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>Magister Scientiae - MSc / Chromosome doubling has become an important tool in breeding programmes as it offers the ability of introducing novel traits into existing plants. Doubled haploid plants are highly valued by both consumers and breeders as these plants usually show larger flower, leaves and fruit, thus making them more marketable. Marianna open pollinated plum rootstocks’ adaptability to different soil types and moisture conditions has been favoured in polyploidy studies as parental material in breeding programmes. The potential of the microtubule depolymerizing herbicide (oryzalin) for in vitro chromosome doubling were investigated by optimizing the concentration and incubation time of plant shoots to the antimitotic agent. Meristem tissues were treated for two time intervals (24 and 48 h) with five different concentrations of oryzalin (50, 75, 100, 150 or 200 μM) in liquid Murashige and Skoog (MS) medium. After treatment, plants were allowed to grow under a 16/8 h light/dark photoperiod at 24±2˚C for 4 weeks. One and two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) was used to separate, visualise and identify differently expressed proteins. Furthermore, changes in ROS accumulation, photosynthetic pigmentation, lipid peroxidation and antioxidant enzyme activity (SOD, APX and GR) were investigated. Flow cytometry results revealed that treatment of plants with oryzalin concentrations ranging from 75 to 150 μM induced ploidy after 24 h exposure whereas, 200 μM produced mixoploids containing both tetraploid and octoploids plants after 24 h exposure. Longer incubations of 48 h were detrimental to plant tissues as complete mortality was observed in the higher concentration (100 to 200 μM) treatments. Mass spectrometry analysis identified 14 differentially expressed protein spots that were characterized into different functional categories. ROS accumulation, the extent of lipid peroxidation and antioxidant capacity were differentially regulated in response to oryzalin treatment whereas photosynthetic pigments were significantly enhanced. The results suggests that oryzalin-induced proteins may act as potential biomarkers to improve fruit characteristics in future breeding programs whereas antioxidant enzymes play an important role in scavenging ROS in plants to enhance their adaptability to different environmental conditions.

Edible insects have been part of the diet of many countries across the world for several decades. It is becoming increasingly popular in the europian developed countries, mainly because of the content of nutritionally important substances. The theoretical part of the thesis deals with the characterization of edible insects in terms of nutritional benefits, explains the reasons and method of breeding edible insects. For the analysis of nutritionally active substances, flour of house cricket (Acheta domesticus) was used. A. domesticus belongs to the representatives of insects legalized from 1.1. 2018 on the EU market. Of the significantly nutritional substances, proteins, lipids, fatty acids, fiber and selected minerals were analyzed. In this study, optimalization of vertical electrophoresis SDS-PAGE was performed to determine protein fragment sizes. In the second part the influence of acricket flour addition protein bars was investigated by sensory analysis. In the experimental part that was found that cricket flour contains protein, lipids and fatty acids in significant amounts. It also contains important minerals for human health such as magnesium, potassium, iron or zinc. However, the sensory properties of protein bars for consumers were not very attractive.

This diploma thesis deals with the analysis of low molecular weight proteins in barley during malting by SDS-PAGE method. Attention was paid to PR proteins, specifically LTP proteins and thionins, considered to be connected with gushing effect. Barley samples, malt intermediates and malt samples taken in 10 consecutive days to cover the whole malting process were used for the experiment. In total, 5 samplings were used for the analysis. Proteins extracted from the samples were separated by SDS-PAGE using a Tris-tricine buffer system. The protein lines of LTP proteins and thionins were identified in the resulting gels. The relative optical density values of the selected protein bands were obtained to assess the effect of malting on these proteins. A similar pattern of change in the content of mentioned low molecular weight proteins during the malting process was observed. This was confirmed by finding a statistically significant positive correlation between the relative optical density values of LTP proteins and thionins. Furthermore, the relationship between the low molecular weight protein content and the gushing potential and the microbiological contamination of the samples was investigated, but was not confirmed.

The MS-818 compound is used in the proliferation process of neuronal cells and many biological activities that accompany this process such as astrocyte differentiation, inhibition of neuronal apoptosis, and fraction repairs. We do know the effects of this compound, but the mechanism of action remained uncertain until now. To determine the pathway of this compound, NT2 cells were cultured and lysed to isolate the proteins. Affinity Chromatography was performed in order to immobilize the MS-818 compound to a Hi-Trap NHS column. The NT2 protein sample was injected through the column and eluted with a MS-818 concentrated, high salt content elution buffer. SDS-PAGE was then performed to isolate the proteins that bound to MS-818. The gel was visualized using Coomassie Blue. The results indicate that there are two proteins associated in the mechanism of this compound. A standard protein marker ranging from 10 kDa to 250 kDa was used to compare the bands. The findings indicate that one of the protein bands is slightly less than 250kDa and the other is between 50-75 kDa. When the proteins are confirmed by mass spectrometry sequencing, this will help to promote this compound as a drug candidate.

