Algorithmic Techniques in Gene Expression Processing. From Imputation to Visualization

The amount of biological data has grown exponentially in recent decades. Modern biotechnologies, such as microarrays and next-generation sequencing, are capable to produce massive amounts of biomedical data in a single experiment. As the amount of the data is rapidly growing there is an urgent need for reliable computational methods for analyzing and visualizing it. This thesis addresses this need by studying how to efficiently and reliably analyze and visualize high-dimensional data, especially that obtained from gene expression microarray experiments. First, we will study the ways to improve the quality of microarray data by replacing (imputing) the missing data entries with the estimated values for these entries. Missing value imputation is a method which is commonly used to make the original incomplete data complete, thus making it easier to be analyzed with statistical and computational methods. Our novel approach was to use curated external biological information as a guide for the missing value imputation. Secondly, we studied the effect of missing value imputation on the downstream data analysis methods like clustering. We compared multiple recent imputation algorithms against 8 publicly available microarray data sets. It was observed that the missing value imputation indeed is a rational way to improve the quality of biological data. The research revealed differences between the clustering results obtained with different imputation methods. On most data sets, the simple and fast k-NN imputation was good enough, but there were also needs for more advanced imputation methods, such as Bayesian Principal Component Algorithm (BPCA). Finally, we studied the visualization of biological network data. Biological interaction networks are examples of the outcome of multiple biological experiments such as using the gene microarray techniques. Such networks are typically very large and highly connected, thus there is a need for fast algorithms for producing visually pleasant layouts. A computationally efficient way to produce layouts of large biological interaction networks was developed. The algorithm uses multilevel optimization within the regular force directed graph layout algorithm.

The application of cDNA and tissue microarray methods in the study of human carcinomas

Currently, numerous high-throughput technologies are available for the study of human carcinomas. In literature, many variations of these techniques have been described. The common denominator for these methodologies is the high amount of data obtained in a single experiment, in a short time period, and at a fairly low cost. However, these methods have also been described with several problems and limitations. The purpose of this study was to test the applicability of two selected high-throughput methods, cDNA and tissue microarrays (TMA), in cancer research. Two common human malignancies, breast and colorectal cancer, were used as examples. This thesis aims to present some practical considerations that need to be addressed when applying these techniques. cDNA microarrays were applied to screen aberrant gene expression in breast and colon cancers. Immunohistochemistry was used to validate the results and to evaluate the association of selected novel tumour markers with the outcome of the patients. The type of histological material used in immunohistochemistry was evaluated especially considering the applicability of whole tissue sections and different types of TMAs. Special attention was put on the methodological details in the cDNA microarray and TMA experiments. In conclusion, many potential tumour markers were identified in the cDNA microarray analyses. Immunohistochemistry could be applied to validate the observed gene expression changes of selected markers and to associate their expression change with patient outcome. In the current experiments, both TMAs and whole tissue sections could be used for this purpose. This study showed for the first time that securin and p120 catenin protein expression predict breast cancer outcome and the immunopositivity of carbonic anhydrase IX associates with the outcome of rectal cancer. The predictive value of these proteins was statistically evident also in multivariate analyses with up to a 13.1- fold risk for cancer specific death in a specific subgroup of patients.

A cell spot microarray method for high-throughput biology

High-throughput screening of cellular effects of RNA interference (RNAi) libraries is now being increasingly applied to explore the role of genes in specific cell biological processes and disease states. However, the technology is still limited to specialty laboratories, due to the requirements for robotic infrastructure, access to expensive reagent libraries, expertise in high-throughput screening assay development, standardization, data analysis and applications. In the future, alternative screening platforms will be required to expand functional large-scale experiments to include more RNAi constructs, allow combinatorial loss-of-function analyses (e.g. genegene or gene-drug interaction), gain-of-function screens, multi-parametric phenotypic readouts or comparative analysis of many different cell types. Such comprehensive perturbation of gene networks in cells will require a major increase in the flexibility of the screening platforms, throughput and reduction of costs. As an alternative for the conventional multi-well based high-throughput screening -platforms, here the development of a novel cell spot microarray method for production of high density siRNA reverse transfection arrays is described. The cell spot microarray platform is distinguished from the majority of other transfection cell microarray techniques by the spatially confined array layout that allow highly parallel screening of large-scale RNAi reagent libraries with assays otherwise difficult or not applicable to high-throughput screening. This study depicts the development of the cell spot microarray method along with biological application examples of high-content immunofluorescence and phenotype based cancer cell biological analyses focusing on the regulation of prostate cancer cell growth, maintenance of genomic integrity in breast cancer cells, and functional analysis of integrin protein-protein interactions in situ.

