Durante o desenvolvimento deste projeto adotou-se como estratégia o sequenciamento de ESTs, com a finalidade de encontrar mensagens relacionadas com desenvolvimento, metabolismo e principalmente amplificação/politenização em glândulas salivares de Rhynchosciara americana, um díptero (Sciarídeo) que apresenta cromossomos politênicos e amplificação gênica rigidamente regulada ao longo do desenvolvimento larval, tanto neste tecido quanto em outros. Um total de 8193 ESTs foi gerado, estas foram anotadas e categorizadas segundo os termos do Gene Ontology Consortium, proporcionando uma visão geral do status metabólico, como em um Northern eletrônico, de um ponto importante no desenvolvimento desta espécie, quando surgem amplificações gênicas específicas e a glândula salivar necessita secretar as proteínas do casulo. Outros frutos deste seqüenciamento foram a determinação de 91 polimorfismos e a criação de uma tabela de códon usage. Diversos ESTs foram identificados com potencial envolvimento com os endociclos observados neste tecido, destes, RaDup e RaMCM5 foram selecionados para estudo. Suas regiões genômicas foram isoladas e suas localizações cromossômicas foram identificadas, em relação a RaDup, toda a porção codificante de seu mensageiro e 12kb de DNA genômico contendo seu gene foram seqüenciados, revelando sua estrutura gênica. Anticorpos foram produzidos para detectar esta proteína, gerando evidências de sua participação tanto na replicação mitótica como nos endociclos presentes nas glândulas salivares. A localização cromossômica de RaDup é um dado muito interessante, pois pela primeira vez um pufe amplificado é relacionado com um gene regulatório. / In this work EST sequencing was used as strategy to find messages related to development, metabolism and polyteny/amplification in salivary glands of Rhynchosciara americana, a dipteran (Sciaridae) that shows in this tissue giant polytene chromosomes and gene amplification tightly regulated throughout development of the larvae. A total of 8193 EST sequences were generated, annotated and categorized using Gene Ontology Consortium terms, providing a general view of the metabolic status, like an electronic Northern, of an important point in development of the larvae, that shows where specific genes are amplified and the salivary gland needs to secrete the proteins to form the cocoon. Other data include determination of 91 SNPs and a statistic of codon usage. Several ESTs were identified with potential connection to endocicles, from these RaDup and RaMCM5 were selected for further studies. Both chromosomal loci were identified and genomic regions isolated, for RaDup the coding region of its mRNA and 12kb of genomic region were completely sequenced, revealing its gene structure, and antibodies were raised against this protein, making evident data about its involvement in replication in mitotic cells and in endocicles in salivary glands. About the chromosomal locus of RaDup, it becomes very interesting, because for the first time one amplified puff can be related to a regulatory gene.

DuMPLING is a newly developed high-throughput method to study singlecellphenotypes in a pooled and barcoded library using a microfluidicchip. The chip enables parallel biophysical measurements of singlecells, after which in-situ genotyping connects the cells to a certainstrain of the library. The method has been previously applied with abarcoded library, where genotyping was performed on barcodes presenton high copy number plasmids. In this project, I apply and developthe Rolling Circle Amplification method to amplify the signal frombarcodes present on the E. coli chromosome. A small librarycontaining three different chromosomal barcodes is investigated. Veryhigh efficiency of signal generation is achieved for the firstbarcode, good efficiency is achieved for the second, and no signal isachieved for the third. Genotyping is also successfully performed ona strain with two different barcodes present on the chromosome. Thegenotyping method described herein can be applied to screen foradditional barcodes that may be incorporated in a larger library thatin turn can be used to ask important biological questions, forexample using the high throughput DuMPLING method.

