Masters of Science / Thermophilic bacteriophages provide simple model systems for understanding biochemical and biological adaptation mechanisms at elevated temperatures. The essential objectives of this study were to characterise the physicochemical properties of select Geobacillus bacteriophages and to sequence their complete genomes. The later objective is believed to be an essential prerequisite to the engineering of a sitespecific integration vector for the stable cloning of exogenous genes into host bacteria. Bacteriophages were assayed at 55oC by the agar overlay technique using dry Karoo soils as source material. A pure strain of bacteriophage called GV1 (for Geobacillus stearothermophilus virus 1) was isolated with the strain Geobacillus stearothermophilus TAU3A1. Plaques were medium sized (2 to 4 mm diameters), with regular contour, clear, and without resistant cells. Host range specificity study showed that GV1 was lytic on thirteen thermophilic Bacillus-like strains tested, including strains of Geobacillus stearothermophilus, G. thermoglucosidasius, B. licheniformis, Anoxybacillus idirlerensis, and A. kuwalawohkensis. However, GV1 failed to infect a mesophilic strain of Bacillus megaterium. TEM analysis of semipurified particles revealed that the phage belongs to the family of Siphoviridae. Morphological characteristics included a long tail of approximately 100 nm and a hexagonal head of approximately 50 nm diameter. Viability and stability studies showed that the phage was best maintained at -80oC in PMN buffer supplemented with 20% glycerol. It was stable at a pH range of 5.5 to 7.5 and MgCl2 and CaCl2 concentration of 0.001 M. hermostability experiments, conducted over short periods of time, showed that GV1 was stable over the temperature range 50 to 75oC, with optimum at 55oC. The study of phage-host interactions showed that phage articles inhibited the initial growth of infected cultures in the first six hours post-infection, presumably while mature phages were released. This was followed by a steady recovery of the growth rate. Atempts to obtain pure particles and to extract and sequence phage DNA were unsuccessful due to the low titer nature of the phage.

In the age of big data, thoughtful management and harmonization of clinical metadata and sample processing in translational research is a critical for effective data generation, integration, and analysis. These steps enable the cutting edge discoveries and enhance overall conclusions that may come from complex multi-omic translational research studies. The focus of my thesis has been on harmonizing the clinical metadata collected as part of the lung Pre Cancer Genome Atlas (PCGA) in addition to expanding the use of banked samples. The lung PCGA study included longitudinal collected samples and data from participants in a high-risk lung cancer-screening program at Roswell Park Comprehensive Cancer Center (Roswell) in Buffalo, NY. Clinical metadata for this study was collected over many years at Roswell and subsets of this data were shared with Boston University Medical Campus (BUMC) for the lung PCGA study. During the study, additional clinical metadata was acquired and shared with BUMC to complement the analysis of genomic profiling of DNA and RNA, as well as protein staining of tissue. With regards to the PCGA study, my thesis has two aims: 1) Curate the clinical metadata from received from Roswell during the PCGA study to enhance both its accessibility to current investigators and collaborators and reproducibility of results 2) Test methods to isolate DNA from remnant samples to expand the use of banked samples for genomic profiling. We hypothesized that the accomplishment of these goals would allow for increased use of the clinical metadata, enhanced reproducibility of the results, and expansion of samples available for DNA sequencing The clinical metadata received from Roswell was consolidated into a singular source that is continually updated and available for export for future research use. These metadata management efforts led to increased use among the members of our laboratory and collaborators working with the lung PCGA cohort. Additionally, the curation of metadata has allowed for improved analysis, reproducibility, and increased awareness of the current inventory of remaining samples. During the process of lung PCGA clinical metadata curation, physical inventory of the remaining samples revealed remnant organic phase samples. Therefore, in addition to my work associated with clinical metadata, the second goal of my thesis focuses on DNA isolation from remnant banked biological samples from the lung PCGA cohort. In the first phase of the lung PCGA, nucleic acid isolation of RNA was intended to be collected exclusively from fresh frozen endobronchial biopsy samples, and formalin-fixed paraffin embedded (FFPE) biopsy samples were to be used for DNA isolation. DNA isolation from the FFPE samples was unsuccessful. However, from the RNA isolation, the remaining organic phase was banked and could potentially serve as a source of DNA. The organic phase of this isolation contained cell debris, proteins, and, as previously mentioned, DNA. We hypothesized that current protocols for organic phase DNA isolation might yield adequate quantities of DNA for genomic profiling. Utilizing immortalized cell culture lines to establish methodology, numerous organic phase DNA isolation protocols were tested. During subsequent validation using the remaining organic phase samples from the lung PCGA cohort, the protocol yielded varied results, suggesting that further optimization to increase DNA purity is required. The ability to isolate DNA from these valuable samples will enhance progress in the lung PCGA study. The aims of this thesis involving curation of clinical metadata and generation of additional DNA samples for DNA profiling has had significant impact on the PCGA study and future expansions of this work.

