abstract: Radioactive cesium (137Cs), released from nuclear power plants and nuclear accidental releases, is a problem due to difficulties regarding its removal. Efforts have been focused on removing cesium and the remediation of the contaminated environment. Traditional treatment techniques include Prussian blue and nano zero-valent ion (nZVI) and nano-Fe/Cu particles to remove Cs from water; however, they are not efficient at removing Cs when present at low concentrations of about 10 parts-per-billion (ppb), typical of concentrations found in the radioactive contaminated sites. The objective of this study was to develop an innovative and simple method to remove Cs+ present at low concentrations by engineering a proteoliposome transporter composed of an uptake protein reconstituted into a liposome vesicle. To achieve this, the uptake protein, Kup, from E. coli, was isolated through protein extraction and purification procedures. The new and simple extraction methodology developed in this study was highly efficient and resulted in purified Kup at ~1 mg/mL. A new method was also developed to insert purified Kup protein into the bilayers of liposome vesicles. Finally, removal of CsCl (10 and 100 ppb) was demonstrated by spiking the constructed proteoliposome in lab-fortified water, followed by incubation and ultracentrifugation, and measuring Cs+ with inductively coupled plasma mass spectrometry (ICP-MS). The ICP-MS results from testing water contaminated with 100 ppb CsCl, revealed that adding 0.1 – 8 mL of Kup proteoliposome resulted in 0.29 – 12.7% Cs removal. Addition of 0.1 – 2 mL of proteoliposome to water contaminated with 10 ppb CsCl resulted in 0.65 – 3.43% Cs removal. These removal efficiencies were greater than the control, liposome with no protein. A linear relationship was observed between the amount of proteoliposome added to the contaminated water and removal percentage. Consequently, by adding more volumes of proteoliposome, removal can be simply improved. This suggests that with ~ 60-70 mL of proteoliposome, removal of about 90% can be achieved. The novel technique developed herein is a contribution to emerging technologies in the water and wastewater treatment industry. / Dissertation/Thesis / Doctoral Dissertation Civil, Environmental and Sustainable Engineering 2018

A gel is a cross-linked polymer network that spans an entire liquid medium; its properties depend strongly on the interaction of the polymer and the liquid medium. There are various ways to induce gelation in different systems such as altering temperature or pH. In this study, phenol extracted protein fractions from non-acclimated (NA) and cold-acclimated (CA) winter rye (Secale cereale L. cv Musketeer) leaf tissue were subjected to freeze-thaw treatment. Gelation was induced in the NA and CA extracts after repeated freeze-thaw treatments, accompanied by a change in sample rheological properties. Further experimentation revealed that gel formation only occurred at high pH (pH 12.0) and that a minimum of 3 to 4 freeze-thaw cycles were required. The viscosity of the protein gel increased 5.7- to 9.5-fold in the NA and CA extracts respectively upon freeze-thaw. Experiments optimizing the extraction conditions and protein concentration were also performed. The gel was stable and only a specific combination of chaotropic agent, anionic surfactant and reducing agent such as urea, sodium docecyl sulfate (SDS) and â-mercaptoethanol (â-ME) with heating could disrupt the gel network. The gel was composed of several proteins in the extracts as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Based on SDS-PAGE analysis, ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) was identified as the major protein component in the gel. Various experiments were performed to assess the role of Rubisco in gel formation; however, the results were inconclusive. It is suggested that these extracts may contain antifreeze proteins (AFPs) that have been demonstrated to form amyloid gels upon freeze-thaw. Further studies examining the composition and mechanism of gel formation may result in a future role for this material in the food industry.