Experiments were conducted to test for ricin content in tissue samples from four castor cultivars, developing castor seed, germinating castor seedlings, and chemically and heat treated seed meal. Ricin content of each sample was examined via Western blotting with ricin A-chain specific antibodies. Results indicate that ricin is present solely within castor endosperm and is not present any other tissues. Samples from developing seed and germinating seedlings indicate ricin production begins around day 28 post pollination, and ricin is absent from the seedling 6 days after the onset of radicle emergence. This would seem to indicate that the purpose of ricin is to protect the seed and not the entire plant. Ricin content of seed meal treated separately with heat and chemicals was tested. It was found that hot-pressing of the seed was sufficient to denature ricin in the seed meal.

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Made available in DSpace on 2017-07-10T17:37:45Z (GMT). No. of bitstreams: 1 Patricia_Paro.pdf: 402899 bytes, checksum: cb9972fdc35b447bcb1020e13a07782b (MD5) Previous issue date: 2011-04-08 / Fundação Araucária / The wheat is a species of great importance in human nutrition, wheat grain is extracted from wheat flour used in the preparation of various food products. The rheological properties of wheat flour is a feature that determines the fate of the flour, being of great importance to this assessment. In breeding programs for wheat determining the quality of flour is essential for the development of cultivars, and it is necessary that the reading of the quality parameters are carried out early, with the use of small amounts of sample, reliable and the level of genotype. The development of this work aimed at the validation of molecular markers for selection of genotypes from high molecular weight glutenin, given their great influence on baking quality of wheat flour and check the correlation between parameters of quality of flour and the high molecular weight glutenin. Analyses were performed in the laboratory of biotechnology COODETEC using 77 samples of wheat (cultivars and lines) from program for wheat breeding of the institution, with data quality technology of wheat flour has characterized. The set of primers called Glu1-DX2-DX5 was used to select individuals that contained subunits Glu1-DX5 + Dy10 by PCR, nevertheless was necessary to make adjustments so that the marker amplified a fragment corresponding to allele Glu1-DX5. To check the reliability of molecular marker, all samples were characterized by electrophoresis of proteins by SDS-PAGE method and scores as a function of the subunits of glutenin high molecular weight they have. The results of the analysis of protein and molecular differ, indicating that the use of molecular markers should be used with any control in the PCR reaction. From the data collected and the quality of flour, the results were submitted to Pearson correlation analysis. It appears that the score of the protein is positively correlated with W, P and P / L, indicating that the composition of glutenin high molecular weight have significant influence on quality of wheat flour. Alleles Glu1-DX5 and the subunits of the A genome are positively correlated with protein scores and consequently with the wheat breadmaking. The allelic variants of the A genome were positively correlated with W and P. From the results it is concluded that the selection of genotypes with superior quality of flour can be made taking into account the high molecular weight glutenin encoded by the A and D genomes of wheat / O trigo é uma espécie de grande importância na alimentação humana, de seus grãos é extraída a farinha de trigo utilizada no preparo de diversos produtos alimentícios. As propriedades reológicas da farinha de trigo são características que determinam o destino final da farinha, sendo de grande importância a sua avaliação. Em programas de melhoramento genético do trigo a determinação da qualidade de farinha é indispensável para o desenvolvimento de cultivares, e é necessário que a leitura dos parâmetros de qualidade sejam realizados precocemente, com a utilização de pouca quantidade de amostra, confiáveis e a nível de genótipo. Este trabalho teve por objetivo a validação de marcadores moleculares para a seleção de genótipos a partir de gluteninas de alta massa molecular, visto que apresentam grande influencia na qualidade de panificação da farinha de trigo e verificar a correlação existente entre parâmetros de qualidade de farinha e as gluteninas de alta massa molecular. As análises foram realizadas no laboratório de biotecnologia da COODETEC utilizando 77 amostras de trigo (linhagens e cultivares) provenientes do programa de melhoramento genético de trigo da instituição, com dados de qualidade tecnológica da farinha de trigo já caracterizados. O conjunto de primers denominados Glu1-Dx5-Dx2 foi utilizado para selecionar indivíduos que continham o conjunto de subunidades Glu1-Dx5+Dy10 através da técnica de PCR, porém foram necessário ajustes para que o marcador amplificasse o fragmento correspondente ao alelo Glu1-Dx5. Para verificar a confiabilidade do marcador molecular todas as amostras foram caracterizadas através de eletroforese de proteínas pelo método SDS-PAGE, e escoreadas em função das subunidades de gluteninas de alta massa molecular que apresentam. Os resultados entre a análise de proteína e molecular apontam divergências, indicando que para a utilização do marcador molecular deve ser utilizada com algum controle na reação de PCR. A partir dos dados coletados e os de qualidade de farinha os resultados foram submetidos a análise de correlação de Pearson. Verifica-se que o escore de proteína é positivamente correlacionado com W, P e P/L, indicando que a composição de gluteninas de alta massa molecular apresentam influencia significativa na qualidade de farinha de trigo. Os alelos Glu1-Dx5 e as subunidades do genoma A estão positivamente correlacionados com escore de proteína e conseqüentemente com a qualidade e panificação do trigo. Os variantes alélicos do genoma A foram positivamente correlacionados com W e P. A partir dos resultados conclui-se que a seleção de genótipos de trigo com qualidade superior de farinha podem ser realizados levando em consideração as gluteninas de alta massa molecular codificadas pelos genomas A e D do trigo

