The thesis based on analysis of international community peacebuilding policy and consequent reaction by local actors assesses the influence of this interaction onto the liberal peace and changes in missions' operation. The extent of local ownership and the real agency of local actors is then dependent on the time of mission occurrence, power related interests of international community and the force and accessibility of structures that the international community tries to influence. Key words: peacebuilding, hybridization, local ownership, Bosnia and Herzegovina, Afghanistan, Sierra Leone

The emerging potential of miRNA molecules as diagnostic biomarkers calls for the development of a new quantification method. Current approaches usually require time-consuming and costly miRNA isolation for proper sample analysis. In this thesis, a new, isolation-free, oligonucleotide- modified gold nanoparticle (AuNP/DNA) system is proposed and designed for miRNA detection and quantification in living cells. This cascade, entropy-driven, and enzyme-free amplification system provides fluorescence signal upon selective interaction with the target miRNA. For this purpose, citrate-stabilized gold nanoparticles were synthesized, and their diameters were determined by dynamic light scattering and transmission electron microscopy. The AuNP/DNA conjugates were prepared following a recently published "freezing method". Their reaction kinetics with the target miRNA and selectivity to various miRNAs were compared with those of an analogous DNA system without AuNPs in a series of fluorescence measurements. Furthermore, stability experiments in glutathione environment were conducted, as well as DNA electrophoresis, demonstrating the mechanistic aspects of the reaction. The reaction yields and selectivity to target miRNA of 42.31 ± 2.91 nm AuNP/DNA constructs, containing approximately 25 DNA complexes per AuNP,...

(in English): The aim of this diploma thesis is to analyse the Dominican identity in the works of the Dominican author Marcio Veloz Maggiolo. At first, the thesis briefly summarizes the evolution of the Dominican literature and mentions its most significant figures. Secondly, it deals with the evolution of the dominicaness from the colonial period up to the present and explains the reasons of the rooted antihaitianism. The second part consists of a detailed analysis of the novels The Diffuse Biography of Sombra Castañeda and The Accordion Man by Marcio Veloz Maggiolo, emphasizing the symbols of the official government's discourse, and also a more opened perspective of the dominicaness.

Rapidly diagnosing the precise drug resistance present in sepsis-inducing bacteria is a continuing need to maintain the efficacy of our medical systems. Diagnostics currently being developed for such scenarios are either sensitive or rapid, but not both. Sequence-specific single DNA molecule analysis could fill this gap if it could be adapted to work on smaller targets, similar to those produced by classical biological methods. In this work, I demonstrate that immobilized ssDNA in the appropriate hybridization buffer can rapidly pull its complementary sequence out of solution. I also demonstrate that such systems in a microfluidic chip can be used to capture bacterial plasmids as a step toward subsequent multiplexed analysis. Finally, I demonstrate that a 120 bp double stranded polynucleotide with an overhanging single stranded 25 bp probe sequence can be modified with multiple fluorophores and used to label captured targets in a sequence-specific manner. This system shows that it is possible to label bacterial plasmids in a manner that can bridge the technological gap between single molecule counting and small oligonucleotide targets. Such a system can achieve lower limits of detection for clinically relevant samples while maintaining rapid processing times.

Phylogenetics is crucial in the study of evolutionary processes and the determination of appropriate conservation units, and often these efforts are complicated by hybridization and introgression. Aliciella subsection Subnuda consists of seven species of herbaceous plants occurring in Utah and the Four Corners region of North America. Previous molecular and morphological work left relationships in the subsection unresolved. Here, we use comparative DNA sequencing of ITS and cpDNA regions and RAD-seq data to clarify phylogenetic relationships and examine the role of hybridization in the subsection. We construct haplotype and nucleotype networks from the cpDNA and ITS sequence matrices and compare nuclear and chloroplast phylogenies to identify multiple putative chloroplast capture events. The RAD-seq maximum likelihood phylogeny robustly resolves relationships between six clades, supportive of merging of two species. We employ STRUCTURE and HyDe on the RAD-seq data to evaluate the influence of hybridization within the subsection. The HyDe results provide evidence of hybridization among and between all species in the subsection. Our study robustly resolves relationships in Aliciella subsection Subnuda and provides a framework for discussing its speciation despite a history of hybridization and introgression.