A gel is a cross-linked polymer network that spans an entire liquid medium; its properties depend strongly on the interaction of the polymer and the liquid medium. There are various ways to induce gelation in different systems such as altering temperature or pH. In this study, phenol extracted protein fractions from non-acclimated (NA) and cold-acclimated (CA) winter rye (Secale cereale L. cv Musketeer) leaf tissue were subjected to freeze-thaw treatment. Gelation was induced in the NA and CA extracts after repeated freeze-thaw treatments, accompanied by a change in sample rheological properties. Further experimentation revealed that gel formation only occurred at high pH (pH 12.0) and that a minimum of 3 to 4 freeze-thaw cycles were required. The viscosity of the protein gel increased 5.7- to 9.5-fold in the NA and CA extracts respectively upon freeze-thaw. Experiments optimizing the extraction conditions and protein concentration were also performed. The gel was stable and only a specific combination of chaotropic agent, anionic surfactant and reducing agent such as urea, sodium docecyl sulfate (SDS) and â-mercaptoethanol (â-ME) with heating could disrupt the gel network. The gel was composed of several proteins in the extracts as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Based on SDS-PAGE analysis, ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) was identified as the major protein component in the gel. Various experiments were performed to assess the role of Rubisco in gel formation; however, the results were inconclusive. It is suggested that these extracts may contain antifreeze proteins (AFPs) that have been demonstrated to form amyloid gels upon freeze-thaw. Further studies examining the composition and mechanism of gel formation may result in a future role for this material in the food industry.

Staphylococcus aureus is a bacterial pathogen that is believed to be the most common agent of human infectious disease, causing conditions ranging from common skin lesions to life-threatening illnesses. S. aureus has also shown a remarkable ability to develop resistance to antimicrobial treatment, making infections difficult to treat. In the post-genomic era, proteomic studies analyzing the protein complement of a genome in a particular organism at any given time, have gained real significance. This result is largely due to dynamic changes in protein expression profiles which can lead wide alterations in physiology and behavior. For proteomics, it is necessary to maximize protein concentration and to devise a method that can be easily employed and provide reproducible results. Most proteomic studies of S. aureus involve 2D gel electrophoresis (2-DE); however, 2-DE has many drawbacks. Proteins that are too large, hydrophobic, acidic, or basic are poorly resolved. Multi-dimensional protein identification (MudPIT) allows complex protein samples to be analyzed in solution. As yet, there has not been a study involving solely 2D liquid chromatography followed by mass spectrometric analysis in S. aureus ; therefore we sought to catalogue the intracellular proteome and secretome of a commonly used and well-studied lab strain, SH1000. This was conducted during post-exponential and stationary phases of growth so as to understand its adaptation over time by utilizing differential protein synthesis. We found cytoplasmic proteins involved in glycolysis to be highly expressed in post-exponential phase while proteins involved in tricarboxylic acid cycle to be prevalent in stationary phase. We also found production of agr-regulated secreted toxins and proteases to be upregulated in stationary phase. In addition to this we employed proteomic approaches to quantitatively profile the secretomes of leading clinical isolates of S. aureus, as such a study is currently lacking. These included the two most common hospital-associated S. aureus strains (USA100 and USA200), and the two most common community-associated S. aureus strains (USA300 and USA400). We found agr-regulated proteins are generally upregulated in CA-MRSA strains USA300 and USA400 and surface-associated proteins to be upregulated in HA-MRSA strains USA100 and USA200. This finding concurs with literature regarding transcriptomic studies showing a hyperactive agr in CA-MRSA strains compared to HA-MRSA strains.

No description available.

Syftet med studien var att etablera Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry(MALDI-TOF MS) och onlinedatabasen Mass Spectrometry identification platform(MSI) som metod för identifiering av dermatofyter och mögelsvamp. Svamparna inkuberades 3 eller 6 dagar aerobt i 30°C på Sabouraud dextros agar. Två olika extraktionsmetoder användes innan analys med MALDI-TOF MS. Spektra analyserades med Flexcontrol version 3.4 och resulterade i scorevärde. De spektra som framkom skickades även till MSI för ytterligare analys där resultat erhölls som sannolikhet i procent. Av de ursprungliga 23 st prov identifierades totalt 22st(95,6%) efter 3 eller 6 dagars inkubation och med extraktionsmetod 1 eller 2. MSI databasen var mer omfattande än den interna databasen, men använde sig inte av de aktuella namnen. Behovet av artidentifiering kan ifrågasättas, då många dermatofyter och mögel inom samma släkte behandlas på samma sätt. Artbestämning för immunsupprimerade patienter kan däremot vara viktigt. Studien var lyckad och metoderna kommer tillämpas inom en snar framtid på Klinisk Mikrobiologi i Halmstad. / The purpose of this study was to establish Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry(MALDI-TOF MS) and the online database Mass Spectrometry identification platform(MSI) as a method for identification of dermatophytes and molds. The fungi were incubated 3 or 6 days aerobic in 30°C on Sabouraud dextrose agar. Two different extraction methods were used before analysis with MALDI-TOF MS. Spectra analyzed with Flexcontrol version 3.4 resulted in score values. The same spectra was later sent for analysis with MSI and the result obtained was seen as probability in percent. Of the original 23 samples a total of 22(95,6%) samples were identified after 3 or 6 days of incubation and by extraction method 1 or 2.  The MSI database was more comprehensive than the internal database, but did not use the current fungi names. The necessity of species identification can be questioned, since dermatophytes and molds within the same genera is treated the same way. Species identification for immunosuppressed patients could however be significant. The study was successful and the methods will be applied within a near future in the Clinical Microbiology laboratory in Halmstad.