<p>Obesity is a globally increasing disease particularly in developing countries and among children. It is mainly caused by intake of diets high in fat and the lack of physical activity. Obesity is a risk factor for diseases such as type II diabetes, high blood pressure, high cholesterol and certain cancers. Prediabetes is a condition where blood glucose levels are above normal but have not&nbsp / reached those of diabetes. It is difficult to diagnose, as there are no signs or symptoms. Some type II diabetes patients bear no symptoms at all and the disease is discovered late. Proteomics is a field that can provide opportunities for early diagnosis of diseases through biomarker discovery. The early diagnosis of diabetes can assist in the prevention and treatment of diabetes. Therefore there is a need for the early diagnosis of diabetes. Twenty Wistar rats were used. The rats were initially fed a CHOW diet, which is the standard balanced diet for rats, for 4 weeks. The rats were then divided into 2 groups of 10 where 1 group was fed CHOW and another was fed a high fat (HF) diet in order to induce obesity. The two groups were fed their respective diets for 18 weeks. Rats were weighed. Rats were placed in metabolic chambers and 24 hour urine samples were collected. Ketone levels were measured by Ketostix. Urine proteins were precipitated by acetone, quantified and separated on both the 1D SDS-PAGE and the 2D SDS-PAGE. Protein expression changes between CHOW and HF fed rats were determined and identified using MALDI-TOF mass spectrometry. Protein spots intensities increased and decreased between the CHOW and HF fed rats. Transducin (beta)-like 3 was identified as the only differentially expressed protein, which might serve as a potential biomarker for prediabetes.</p>

<p>Obesity is a globally increasing disease particularly in developing countries and among children. It is mainly caused by intake of diets high in fat and the lack of physical activity. Obesity is a risk factor for diseases such as type II diabetes, high blood pressure, high cholesterol and certain cancers. Prediabetes is a condition where blood glucose levels are above normal but have not&nbsp / reached those of diabetes. It is difficult to diagnose, as there are no signs or symptoms. Some type II diabetes patients bear no symptoms at all and the disease is discovered late. Proteomics is a field that can provide opportunities for early diagnosis of diseases through biomarker discovery. The early diagnosis of diabetes can assist in the prevention and treatment of diabetes. Therefore there is a need for the early diagnosis of diabetes. Twenty Wistar rats were used. The rats were initially fed a CHOW diet, which is the standard balanced diet for rats, for 4 weeks. The rats were then divided into 2 groups of 10 where 1 group was fed CHOW and another was fed a high fat (HF) diet in order to induce obesity. The two groups were fed their respective diets for 18 weeks. Rats were weighed. Rats were placed in metabolic chambers and 24 hour urine samples were collected. Ketone levels were measured by Ketostix. Urine proteins were precipitated by acetone, quantified and separated on both the 1D SDS-PAGE and the 2D SDS-PAGE. Protein expression changes between CHOW and HF fed rats were determined and identified using MALDI-TOF mass spectrometry. Protein spots intensities increased and decreased between the CHOW and HF fed rats. Transducin (beta)-like 3 was identified as the only differentially expressed protein, which might serve as a potential biomarker for prediabetes.</p>