The thesis has optimized DNA isolation from pure yoghurt cultures and yoghurt products. The isolated DNA was than subjected to RT-PCR analysis. In the first part of the thesis, DNA isolation from pure yoghurt cultures using a commercial kit was evaluated as more effective than isolation by phenol extraction and magnetic microparticles. To assess the quality and quantity of DNA obtained the spectrophotometric determination of concentration and purity and qPCR were used. DNA of a total of ten pure yoghurt cultures in a quality suitable for PCR was obtained using the commercial kit. In the second part of the thesis, bacterial DNA was isolated from yoghurt products using the same commercial kit with a previous sample washing by lysation solution. DNA of six yoghurt products was isolated this way. Furthermore, two packages of homemade yoghurt were mad of each product, of which DNA was isolated in the same way. DNA obtained from yoghurts was subjected to RT-PCR using six pairs of primers (V3_F a V3_R, V6_F a V6_R, V1_F a V1_R, GroHRM_F a GroHRM_R, UPF a UPR, P1V1 a P2V1) and using the pure cultures DNA as a positive controls. The results confirmed the presence of cultures declared in each yoghurt and their ability to multiply after inoculation into a new medium (milk).

Different species of genus Bifidobacterium are part of human and animal intestinal flora. These bacteria have benefit effects and therefore they are used in foods and pharmaceutical products as probiotics. Cheese is now suitable as a probiotic matrix except yoghurts and fermentated milks. This diploma thesis was focused on optimalization of DNA isolation from bacteria of genus Bifidobacterium. Magnetic microparticles (P(HEMA-co¬-GMA)) were used for DNA isolation in presence of 8% polyethyleneglycol PEG 6000 and 5 M sodium chloride. Phenol extraction weas also used as an isolation method. Isolated DNA was used for amplification in domain, genus and species specific PCRs. Optimized method was tested for detection of bacteria of genus Bifidobacterium in experimentaly prepared probiotic cheeses. These cheeses contained potential probiotic bacteria from Laktoflóra collection. Bacteria were identified into species using species specific PCR. Species Bifidobacterium animalis was identified in all samples of probiotic cheeses.

Among the most common endoparasites who infected small crustacean Daphnia pulex include microsporidia. These intracellular parasites appear to look like a simple single- celled organisms, but their cell structure and lifecycle prove the opposite. Microsporidia are species-specific. Although they infected most organisms of the animal kingdom, they are not yet sufficiently understood. This theses is inderectly followed up to the studies of waterflea Daphnia longispina and microsporidia Berwaldia schaefernai from the dam reservoirs The aim was to analyze closer microsporidian infection on host Daphnia pulex in a forest pool with simple enviroment, which included monitoring time dynamics of Daphnia population and identification infection caused by microsporidia Berwaldia singularis and yet unknown microsporidia labor-marked "HVH". Laboratory work included determination of zooplankton and parasites, calculation of prevalence, laboratory experiments with transmission of microsporidian infection between healthy and infected flea culture or by isolated spores. Genetic analysis of aquatic invertebrates from the forest pool, especially larvae of mite and mosquitos, helped make closer microsporidian life cycle and hypothesis about secondary hosts, for the presence of pathogen DNA using specific DNA...

This thesis deals with the design of a stretcher and compressor systems used for the chirped pulse amplification method for the L4 beamline. The L4 beamline is being developed for the ELI Beamlines project and aims to deliver pulses with peak power of 10 petawatt, central wavelength of 1060 nanometers, pulse duration of 150 femtoseconds and energy of 1500 Joules. Since the laser induced damage threshold and aperture of commercial diffraction gratings is currently a limiting factor in reaching higher peak powers, it was necessary to increase the effective aperture of the compressor using either tiled grating or object-image-grating self tiling methods. These two methods are compared for two compressor configurations using either 1740 ln/mm or 1136 ln/mm diffraction gratings, methods for their alignment are discussed and the selected alignment method is experimentally tested. Moreover, an analytical theory connecting the Seidel aberrations of the stretcher imaging system with the spectral phase deviation of the stretched pulse is presented. This theory is applied to commonly used Banks and Offner stretcher designs and it is demonstrated how it can be employed for the suppression of residual spectral phase of compressed pulses. Next, the design of the stretcher for the L4 beamline based on this theory is...