Submitted by CAROLINE SCOTT null (loraclol@yahoo.com.br) on 2017-08-30T16:56:06Z No. of bitstreams: 1 tese com ficha.pdf: 1676779 bytes, checksum: 1003b0a00f4220379e36033ae715d740 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-08-30T18:05:48Z (GMT) No. of bitstreams: 1 scott_c_dr_bot.pdf: 1676779 bytes, checksum: 1003b0a00f4220379e36033ae715d740 (MD5) / Made available in DSpace on 2017-08-30T18:05:48Z (GMT). No. of bitstreams: 1 scott_c_dr_bot.pdf: 1676779 bytes, checksum: 1003b0a00f4220379e36033ae715d740 (MD5) Previous issue date: 2017-08-25 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O estudo proteômico é utilizado como ferramenta na reprodução animal como auxílio para compreesão da fisiologia das células. Neste sentido esta técnica é empregada em espermatozoides na tentativa de elucidar os processos biológicos e assim determinar os mecanismos moleculares envolvidos na separação do sexo. Assim, o objetivo deste estudo foi descrever as proteínas das células espermáticas de bovinos em diferentes aspectos. No estudo 1, investigou-se a influência do tampão de extração, associado ou não ao método mecânico (flash-frozen), e da concentração celular sobre a quantidade de proteínas extraídas de espermatozoides de bovinos. Foram utilizados como tampões TRIS contendo Nonidet P-40 (NP), RIPA modificado (RP) e uréia/tiuréia/CHAPS (UT), e as concentrações de 2, 4, 6, 8 e 10 x 106 espermatozoides/mL em grupos submetidos ou não ao flash-frozen. As concentrações de proteína total foram quantificadas e gel de eletroforese SDS-1D foi confeccionado. O tratamento UT recuperou maior concentração de proteínas, porém no RP as proteínas apresentaram melhor resolução no gel de eletroforese. A concentração protéica aumentou de acordo com a concentração de células no NP e UT. A influência do flash-frozen variou de acordo com o tratamento. No estudo 2, o objetivo foi traçar o perfil proteico de células espermáticas sexadas (X e Y) de bovinos. Foram utilizadas amostras comerciais de espermatozoides sexados X (n = 6) e Y (n = 6). As proteínas foram solubilizadas, submetidas a espectromia de massas, análise SWATH. Foram identificadas 459 proteínas comuns aos grupos, 10 variaram a abundância relativa (p < 0,05) De acordo com os resultados obtidos nos 2 estudos conclui-se que a concentração de proteína recuperada em uma amostra varia de acordo com o tratamento e concentração celular. Não foram identificadas proteinas exclusivas presentes nos espermatozoides sexados para X ou Y, entretanto deve-se ressaltar que há uma contaminação de 10% com espermatozoides portadores dos cromossomos opostos, ainda assim, este estudo poderá ser utilizado como embasamento científico para novas pesquisas em busca de marcadores sexo-específicos. / The proteomic study is used as a tool in animal reproduction as an aid to understanding of the cells physiology. In this regard, this technique is used in spermatozoa in an attempt to elucidate the biological processes and thus determine the molecular mechanisms involved in the sex separation. Therefore the aim of this study was to describe the bovine sperm cell proteins in different aspects. In study 1, the influence of the extraction buffer, associated or not to mechanical method (flash-frozen), and cellular concentration on the amount of proteins extracted from bovine spermatozoa were investigated. TRIS buffers containing Nonidet P-40 (NP), modified RIPA (RP) and urea/thiourea/CHAPS (UT) were used as well as concentrations of 2, 4, 6, 8 and 10 x 106 spermatozoa/mL in groups submitted or not to flash-frozen. Total protein concentrations were quantified and SDS-1D gel electrophoresis was prepared. The UT treatment recovered a higher concentration of proteins, but in RP the proteins showed better resolution in electrophoresis gel. The protein concentration increased according to the concentration of cells in the NP and UT. The influence of flash-frozen varied according to the treatment. In study 2, the objective was to map the protein profile of sexed sperm cells (X and Y) of cattle. Commercial samples of sexed spermatozoa X (n = 6) and Y (n = 6) were used. The proteins were solubilized, submitted to mass spectrometry SWATH analisys. 