Indiana University-Purdue University Indianapolis (IUPUI) / The single cell gel electrophoresis assay (comet assay) is a useful tool for monitoring individuals who may be at risk of DNA damage and the ensuing process of carcinogenesis or other disease states. Leukocytes in blood samples provide a means of obtaining cells for use in the comet assay. However instances may arise when samples must be stored for later analysis. The present study investigated the effects of storage conditions on DNA damage in the form of strand breaks and oxidized bases in rat and human leukocytes using the comet assay. Whole blood and buffy coat samples were stored at room temperature or 4ºC for 1, 2, 24, and 48 hours or cryopreserved at -80ºC for 1 day and 1, 2, 3, and 4 weeks. The results show that the time of storage is limited if the whole blood or buffy coat samples are stored at room temperature or 4ºC. However, if cryopreserved using glycerol or DMSO as the cryoprotectant, the samples may be stored for at least 4 weeks without DNA strand breaks or oxidative damage deviating significantly from the fresh samples.

Made available in DSpace on 2014-08-20T13:44:42Z (GMT). No. of bitstreams: 1 dissertacao_lilian_tunes.pdf: 3629520 bytes, checksum: c63e3ee7e985806eda7edeb195ebba76 (MD5) Previous issue date: 2009-01-17 / The barley culture (Hordeum vulgare L.) for the beer production it is constituting in one of the more promising and safe agricultural businesses of the country, because it already comes linked to the commercialization process, by previous contract between the producer and the malt industry. In view of this and of the economical answer in relation to other winter cultures, many producers show interest in the inclusion of the barley in their production systems. The objective of this work was to evaluate physiologic attribute of quality about different harvesting periods of barley seeds and during the storage. The seeds used were of the cultivars MN 721 and Scarlett. Harvesting was accomplished when the plants were with 118, 129 and 140 days after the seeding. The seeds were dried in an oven with forced air circulation, until reaching 13% of moisture and then, stored in a cold room and out of the room. After harvesting different methods were tested for overcoming dormancy. The seeds were put for the germination tests, seedling length, mass dry weight, tetrazolium, accelerated aging, seed emergence, electrical conductivity, weight of a thousand seeds, hectolitric weight and also isoenzimatic diferentitation [malato desidrogenase (MDH), alcohol desidrogenase (ADH), acid fosfatase (FAC), glutamate oxalacetato transaminase (GOT) and esterase (EST)]. The results obtained showed that the intensity of dormancy and harvest periods interferes, directly, in the efficiency of the treatments used for overcoming dormancy. Harvesting should be accomplished when the seeds reach the humidity between 26 and 18%. The weight of a thousand seeds influences in a positive way, demonstrating that heavier seeds presented better quality than the light ones. The hectolitric weight, germination and viability tend to decrease as the harvesting period is delayed. There are variations in the pattern of expression of the enzymes EST, ACP, MDH, ADH and GOT between the seeds and seedlings. In the germination process the enzyme EST showed difference between cultivars MN 721 and Scarlett, however it had small variation in the different harvesting periods. In the germination process the enzyme GOT showed variation in the intensity of bands in the different moisture percentages. During storage it was evaluated the physiologic quality of the seeds, at the 3rd and the 6th months, analyzing two atmospheres (cold room and natural conditions). The results were that, the capacity of conservation of barley seeds it is just according to its initial quality. / A cultura de cevada (Hordeum vulgare L.) para a produção de cerveja vem se constituindo em um dos negócios agrícolas mais promissores e seguros do país, pois já vem vinculado ao processo de comercialização, mediante contrato prévio entre o produtor e a indústria de malte. Em vista disto e da resposta econômica em relação a outras culturas de inverno, muitos produtores mostram interesse na inclusão da cevada em seus sistemas de produção. O objetivo do trabalho foi avaliar atributos fisiológicos de qualidade de sementes de cevada sobre diferentes épocas de colheita e durante o armazenamento. Foram utilizadas sementes das cultivares MN 721 e Scarlett. As sementes foram secadas em estufa com circulação de ar forçado, até atingir 13% de umidade e então, armazenadas em câmara fria e seca e ambiente natural. Após a colheita foram testados diferentes métodos para a superação de dormência. As sementes foram avaliadas pelos testes de germinação, comprimento de plântulas, massa seca, tetrazólio, envelhecimento acelerado, emergência de plântulas, condutividade elétrica, peso de mil sementes, peso hectolítrico e também diferenciação isoenzimática [malato desidrogenase (MDH), álcool desidrogenase (ADH), fosfatase ácida (FAC), glutamato oxalacetato transaminase (GOT) e esterase (EST)]. Dos resultados obtidos, a intensidade da dormência e época de colheita das sementes de cevada interfere, diretamente, na eficiência dos tratamentos utilizados para sua superação. A colheita da cevada deve ser realizada quando as sementes atingirem a umidade entre 26 e 18%. O peso de mil das sementes influi de maneira positiva, demonstrando que sementes mais pesadas apresentaram melhor desempenho que as leves. O peso hectolítrico, a germinação e a viabilidade tendem a decrescer com o processo de retardamento da colheita. Há variações no padrão de expressão das enzimas EST, ACP, MDH, ADH e GOT entre as sementes e as plântulas. No processo de germinação a enzima EST apresentaram diferença entre as cultivares MN 721 e a Scarlett, no entanto teve pequena variação nos diferentes períodos de colheita. No processo de germinação a enzima GOT apresentou variação na intensidade de bandas nas diferentes percentagens de umidade. Durante o armazenamento foram avaliados a qualidade fisiológicas das sementes, por um período de 3 e seis meses, analisando dois ambientes (câmara fria e seca e ambiente em condições naturais). Os resultados encontrados foram que, a capacidade de conservação de sementes de cevada relaciona-se com a sua qualidade inicial.