This dissertation is concerned with the unconstrained global optimization of nonlinear problems. These problems are not easy to solve because of the multiplicity of local and global minima. In this dissertation, we first study the pattern search method for local optimization. We study the pattern search method numerically and provide a modification to it. In particular, we design a new pattern search method for local optimization. The new pattern search improves the efficiency and reliability of the original pattern search method. We then designed two simulated annealing algorithms for global optimization based on the basic features of pattern search. The new methods are therefore hybrid. The first hybrid method is the hybrid of simulated annealing and pattern search. This method is denoted by MSA. The second hybrid method is a combination of MSA and the multi-level single linkage method. This method is denoted by SAPS. The performance of MSA and SAPS are reported through extensive experiments on 50 test problems. Results indicate that the new hybrids are efficient and reliable.

The thesis based on analysis of international community peacebuilding policy and consequent reaction by local actors assesses the influence of this interaction onto the liberal peace and changes in missions' operation. In accordance with O. Richmond the conclusion of post-liberal peace coming to the fore is accepted if only in contextually based forms. The extent of local ownership and the real agency of local actors is then dependent on the time of mission occurrence, power related interests of international community and the force and accessibility of structures that the international community tries to influence. Key words: peacebuilding, hybridization, local ownership, Bosnia and Herzegovina, Afghanistan, Sierra Leone

Modern high-throughput technologies like DNA microarrays are powerful tools that are widely used in biomedical research. They target a variety of genomics applications ranging from gene expression profiling over DNA genotyping to gene regulation studies. However, the recent discovery of false positives among prominent research findings indicates a lack of awareness or understanding of the non-biological factors negatively affecting the accuracy of data produced using these technologies. The aim of this thesis is to study the origins, effects and potential correction methods for selected methodical biases in microarray data. The two-species Langmuir model serves as the basal physicochemical model of microarray hybridization describing the fluorescence signal response of oligonucleotide probes. The so-called hook method allows to estimate essential model parameters and to compute summary parameters characterizing a particular microarray sample. We show that this method can be applied successfully to various types of microarrays which share the same basic mechanism of multiplexed nucleic acid hybridization. Using appropriate modifications of the model we study RNA quality and sequence effects using publicly available data from Affymetrix GeneChip expression arrays. Varying amounts of hybridized RNA result in systematic changes of raw intensity signals and appropriate indicator variables computed from these. Varying RNA quality strongly affects intensity signals of probes which are located at the 3\'' end of transcripts. We develop new methods that help assessing the RNA quality of a particular microarray sample. A new metric for determining RNA quality, the degradation index, is proposed which improves previous RNA quality metrics. Furthermore, we present a method for the correction of the 3\'' intensity bias. These functionalities have been implemented in the freely available program package AffyRNADegradation. We show that microarray probe signals are affected by sequence effects which are studied systematically using positional-dependent nearest-neighbor models. Analysis of the resulting sensitivity profiles reveals that specific sequence patterns such as runs of guanines at the solution end of the probes have a strong impact on the probe signals. The sequence effects differ for different chip- and target-types, probe types and hybridization modes. Theoretical and practical solutions for the correction of the introduced sequence bias are provided. Assessment of RNA quality and sequence biases in a representative ensemble of over 8000 available microarray samples reveals that RNA quality issues are prevalent: about 10% of the samples have critically low RNA quality. Sequence effects exhibit considerable variation within the investigated samples but have limited impact on the most common patterns in the expression space. Variations in RNA quality and quantity in contrast have a significant impact on the obtained expression measurements. These hybridization biases should be considered and controlled in every microarray experiment to ensure reliable results. Application of rigorous quality control and signal correction methods is strongly advised to avoid erroneous findings. Also, incremental refinement of physicochemical models is a promising way to improve signal calibration paralleled with the opportunity to better understand the fundamental processes in microarray hybridization.

In situ hybridization (ISH) has played a crucial role in developing a spatial transcriptomic understanding of emerging model organisms in the past, but advancing high-throughput RNA-sequencing (RNA-seq) technology has pushed this method into the shadows, leading to a loss of data resolution. This shift in research towards the exclusive use of RNA-seq neglects essential considerations for transcriptomic studies including the spatial and temporal expression of transcripts, available budget, experimental design needs, and validation of data. A synergy of spatiotemporal transcriptomic techniques is needed, using the bulk and unbiased analysis of RNA-seq and the visual validation and spatiotemporal resolution of ISH. Integration of this synergistic approach can improve our molecular understanding of non-model organisms and establish the background data needed for advancing research techniques. A prime example lies within an emerging model of the marine science and symbiosis fields, where I present a case study on a threatened coral reef keystone – the cnidarian-dinoflagellate symbiosis. Establishing a whole-mount ISH protocol for the emerging cnidarian model Aiptasia (sea anemone) will help future studies reveal the gene regulation underpinning the establishment, persistence, and breakdown of this complex symbiotic relationship.

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non- selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32p or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).