Arrangement of experiments for simulating the effects of elevated temperatures and elevated CO2 levels on field-sown crops in Finland

Selostus: Koejärjestelyt kohonneen lämpötilan ja CO2-tason vaikutusten simuloimiseksi peltokasveilla Suomessa

Magnetic Resonance Experiments with Atomic Hydrogen

In this thesis three experiments with atomic hydrogen (H) at low temperatures T<1 K are presented. Experiments were carried out with two- (2D) and three-dimensional (3D) H gas, and with H atoms trapped in solid H2 matrix. The main focus of this work is on interatomic interactions, which have certain specific features in these three systems considered. A common feature is the very high density of atomic hydrogen, the systems are close to quantum degeneracy. Short range interactions in collisions between atoms are important in gaseous H. The system of H in H₂ differ dramatically because atoms remain fixed in the H₂ lattice and properties are governed by long-range interactions with the solid matrix and with H atoms. The main tools in our studies were the methods of magnetic resonance, with electron spin resonance (ESR) at 128 GHz being used as the principal detection method. For the first time in experiments with H in high magnetic fields and at low temperatures we combined ESR and NMR to perform electron-nuclear double resonance (ENDOR) as well as coherent two-photon spectroscopy. This allowed to distinguish between different types of interactions in the magnetic resonance spectra. Experiments with 2D H gas utilized the thermal compression method in homogeneous magnetic field, developed in our laboratory. In this work methods were developed for direct studies of 3D H at high density, and for creating high density samples of H in H₂. We measured magnetic resonance line shifts due to collisions in the 2D and 3D H gases. First we observed that the cold collision shift in 2D H gas composed of atoms in a single hyperfine state is much smaller than predicted by the mean-field theory. This motivated us to carry out similar experiments with 3D H. In 3D H the cold collision shift was found to be an order of magnitude smaller for atoms in a single hyperfine state than that for a mixture of atoms in two different hyperfine states. The collisional shifts were found to be in fair agreement with the theory, which takes into account symmetrization of the wave functions of the colliding atoms. The origin of the small shift in the 2D H composed of single hyperfine state atoms is not yet understood. The measurement of the shift in 3D H provides experimental determination for the difference of the scattering lengths of ground state atoms. The experiment with H atoms captured in H2 matrix at temperatures below 1 K originated from our work with H gas. We found out that samples of H in H2 were formed during recombination of gas phase H, enabling sample preparation at temperatures below 0.5 K. Alternatively, we created the samples by electron impact dissociation of H₂ molecules in situ in the solid. By the latter method we reached highest densities of H atoms reported so far, 3.5(5)x10¹⁹ cm^-3. The H atoms were found to be stable for weeks at temperatures below 0.5 K. The observation of dipolar interaction effects provides a verification for the density measurement. Our results point to two different sites for H atoms in H2 lattice. The steady-state nuclear polarizations of the atoms were found to be non-thermal. The possibility for further increase of the impurity H density is considered. At higher densities and lower temperatures it might be possible to observe phenomena related to quantum degeneracy in solid.

Software for design of experiments and response modelling of cake ltration applications

Filtration is a widely used unit operation in chemical engineering. The huge variation in the properties of materials to be ltered makes the study of ltration a challenging task. One of the objectives of this thesis was to show that conventional ltration theories are di cult to use when the system to be modelled contains all of the stages and features that are present in a complete solid/liquid separation process. Furthermore, most of the ltration theories require experimental work to be performed in order to obtain critical parameters required by the theoretical models. Creating a good overall understanding of how the variables a ect the nal product in ltration is somewhat impossible on a purely theoretical basis. The complexity of solid/liquid separation processes require experimental work and when tests are needed, it is advisable to use experimental design techniques so that the goals can be achieved. The statistical design of experiments provides the necessary tools for recognising the e ects of variables. It also helps to perform experimental work more economically. Design of experiments is a prerequisite for creating empirical models that can describe how the measured response is related to the changes in the values of the variable. A software package was developed that provides a ltration practitioner with experimental designs and calculates the parameters for linear regression models, along with the graphical representation of the responses. The developed software consists of two software modules. These modules are LTDoE and LTRead. The LTDoE module is used to create experimental designs for di erent lter types. The lter types considered in the software are automatic vertical pressure lter, double-sided vertical pressure lter, horizontal membrane lter press, vacuum belt lter and ceramic capillary action disc lter. It is also possible to create experimental designs for those cases where the variables are totally user de ned, say for a customized ltration cycle or di erent piece of equipment. The LTRead-module is used to read the experimental data gathered from the experiments, to analyse the data and to create models for each of the measured responses. Introducing the structure of the software more in detail and showing some of the practical applications is the main part of this thesis. This approach to the study of cake ltration processes, as presented in this thesis, has been shown to have good practical value when making ltration tests.