459 proteins common to the groups were identified, 10 varied in relative abundance (p <0.05). According to the results obtained in the 2 studies it is concluded that the concentration of recovered protein in a sample varies according to the treatment and cellular concentration. No exclusive proteins were identified in spermatozoa sexed for X or Y, but it should be noted that there is a 10% contamination with spermatozoa bearing the opposite chromosomes, however, this study could be used as a scientific basis for further research in search of sex-specific markers. / FAPESP: 2013/23351-3

The analysis of protein residues recovered from archaeological artifacts provides a unique opportunity to reveal new information about past societies. However, many scientists are currently unwilling to accept protein-based results due to problems in method development and a basic lack of agreement regarding the ability of proteins to bind to, and preserve within, artifacts such as pottery. In this paper, I address these challenges by conducting a two-phase experiment. First, I quantitatively evaluate the tendency of proteins to sorb to ceramic matrices by using total organic carbon analysis and spectrophotometric assays to analyze samples of experimentally cooked ceramic. I then test a series of solvent and physical parameters in order to develop an optimized method for extracting and preparing protein residues for identification via mass spectrometry. Results demonstrate that protein strongly sorbs to ceramic and is not easily removed, despite repeated washing, unless an appropriate extraction strategy is used. This has implications for the future of paleodietary, conservation ecology and forensic research in that it suggests the potential for recovery of aged or even ancient proteins from ceramic matrices.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2016-10-04T19:57:29Z No. of bitstreams: 1 2006 - Elisangela Sousa de Araujo.pdf: 1061759 bytes, checksum: 865d0167431190cf5627ccdf21cf91fb (MD5) / Made available in DSpace on 2016-10-04T19:57:29Z (GMT). No. of bitstreams: 1 2006 - Elisangela Sousa de Araujo.pdf: 1061759 bytes, checksum: 865d0167431190cf5627ccdf21cf91fb (MD5) Previous issue date: 2006-02-23 / FAPERJ / CAPES / Brazil is the country with the greatest plant genetic diversity in the world with the potential still underestimated what makes it necessary to characterize local genetic material. Genetic resources are studie in several stages and identify traits of agronomic value in hardy varieties is one of the roles of gene banks (BAG's). In rice, because it is a very important crop for the diet of the brazilian population, selecting genotypes with high yield, nutritional value and adapted to the most diverse environmental conditions is a continuous search for improvement programs. In this sense, the present study aimed to adapt methodologies for protein extraction reserve and DNA of rice seeds to be used in routine laboratory for characterization of germplasm banks. After optimization of methodologies, seeds of twenty local varieties of rice grown in the state of Maranh?o different times (Experiment I: Aug-2002 to Mar-2003 and Experiment II: Nov-2002 to Jun-2003) were analyzed for study of diversity genetic (DNA and protein) and seasonality effect on its accumulation of reserves and gross protein. The analysis of variance or protein content and protein fractions was highly significant (P <0.01) between genotypes and experiments. The mean protein fractions albumin+globulin and glutelin were higher in experiment I, whose average temperatures were 330C while the average gross protein (CP) was higher than in Experiment II in which the average temperature was 410C.Varieties analyzed, the Pingo d? ?gua (220019) and Jatob? (220012) showed the highest values for PB (11.32 and 11.13%) and glutelin (83.73 and 92.86 mg flour). The