The aim of this thesis was to determine the authenticity of fruit food for infants using molecular and instrumental methods. In the experimental part, plant DNA isolations from fruit leaves (peaches, apricots, plums and apples) and bananas were performed. Further, DNA was isolated also from five commercial products, and from model mixtures that were prepared in terms of content identical to the commercial mixtures. The isolated DNA was characterized and verified by qPCR with plant DNA-specific ITS2 primers. Three triple primer pairs were selected, and their specificity was evaluated when performing multiplex PCR. This method makes it possible to detect more types of fruit in one reaction, reducing the economic and time requirements for detection. As none of the selected primer pairs were sufficiently specific for the apricot, the evidence from the plum and peach was further realized using duplex PCR. High resolution melting curve analysis was used for better DNA type recognition. Subsequently, agarose gel electrophoresis was performed to analyse the fragment lengths. Furthermore, experiments have been made to identify some specific phenolic substances in commercial and model fruit mixtures by HPLC. Since phenolic substances are degradable under unsuitable storage conditions, the presence of individual compounds was not detected by this method.

Submitted diploma thesis deals with an effect of antimicrobial substances on raw food durability. Nowadays, the raw food has become very required style of nutrition, which is being practised by more and more people. The issue is that this kind of alimentation, especially vegetable raw food is not prepared in heat treatment which is higher than 42–45 °C. The main aim of the thesis is to study antioxidant and antibacterial properties of the herbal and spice extracts and their following implementation into raw desserts to prolong their durability. At the beginning of the theoretical part, there is an analysis of the issue of raw food and the description of micro-organisms, which are mostly occurred on plant materials. Afterwards, selected herbs and spices, and mechanisms of their antimicrobial effects, and also the methods used to test the products are characterised. The experimental part focuses on characterization of ethanol extracts from 13 selected kinds of spices and herbs. The determination of biologically active substance was accomplished, specifically, polyphenols, flavonoids, anthocyanins and their antioxidant properties. Moreover, these extracts were tested to antimicrobial effect towards gram-positive species of germs Micrococcus luteus, gram-negative species of germs Escherichia coli and yeast Candida glabrata. The molecular part of the thesis contains the DNA isolation from the tested raw material, which was enriched of combination of the selected herbs and spices. Change of expiry date of selected samples of raw desserts was monitored. The presence of chosen micro-organisms was confirmed by the PCR method.

The effectiveness of magnetic microparticles in isolation of DNA from Lactobacillus rhamnosus CCM 1825T and DNA from chicken erythrocytes were studied in diploma thesis. Magnetic HEMA based microparticles coated by carboxylic groups and hyperbranched styrene-divinylbenzene particles (IMC AS ČR, Prague, Czech Republic) were used for DNA isolation. Magnetic microparticles Dynabeads® DNA DIRECT™ Universal (Dynal, Norway) based on polystyrene and MPG® Uncoated (PureBiotech, USA) based on magnetic glass were used as a control. The dependence of amount of eluted DNA on concentration of DNA in the base solution and the dependence of amount of eluted DNA on concentration of magnetic microparticles were studied. The affinity of magnetic microparticles to RNA for various concentrations of RNA solution was studied, too. The ability of tested particles to isolate DNA from real samples was validated using milk product Actimel. The quality of isolated DNA of Lactobacillus genus was proved using genus specific PCR.