Software for design of experiments and response modelling of cake filtration applications

Filtration is a widely used unit operation in chemical engineering. The huge variation in the properties of materials to be ltered makes the study of ltration a challenging task. One of the objectives of this thesis was to show that conventional ltration theories are di cult to use when the system to be modelled contains all of the stages and features that are present in a complete solid/liquid separation process. Furthermore, most of the ltration theories require experimental work to be performed in order to obtain critical parameters required by the theoretical models. Creating a good overall understanding of how the variables a ect the nal product in ltration is somewhat impossible on a purely theoretical basis. The complexity of solid/liquid separation processes require experimental work and when tests are needed, it is advisable to use experimental design techniques so that the goals can be achieved. The statistical design of experiments provides the necessary tools for recognising the e ects of variables. It also helps to perform experimental work more economically. Design of experiments is a prerequisite for creating empirical models that can describe how the measured response is related to the changes in the values of the variable. A software package was developed that provides a ltration practitioner with experimental designs and calculates the parameters for linear regression models, along with the graphical representation of the responses. The developed software consists of two software modules. These modules are LTDoE and LTRead. The LTDoE module is used to create experimental designs for di erent lter types. The lter types considered in the software are automatic vertical pressure lter, double-sided vertical pressure lter, horizontal membrane lter press, vacuum belt lter and ceramic capillary action disc lter. It is also possible to create experimental designs for those cases where the variables are totally user de ned, say for a customized ltration cycle or di erent piece of equipment. The LTRead-module is used to read the experimental data gathered from the experiments, to analyse the data and to create models for each of the measured responses. Introducing the structure of the software more in detail and showing some of the practical applications is the main part of this thesis. This approach to the study of cake ltration processes, as presented in this thesis, has been shown to have good practical value when making ltration tests.

Utilization of Particle Image velocimetry in PPOOLEX condensation experiments

Particle Image Velocimetry, PIV, is an optical measuring technique to obtain velocity information of a flow in interest. With PIV it is possible to achieve two or three dimensional velocity vector fields from a measurement area instead of a single point in a flow. Measured flow can be either in liquid or in gas form. PIV is nowadays widely applied to flow field studies. The need for PIV is to obtain validation data for Computational Fluid Dynamics calculation programs that has been used to model blow down experiments in PPOOLEX test facility in the Lappeenranta University of Technology. In this thesis PIV and its theoretical background are presented. All the subsystems that can be considered to be part of a PIV system are presented as well with detail. Emphasis is also put to the mathematics behind the image evaluation. The work also included selection and successful testing of a PIV system, as well as the planning of the installation to the PPOOLEX facility. Already in the preliminary testing PIV was found to be good addition to the measuring equipment for Nuclear Safety Research Unit of LUT. The installation to PPOOLEX facility was successful even though there were many restrictions considering it. All parts of the PIV system worked and they were found out to be appropriate for the planned use. Results and observations presented in this thesis are a good background to further PIV use.

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.

Surface studies of limestones and dolostones : characterisation using various techniques and batch dissolution experiments with hydrochloric acid solutions