molecular characterization by RAPD was based on 37 polymorphic band sand phylogenetic analysis revealed a genetic diversity at 50% training with four groups and three separate varieties. Groups based on the varieties named Lageado and Zebu branco seems to have been the shape of the grains and reason C/L. / O Brasil ? o pa?s com a maior diversidade gen?tica vegetal do mundo com potencial ainda subestimado o que torna fundamental a caracteriza??o do material gen?tico local. Os recursos gen?ticos s?o estudados em v?rias etapas e identificar caracter?sticas de valor agron?mico em variedades r?sticas ? um dos pap?is dos bancos de germoplasma (BAG?s). Em arroz, por se tratar de uma cultura muito relevante para a dieta da popula??o brasileira, selecionar gen?tipos com alta produtividade, valor nutricional e adaptados ?s mais diversificadas condi??es ambientais ? uma busca cont?nua em programas de melhoramento. Nesse sentido, o presente trabalho teve por objetivos adaptar metodologias para extra??o de prote?na de reserva e de DNA de sementes de arroz a fim de serem empregadas na rotina de laborat?rio para caracteriza??o de bancos de germoplasma. Ap?s otimiza??o das metodologias, sementes de vinte variedades locais de arroz do Estado do Maranh?o cultivadas em ?pocas distintas (Experimento I: Ago-2002 a Mar-2003 e Experimento II: Nov-2002 Jun-2003) foram analisadas para fins de estudo de diversidade gen?tica (DNA e prote?na) e efeito da sazonalidade em seu ac?mulo de prote?na bruta e reserva. A an?lise de vari?ncia para teor de prote?na e fra??es proteicas foi altamente significativa (P<0,01) entre os gen?tipos, e os experimentos. As m?dias das fra??es proteicas albumina+globulina e glutelina foram superiores no experimento I, cujas temperaturas m?dias foram de 330C enquanto que a m?dia da prote?na bruta (PB) foi superior no Experimento II em que a m?dia de temperatura foi de 410C. Das variedades analisadas, a Pingo d??gua (220019) e Jatob? (220012) foram as que apresentaram os maiores valores para PB (11,32 e 11,13%) e glutelina (83,73 e 92,86 mg.g farinha). A caracteriza??o molecular pela RAPD foi baseada em 37 bandas polim?rficas e a an?lise filogen?tica revelou uma diversidade gen?tica em 50% com forma??o de quatro grupos e tr?s variedades isoladas. Os grupos formados pelas variedades de nome Lageado e Zebu branco parecem ter sido pela forma dos gr?os e raz?o C/L.

Made available in DSpace on 2017-05-12T14:47:15Z (GMT). No. of bitstreams: 1 Janaina Lima da Silva.pdf: 3461069 bytes, checksum: 86eccae232302ee27032f84e3721edb7 (MD5) Previous issue date: 2007-07-17 / Leaves and aerial part of the cassava are great protein, vitamins, minerals and essential amino acids sources, being these abundant and in low cost. However, this residual is still little explored. The cassava leaves use as protein source is, many times, not stimulated by the presence of anti nutritional factors what indicates the obtainment of proteic concentrates. The objective of this study was to obtain proteic concentrates from cassava leaf and aerial part (leaf, stalk and stem) through 4 extraction methods, besides to evaluate the mineral composition and functional properties of the obtained concentrates. The extraction methods used were: 1) Extraction method by isoeletric precipitation described by CEREDA; VILPOUX (2003); 2) Extraction Method by fermentation described by CHAVES (1987); 3) Extraction Method by fermentation described by CHAVES (1987) with fermentation time shortening described by FERRI (2006); 4) Extraction Method by fermentation described by CHAVES (1987) with pH alteration in the end of fermentation described by FERRI (2006).It was determined for each method the extraction yield, the proteic concentrate yield, the mass and protein loss. In the obtained concentrates were determined the Fe, Mn, Cu, Na, K and Zn levels, besides the functional properties of water and oil absorption. The factorial 2X4 experimental outline was used, being its factors the material kind (leaf and aerial part) and the protein