This work was focused on DNA isolation from cheeses for the use in polymerase chain reaction. First, there was optimised the procedure of homogenisation of different types of cheeses from commercial sources, cell lysis and DNA isolation. DNA was isolated using magnetic microspheres and phenol extraction. It was shown that the DNA was amplified in PCR for domain Bacteria after dilution. Next, there was optimised the procedure of DNA isolation from fresh cheeses and from contaminated fresh cheeses and their pickles. DNA from all samples was amplified in PCR. The presence of DNA of domain Bacteria and yeast DNA was demonstrated. In the last part of the work, there were optimised the preparation of PCR mixtures and bacterial DNA amplification in PCR with primers with clamp (F357-GC and R518). Synthetized PCR products were analysed using DGGE. It was shown that amplicons of DNA isolated from cheeses and pickles differ in positions and numbers. Larger number of bands of different intensities was detected after amplification of DNA isolated from contaminated pickles.

Probiotics products are an integral part of the current market. Products containing probiotics cultures are also cosmetic products. The first part of the study focuses on testing of bacterial survival abilities in the environment of preservatives presented in cosmetic products. Collection strains of genus Lactobacillus were used for these tests. Another part of the study focuses on isolation of bacterial DNA from probiotic cosmetic products Ryor, Yoghurt of Bulgaria, FeminaMed and Lactovit Activit in PCR-ready quality. DNA was isolated by fenol extraction and with magnetic particles. Presence of bacteria was proved by genus and species specific PCRs Lactobacillus. Species specific PCR for identification of Lactobacillus pentosus was optimalized. Species identification was in accord with data declared by producers.

With a development of molecular biology methods it is an increasing interest in new procedures of DNA isolation of high quality. DNA isolation is performed on crude cell lysates by many techniques e.g. phenol extraction, salting out or adsorption on solid phase. Classical DNA isolation, such as phenol extraction is quite complicated and time consuming. New alternative methods of DNA isolation was development using reverse immobilizing DNA to a solid phase. Widespread is the use of the magnetic particles as carriers, which allow the isolation of DNA in high quality directly from crude cells lysates of complex samples. The current method of DNA adsorption onto the surface of magnetic particles does not provided sufficiently pure DNA for analysis of some comlex samples (e.g. food). Some inhibitors of the polymerase chain reaction (PCR) are apparently adsorbed onto the tube wall and the next step of DNA elution leads to their release into the solution and cpnsequent negative effect on quality of DNA (e.g. decreasing of PCR amplification). The principle of the developed procedure is design a device, which utilizes transfer of magnetic particles by paramagnetic newddle from one to another Eppendorf tube, in which further processing of the sample extends. Transfer of magnetic particles with DNA using needle prevents transmission of contaminating impurities. The proposed device allows to realize above-mentioned procedure. The functionality of the device being tested in the isolation of plasmid pUC19 DNA from crude lysates of E. Coli JM 109 (pUC19).

Micromethod of DNA isolation using magnetic particles is one of the modern technological methods used in DNA isolation, and makes the process simpler, more effective and faster. The main aim of this study was to isolate the DNA from various plant (tomato) food products, using different types of magnetic particles. The results were compared and the quantity, purity and the possibility of amplication of the isolated DNA among samples were found to be different. The DNA isolation method using magnetic particles P(HEMA-co-GMA) or HPS B-M-NH2 was shown to be the most effective in achieving the above mentiond parametres. DNAs from the analysed samples of plant food products were isolated in sufficient quantity and quality to be used in the conventional PCR. Differences in the possibility of the amplification of the isolated DNA stored at -20 °C during more than a half year were not found.

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).