because it enhances the optical properties of paper at a relatively low cost. Traditionally, PCC is manufactured from limestone, which is first calcined to calcium oxide, then slaked with water to calcium hydroxide and finally carbonated to PCC. This process emits large amounts of CO2, mainly because of the energy-intensive calcination step. This thesis presents research work on the scale-up of the above-mentioned ammonium salt based calcium extraction and carbonation method, named Slag2PCC. Extending the scope of the earlier studies, it is now shown that the parameters which mainly affect the calcium utilization efficiency are the solid-to-liquid ratio of steel slag and the ammonium salt solvent solution during extraction, the mean diameter of the slag particles, and the slag composition, especially the fractions of total calcium, silicon, vanadium and iron as well as the fraction of free calcium oxide. Regarding extraction kinetics, slag particle size, solid-to-liquid ratio and molar concentration of the solvent solution have the largest effect on the reaction rate. Solvent solution concentrations above 1 mol/L NH4Cl cause leaching of other elements besides calcium. Some of these such as iron and manganese result in solution coloring, which can be disadvantageous for the quality of the PCC product. Based on chemical composition analysis of the produced PCC samples, however, the product quality is mainly similar as in commercial products. Increasing the novelty of the work, other important parameters related to assessment of the PCC quality, such as particle size distribution and crystal morphology are studied as well. As in traditional PCC precipitation process, the ratio of calcium and carbonate ions controls the particle shape; a higher value for [Ca2+]/[CO32-] prefers precipitation of calcite polymorph, while vaterite forms when carbon species are present in excess. The third main polymorph, aragonite, is only formed at elevated temperatures, above 40-50 °C. In general, longer precipitation times cause transformation of vaterite to calcite or aragonite, but also result in particle agglomeration. The chemical equilibrium of ammonium and calcium ions and dissolved ammonia controlling the solution pH affects the particle sizes, too. Initial pH of 12-13 during the carbonation favors nonagglomerated particles with a diameter of 1 μm and smaller, while pH values of 9-10 generate more agglomerates of 10-20 μm. As a part of the research work, these findings are implemented in demonstrationscale experimental process setups. For the first time, the Slag2PCC technology is tested in scale of ~70 liters instead of laboratory scale only. Additionally, design of a setup of several hundreds of liters is discussed. For these purposes various process units such as inclined settlers and filters for solids separation, pumps and stirrers for material transfer and mixing as well as gas feeding equipment are dimensioned and developed. Overall emissions reduction of the current industrial processes and good product quality as the main targets, based on the performed partial life cycle assessment (LCA), it is most beneficial to utilize low concentration ammonium salt solutions for the Slag2PCC process. In this manner the post-treatment of the products does not require extensive use of washing and drying equipment, otherwise increasing the CO2 emissions of the process. The low solvent concentration Slag2PCC process causes negative CO2 emissions; thus, it can be seen as a carbon capture and utilization (CCU) method, which actually reduces the anthropogenic CO2 emissions compared to the alternative of not using the technology. Even if the amount of steel slag is too small for any substantial mitigation of global warming, the process can have both financial and environmental significance for individual steel manufacturers as a means to reduce the amounts of emitted CO2 and landfilled steel slag. Alternatively, it is possible to introduce the carbon dioxide directly into the mixture of steel slag and ammonium salt solution. The process would generate a 60-75% pure calcium carbonate mixture, the remaining 25-40% consisting of the residual steel slag. This calcium-rich material could be re-used in ironmaking as a fluxing agent instead of natural limestone. Even though this process option would require less process equipment compared to the Slag2PCC process, it still needs further studies regarding the practical usefulness of the products. Nevertheless, compared to several other CO2 emission reduction methods studied around the world, the within this thesis developed and studied processes have the advantage of existing markets for the produced materials, thus giving also a financial incentive for applying the technology in practice.