extraction methods, with 3 repetitions, constituting 24 experimental parts. The variable analysis was also made, being its averages compared by the Tukey test in the 5% significance level. The yields obtained on the leaves were 18,31%, 14,11%,14,40% and 13,45% for methods 1, 2, 3 and 4 respectively. On the aerial parts the yields of concentrate obtained were 3,66%, 6,10%, 6,30% and 6,55% for methods 1, 2, 3 and 4 respectively. The leaves presented average extraction yield values three to four times bigger than the values presented by the aerial part, being them more adequate to get proteic concentrate from. The proteic concentrates obtained presented good quantity of nutrients like Fe, Mn, Cu, Na, K and Zn, indicating their application in the food industry, without difference between the concentrates obtained from the leaves or the aerial part. It was observed an elevated capacity of water absorption on the proteic concentrates, indicating its application in foods as meat, bread, soups and sauces. The oil absorption capacity on proteic concentrates was also high, indicating their industrial application in products preparation as sausages, dough, mayonnaise and other salad dressings increasing the good flavor retention of these products. / As folhas e parte aérea da mandioca constituem-se como fontes de proteínas, vitaminas, minerais e aminoácidos essenciais, abundantes e de baixo custo. Contudo este resíduo é ainda pouco explorado. O uso de folhas de mandioca como fonte de proteínas, muitas vezes é desestimulado pela presença de fatores antinutricionais, indicando a obtenção de concentrados protéicos. O objetivo do presente trabalho foi obter concentrados protéicos de folha e parte aérea (folha, haste e caule) de mandioca, por 4 métodos de extração e avaliar a composição mineral e propriedades funcionais dos concentrados obtidos. Os métodos de extração utilizados foram: 1) Método de extração por precipitação isoelétrica descrito por CEREDA; VILPOUX (2003); 2) Método de extração por fermentação descrito por CHAVES (1987); 3) Método de extração por fermentação descrito por CHAVES (1987), com redução no tempo de fermentação descrito por FERRI (2006); 4) Método de extração por fermentação descrito por CHAVES (1987), com alteração de pH no final da fermentação proposto por FERRI (2006). Determinou-se para cada método o rendimento de extração, o rendimento de concentrado protéico, a perda de massa e perda de proteínas. Nos concentrados obtidos determinou-se o teor de Fe, Mn, Cu, Na, K e Zn e as propriedades funcionais de absorção de água e óleo. Utilizou-se delineamento experimental do tipo fatorial 2 x 4, sendo os fatores, tipo de material (folha e parte aérea) e métodos de extração de proteínas, com 3 repetições, constituindo 24 parcelas experimentais. Realizou-se análise de variância, sendo as médias comparadas pelo teste de Tukey, ao nível de 5% de significância. Para as folhas os rendimentos de concentrado obtidos foram 18,31%, 14,11%,14,40% e 13,45% para os métodos 1, 2, 3 e 4, respectivamente. Para a parte aérea os rendimentos de concentrado obtidos foram 3,66%, 6,10%, 6,30% e 6,55% para os métodos 1, 2, 3 e 4, respectivamente. As folhas apresentaram valores médios de rendimento de extração três a quatro vezes maiores que os valores apresentados pela parte aérea, indicando serem mais adequadas para obtenção de concentrado protéico. Os concentrados protéicos obtidos apresentaram bons conteúdos de nutrientes como Fe, Mn, Cu, Na, K e Zn, indicando boa aplicação como suplemento na indústria de alimentos, não havendo diferenças entre os concentrados obtidos de folha ou de parte aérea. Foi observada elevada capacidade de absorção de água nos concentrados protéicos, indicando aplicação em alimentos como carnes, pães, sopas e molhos. A capacidade de absorção de óleo dos concentrados protéicos também foi elevada, indicando boa aplicação industrial para preparação de produtos como salsichas, massas de bolos, maionese e outros molhos para saladas, aumentando a retenção de sabor nestes produtos.