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.

Stochastic approximation methods in stochastic optimization

Stochastic approximation methods for stochastic optimization are considered. Reviewed the main methods of stochastic approximation: stochastic quasi-gradient algorithm, Kiefer-Wolfowitz algorithm and adaptive rules for them, simultaneous perturbation stochastic approximation (SPSA) algorithm. Suggested the model and the solution of the retailer's profit optimization problem and considered an application of the SQG-algorithm for the optimization problems with objective functions given in the form of ordinary differential equation.

Resonant tunneling effects in semiconductor heterostructures

The thesis is devoted to a theoretical study of resonant tunneling phenomena in semiconductor heterostructures and nanostructures. It considers several problems relevant to modern solid state physics. Namely these are tunneling between 2D electron layers with spin-orbit interaction, tunnel injection into molecular solid material, resonant tunnel coupling of a bound state with continuum and resonant indirect exchange interaction mediated by a remote conducting channel. A manifestation of spin-orbit interaction in the tunneling between two 2D electron layers is considered. General expression is obtained for the tunneling current with account of Rashba and Dresselhaus types of spin-orbit interaction and elastic scattering. It is demonstrated that the tunneling conductance is very sensitive to relation between Rashba and Dresselhaus contributions and opens possibility to determine the spin-orbit interaction parameters and electron quantum lifetime in direct tunneling experiments with no external magnetic field applied. A microscopic mechanism of hole injection from metallic electrode into organic molecular solid (OMS) in high electric field is proposed for the case when the molecules ionization energy exceeds work function of the metal. It is shown that the main contribution to the injection current comes from direct isoenergetic transitions from localized states in OMS to empty states in the metal. Strong dependence of the injection current on applied voltage originates from variation of the number of empty states available in the metal rather than from distortion of the interface barrier. A theory of tunnel coupling between an impurity bound state and the 2D delocalized states in the quantum well (QW) is developed. The problem is formulated in terms of Anderson-Fano model as configuration interaction between the carrier bound state at the impurity and the continuum of delocalized states in the QW. An effect of this interaction on the interband optical transitions in the QW is analyzed. The results are discussed regarding the series of experiments on the GaAs structures with a -Mn layer. A new mechanism of ferromagnetism in diluted magnetic semiconductor heterosructures is considered, namely the resonant enhancement of indirect exchange interaction between paramagnetic centers via a spatially separated conducting channel. The underlying physical model is similar to the Ruderman-Kittel-Kasuya-Yosida (RKKY) interaction; however, an important difference relevant to the low-dimensional structures is a resonant hybridization of a bound state at the paramagnetic ion with the continuum of delocalized states in the conducting channel. An approach is developed, which unlike RKKY is not based on the perturbation theory and demonstrates that the resonant hybridization leads to a strong enhancement of the indirect exchange. This finding is discussed in the context of the known experimental data supporting the phenomenon.

To Divide or Not to Divide; MicroRNAs and Small Compounds as Modulators of Mitosis

Mitosis is under the stringent quality control of the spindle assembly checkpoint (SAC). However, in cancer cells this control can fail, leading to excessive cellular proliferation and ultimately to the formation of a tumor. Novel cancer cell selective therapies are needed to stop the uncontrolled cell proliferation and tumor growth. The aim of the research presented in this thesis was to identify microRNAs (miRNAs) that could play a role in cancer cell proliferation as well as low molecular weight (LMW) compounds that could interfere with cell division. The findings could be used to develop better cancer diagnostics and therapies in the future. First, a high-throughput screen (HTS) was performed to identify LMW compounds that possess a similar chemical interaction field as rigosertib, an anti-cancer compound undergoing clinical trials. A compound termed Centmitor-1 was discovered that phenocopied the cellular impact of rigosertib by affecting the microtubule dynamics. Next, another HTS aimed at identifying compounds that would target the Hec1 protein, which mediates the interaction between spindle microtubules and chromosomes. Perturbation of this connection should prevent cell division and induce cell death. A compound termed VTT-006 was discovered that abrogated mitosis in several cell line models and exhibited binding to Hec1 in vitro. Lastly, using a cell-based HTS two miRNAs were identified that affected cancer cell proliferation via Aurora B kinase, which is an important mitotic regulator. MiR-378a-5p was found to indirectly suppress the production of the kinase whereas let-7b showed direct binding to the 3’UTR of Aurora B mRNA and repressed its translation. The miRNA-mediated perturbation of Aurora B induced defects in mitosis leading to abnormal chromosome segregation and induction of aneuploidy. The results of this thesis provide new information on miRNA signaling in cancer, which could be utilized for diagnostic purposes. Moreover, the thesis introduces two small compounds that may benefit future drug research.

Uncertainty of Efficiency Measurements in Electric Drives

The two central goals of this master's thesis are to serve as a guidebook on the determination of uncertainty in efficiency measurements and to investigate sources of uncertainty in efficiency measurements in the field of electric drives by a literature review, mathematical modeling and experimental means. The influence of individual sources of uncertainty on the total instrumental uncertainty is investigated with the help of mathematical models derived for a balance and a direct air cooled calorimeter. The losses of a frequency converter and an induction motor are measured with the input-output method and a balance calorimeter at 50 and 100 % loads. A software linking features of Matlab and Excel is created to process measurement data, calculate uncertainties and to calculate and visualize results. The uncertainties are combined with both the worst case and the realistic perturbation method and distributions of uncertainty by source are shown based on experimental results. A comparison of the calculated uncertainties suggests that the balance calorimeter determines losses more accurately than the input-output method with a relative RPM uncertainty of 1.46 % compared to 3.78 - 12.74 % respectively with 95 % level of confidence at the 93 % induction motor efficiency or higher. As some principles in uncertainty analysis are open to interpretation the views and decisions of the analyst can have noticeable influence on the uncertainty in the measurement result.