Indiana University-Purdue University Indianapolis (IUPUI) / Short tandem repeat (STR) analysis is currently the most common method for processing biological forensic evidence. STRs are highly polymorphic and allow for a strong statistical power of discrimination when comparing deoxyribonucleic acid (DNA) samples. Since sample testing and court proceedings occur months, if not years apart, samples must be stored appropriately in the event additional testing is needed. There are generally accepted methods to store DNA extracts long-term; however, one universally recognized method does not exist. The goal of this project was to examine various methods of storage and make recommendations for a universal storage method that maintained DNA integrity over time. Four variables were evaluated: storage buffer, storage temperature, initial storage concentration and the effects of repeated freeze-thaw cycles. DNA quantity was assessed using real-time polymerase chain reaction and DNA quality was evaluated using STR genotyping. Overall, the Tris-EDTA (TE) buffer outperformed nuclease free water as a long-term storage buffer for DNA extracts. Stock tubes stabilized concentration better than single use aliquots when eluted with TE while tube type was not significant when water was the buffer. For samples stored in TE, temperature had no effect on DNA integrity over time, but samples stored in water were largely affected at room temperature. Additionally, the greater the initial DNA concentration, the less likely it was to degrade in water. As a result of this research, DNA extracts from forensic samples should be stored long-term in TE buffer with a minimum concentration of 0.1 ng/μL. When water is the buffer, frozen storage is recommended.

MSCAGR (Plant Production) / Department of Plant Production / Chickpea (Cicer arietinum L.) is an excellent utilizer of residual soil moisture in agricultural ecosystems. However, its seed quality and hence reproduction is constrained by water stress, seed size and storage conditions. This study was carried out at the University of KwaZulu- Natal (UKZN), Pietermaritzburg Campus. It was conducted to evaluate the performance of chickpea genotypes (Desi-K, Saina-K and ICCV-K) with different seed sizes on seedling emergence (i), seed ageing effect on seed quality and imbibition of genotypes produced under water stressed and non-stressed conditions (ii), and (iii) the effect of water stress during seed development on sugars and protein accumulation, germination and seed vigour. Pot experiments were conducted under glasshouse / tunnel conditions at the Controlled Environment Facilities (CEF). The experiment for objective 1 was laid out as a single factor in completely randomized design (CRD). Data on emergence rate, final hypocotyl and complete emergence was collected. The small seeded Desi-K showed higher and faster emergence compared to medium sized Saina-K and large seeded ICCV-K. In the experiment of the second objective, seeds of the three genotypes were first obtained by production under water stressed and non-stressed growing conditions. They were then aged for 0, 1, 3, 5, or 7 days at 41 ºC and 100% relative humidity to form a 2 x 3 x 5 (water levels x genotypes x ageing) factorial design. Data was collected on germination percentage (GP), mean germination time (MGT), electrical conductivity (EC), tetrazolium chloride test (TZ) and imbibition weight. Seed ageing caused progressive loss of seed viability and vigour in all genotypes, which resulted in lower GP, delayed MGT, reduced TZ staining, cell death and high solute leakage from the seeds produced under the two water regimes. However, the effect was more severe under water stressed conditions. In the experiment for objective 3, seeds of all three genotypes were larger when grown under non-stressed condition compared to those under water stressed condition. These larger seeds had higher seed viability and germination percentage but lower electrical conductivity and mean germination time. Stressed seeds had higher soluble sugars than non-stressed seeds. It was deduced that irrigation during seed development reduces the final sugars and protein content but increases the seed size and physiological quality parameters allied to production of chickpea. Therefore, water provision to chickpea crop is critical during seed development. / NRF