Creating risk measurement model to project portfolio management in construction company

This thesis was carried out as a case study of a company YIT in order to clarify the sev-erest risks for the company and to build a method for project portfolio evaluation. The target organization creates new living environment by constructing residential buildings, business premises, infrastructure and entire areas worth for EUR 1.9 billion in the year 2013. Company has noted project portfolio management needs more information about the structure of project portfolio and possible influences of market shock situation. With interviews have been evaluated risks with biggest influence and most appropriate metrics to examine. The major risks for the company were evaluated by interviewing the executive staff. At the same time, the most appropriate risk metrics were considered. At the moment sales risk was estimated to have biggest impact on company‟s business. Therefore project port-folio evaluation model was created and three different scenarios for company‟s future were created in order to identify the scale of possible market shock situation. The created model is tested with public and descriptive figures of YIT in a one-year-long market shock and the impact on different metrics was evaluated. Study was conducted using con-structive research methodology. Results indicate that company has notable sales risk in certain sections of business portfolio.

Impacts of the Belo Monte hydroelectric dam construction on pioneer vegetation formations along the Xingu River, Pará State, Brazil

There is an important pioneer vegetation formation along the Xingu River in the area where the Belo Monte hydroelectric dam is being constructed that is highly adapted to a seasonally fluctuating water levels. The aim of this study was to examine the habitat and flora of the pioneer formations in the Belo Monte area. The area was divided in three sections for study purposes (Reservoir, Low Flow, and Control) that were expected to experience different degrees of impact from the dam project. The calculations of habitat losses were based on satellite imagery classifications, and a total of 111 plots were established in the three areas for vegetation sampling. Habitat losses of the pioneer formations will total 89.7% when the project is fully functional. Forty-five of the 72 recorded species are restricted to single areas. Species richness and diversity were significantly lower in the control area. The completion of the Belo Monte reservoir will result in habitat reductions and will consequently reduce the richness and diversity of pioneer formations. Studies suggest monitoring the populations located in the reduced flow area to determine possible impacts resulting from changes in the regional hydrological cycle caused by the Xingu River dam.

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

Comparison and problems between Traditional and Agile software development methods

In today’s world because of the rapid advancement in the field of technology and business, the requirements are not clear, and they are changing continuously in the development process. Due to those changes in the requirements the software development becomes very difficult. Use of traditional software development methods such as waterfall method is not a good option, as the traditional software development methods are not flexible to requirements and the software can be late and over budget. For developing high quality software that satisfies the customer, the organizations can use software development methods, such as agile methods which are flexible to change requirements at any stage in the development process. The agile methods are iterative and incremental methods that can accelerate the delivery of the initial business values through the continuous planning and feedback, and there is close communication between the customer and developers. The main purpose of the current thesis is to find out the problems in traditional software development and to show how agile methods reduced those problems in software development. The study also focuses the different success factors of agile methods, the success rate of agile projects and comparison between traditional and agile software development.

Expression of c-erbB-2, p53 and c-myc proteins in male breast carcinoma: Comparison with traditional prognostic factors and survival

There are few data evaluating biological markers for men with breast cancer. The purpose of the present study was to analyze the expression of the oncogenes c-erbB-2 and c-myc and of the suppressor gene p53 by immunohistochemical techniques in archival paraffin-embedded tissue blocks of 48 male breast cancer patients, treated at the A.C. Camargo Cancer Hospital, São Paulo, SP, Brazil. The results were compared with clinicopathological prognostic features. Immunopositivity of c-erbB-2, p53 and c-myc was detected in 62.5, 16.7 and 20.8% of the cases analyzed, respectively. Estrogen and progesterone receptors were positive in 75 and 69% of the cases, respectively. Increasing staging was statistically associated with c-erbB-2 (P = 0.04) and weakly related to p53 positivity (P = 0.06). No significant correlation between specific survival rate (determined by the log rank test) and the molecular markers analyzed was found, whereas the number of compromised lymph nodes and advanced TNM (tumor, node, metastasis) staging were associated with diminished survival.

Development of category planning process in construction business

The aim of this thesis was to develop the category planning process in the case company operating in construction industry. As the interest in the field of research has just recently started to emerge towards the benefits of category management and planning, the theoretical background was derived from literature of subjects with a relation to category planning i.e. procurement strategy, purchasing portfolio model, information flow management and cost analysis. The background for the development of category planning process was derived from retail industry, to where the category planning is more researched. The empirical study was executed with mixed method approach: quantitative data of the categories was analyzed and qualitative data was gathered through semi-structured interview and discussions within the case company. As a result, the category planning process was critically analyzed and development proposals addressed for improving the process description. Additionally a tool was developed based on the empirical study to support the category planning process of the case company.