DNA-encoded peptide libraries are the basis for in vitro selection methods that use various biological systems (phage display; yeast display; mRNA display). Despite the great success of these selection methods, their obvious disadvantage is the limited number of building blocks, which consist of only twenty proteinogenic amino acids. The involvement of other non- proteinogenic amino acids and other building blocks could significantly expand the range of possible applications of these selection methods. For example, the introduction of chemical modifications in amino acid side chains in such libraries would allow the effective study of post-translational modifications (phosphorylation, acylation, glycosylation, methylation, etc.) in living organisms. The aim of this work was to develop a method for preparation of a fully synthetic DNA encoded library of peptides. The basic steps for the preparation were the chemical synthesis of the peptide and associated enzymatic synthesis of encoding DNA. Compatibility of chemical reactions with DNA is essential for the synthesis of DNA-encoded peptide libraries. Because the final acidic deprotection of the side chains in the peptide is not compatible with DNA, two approaches have been tested to overcome this problem. The first was an attempt to develop finer...

<div>Pathogen identification at the point of use is critical in preventing disease transmission and enabling prompt treatment. Current rapid diagnostic tests suffer from high rates of false negatives because they are not capable of detecting the inherently low concentrations of pathogens found in early stages of infection or in environmental reservoirs. The gold standard method for timely pathogen identification is a nucleic acid amplification assay called polymerase chain reaction. Although polymerase chain reaction is extremely sensitive and specific, it requires expensive laboratory equipment and trained personnel to perform the sample preparation, cyclical heating, and amplicon analysis. Isothermal nucleic acid amplification assays are better suited for field use because they operate at a single temperature and are robust to common sample matrix inhibitors. Thus, there is a need to translate isothermal amplification assays to the point of use for rapid and sensitive detection of pathogens in complex samples.</div><div><br></div><div>Here, I outline an approach to bring laboratory-based sample preparation, assays, and analyses to the point of use via portable platforms. First, I characterize a loop-mediated isothermal amplification assay and combine it with lateral flow immunoassay for simple, colorimetric interpretation of results. Next, I optimize an ambient-temperature reagent storage method to eliminate cold-chain requirements and precision pipetting steps. I then incorporate loop-mediated isothermal amplification, lateral flow immunoassay, and reagent drying into two different integrated paperfluidic platforms and demonstrate their ability to separately detect bacteria and viruses in complex sample matrices. Finally, I couple loop-mediated isothermal amplification with particle diffusometry to optically determine pathogen presence by tracking the Brownian motion of particles added to an amplified sample. The combined loop-mediated isothermal amplification and particle diffusometry method is first characterized on a microscope and then translated to a smartphone-based platform. Each of these portable platforms are broadly applicable because they can be easily modified for identification of other pathogens at the point of use.</div>

No description available.

Gliomas are brain tumors arising from neuroglia. In most cases astrocytic or oligodendroglial component is the main element of the tumor. Non-random chromosomal abberations are found in tumor cells as was revealed previously. The aim of this study was a fluorescence in-situ hybridisation analysis (FISH) of tissue samples obtained during neurosurgical procedures, determine the frequence of selected chromosomal abberations, further correlation with morphological and clinical data and statistical analysis of the results. During six years 264 tissue samples were gained in which FISH with defined probes was performed. The acquired results were compared with histological analysis and selected clinical data (age, Karnofsky score, extent of resection, overall survival). The whole series was divided into 7 groups by tumor type for further statistical analysis. In every group median and mean survival time was calculated, Kaplan-Meier analysis was focused on influence of selected parameters to overall survival. In some categories Cox regression model was created to achieve a hazard ratio of selected parameters. In WHO Grade II and III tumors the risk of malignant progression and tumor upgrading is significantly higher in comparison with samples where specific abberations were not found (EGFR amplification, CDKN2A and...