In this thesis, stepwise titration with hydrochloric acid was used to obtain chemical reactivities and dissolution rates of ground limestones and dolostones of varying geological backgrounds (sedimentary, metamorphic or magmatic). Two different ways of conducting the calculations were used: 1) a first order mathematical model was used to calculate extrapolated initial reactivities (and dissolution rates) at pH 4, and 2) a second order mathematical model was used to acquire integrated mean specific chemical reaction constants (and dissolution rates) at pH 5. The calculations of the reactivities and dissolution rates were based on rate of change of pH and particle size distributions of the sample powders obtained by laser diffraction. The initial dissolution rates at pH 4 were repeatedly higher than previously reported literature values, whereas the dissolution rates at pH 5 were consistent with former observations. Reactivities and dissolution rates varied substantially for dolostones, whereas for limestones and calcareous rocks, the variation can be primarily explained by relatively large sample standard deviations. A list of the dolostone samples in a decreasing order of initial reactivity at pH 4 is: 1) metamorphic dolostones with calcite/dolomite ratio higher than about 6% 2) sedimentary dolostones without calcite 3) metamorphic dolostones with calcite/dolomite ratio lower than about 6% The reactivities and dissolution rates were accompanied by a wide range of experimental techniques to characterise the samples, to reveal how different rocks changed during the dissolution process, and to find out which factors had an influence on their chemical reactivities. An emphasis was put on chemical and morphological changes taking place at the surfaces of the particles via X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Supporting chemical information was obtained with X-Ray Fluorescence (XRF) measurements of the samples, and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) and Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) measurements of the solutions used in the reactivity experiments. Information on mineral (modal) compositions and their occurrence was provided by X-Ray Diffraction (XRD), Energy Dispersive X-ray analysis (EDX) and studying thin sections with a petrographic microscope. BET (Brunauer, Emmet, Teller) surface areas were determined from nitrogen physisorption data. Factors increasing chemical reactivity of dolostones and calcareous rocks were found to be sedimentary origin, higher calcite concentration and smaller quartz concentration. Also, it is assumed that finer grain size and larger BET surface areas increase the reactivity although no certain correlation was found in this thesis. Atomic concentrations did not correlate with the reactivities. Sedimentary dolostones, unlike metamorphic ones, were found to have porous surface structures after dissolution. In addition, conventional (XPS) and synchrotron based (HRXPS) X-ray Photoelectron Spectroscopy were used to study bonding environments on calcite and dolomite surfaces. Both samples are insulators, which is why neutralisation measures such as electron flood gun and a conductive mask were used. Surface core level shifts of 0.7 ± 0.1 eV for Ca 2p spectrum of calcite and 0.75 ± 0.05 eV for Mg 2p and Ca 3s spectra of dolomite were obtained. Some satellite features of Ca 2p, C 1s and O 1s spectra have been suggested to be bulk plasmons. The origin of carbide bonds was suggested to be beam assisted interaction with hydrocarbons found on the surface. The results presented in this thesis are of particular importance for choosing raw materials for wet Flue Gas Desulphurisation (FGD) and construction industry. Wet FGD benefits from high reactivity, whereas construction industry can take advantage of slow reactivity of carbonate rocks often used in the facades of fine buildings. Information on chemical bonding environments may help to create more accurate models for water-rock interactions of carbonates.

Data analysis tools and methods for DNA microarray and high-throughput sequencing data

The recent rapid development of biotechnological approaches has enabled the production of large whole genome level biological data sets. In order to handle thesedata sets, reliable and efficient automated tools and methods for data processingand result interpretation are required. Bioinformatics, as the field of studying andprocessing biological data, tries to answer this need by combining methods and approaches across computer science, statistics, mathematics and engineering to studyand process biological data. The need is also increasing for tools that can be used by the biological researchers themselves who may not have a strong statistical or computational background, which requires creating tools and pipelines with intuitive user interfaces, robust analysis workflows and strong emphasis on result reportingand visualization. Within this thesis, several data analysis tools and methods have been developed for analyzing high-throughput biological data sets. These approaches, coveringseveral aspects of high-throughput data analysis, are specifically aimed for gene expression and genotyping data although in principle they are suitable for analyzing other data types as well. Coherent handling of the data across the various data analysis steps is highly important in order to ensure robust and reliable results. Thus,robust data analysis workflows are also described, putting the developed tools andmethods into a wider context. The choice of the correct analysis method may also depend on the properties of the specific data setandthereforeguidelinesforchoosing an optimal method are given. The data analysis tools, methods and workflows developed within this thesis have been applied to several research studies, of which two representative examplesare included in the thesis. The first study focuses on spermatogenesis in murinetestis and the second one examines cell lineage specification in mouse embryonicstem cells.

Scaling analysis of small break experiments in PACTEL

The purpose of this thesis is to study the scalability of small break LOCA experiments. The study is performed on the experimental data, as well as on the results of thermal hydraulic computation performed on TRACE code. The SBLOCA experiments were performed on PACTEL facility situated at LUT. The temporal scaling of the results was done by relating the total coolant mass in the system with the initial break mass flow and using the quotient to scale the experiment time. The results showed many similarities in the behaviour of pressure and break mass flow between the experiments.