Submitted by Luriany Pompeo Ferraz (luriany@hotmail.com) on 2018-05-10T13:54:38Z No. of bitstreams: 1 Tese Luriany Pompeo Ferraz.pdf: 2121530 bytes, checksum: dbc5ecaf4e4d278a002ba09e3ed9af57 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-05-10T18:09:10Z (GMT) No. of bitstreams: 1 ferraz_lp_dr_jabo.pdf: 2121530 bytes, checksum: dbc5ecaf4e4d278a002ba09e3ed9af57 (MD5) / Made available in DSpace on 2018-05-10T18:09:10Z (GMT). No. of bitstreams: 1 ferraz_lp_dr_jabo.pdf: 2121530 bytes, checksum: dbc5ecaf4e4d278a002ba09e3ed9af57 (MD5) Previous issue date: 2018-04-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O fenômeno killler é característico de leveduras que produzem e excretam proteínas ou glicoproteínas que são inibidoras de células microbianas sensíveis. A estabilidade e atividade das toxinas killer são altamente sensíveis a fatores como pH, temperatura de incubação das leveduras, composição e propriedades físico-químicas do meio e concentração de células sensíveis. Sporobolomyces koalae é uma nova espécie de levedura com potencial para produção de toxina killer. No intuito de se conhecer mais sobre as funções desse microrganismo no ecossistema, esse trabalho teve por objetivos: (i) detectar a atividade killer do precipitado proteico de S. koalae, (ii) caracterizar bioquimicamente e funcionalmente o precipitado proteico S. koalae, (iii) purificar parcialmente a toxina killer produzida por S. koalae e, finalmente, (iv) verificar sua ação antagônica sobre Geotrichum citri-aurantii e Penicillium digitatum, patógenos que ocorrem na póscolheita de citros. Pelos resultados obtidos neste trabalho, foi possível detectar a presença de atividade killer no precipitado proteico da levedura S. koalae contra células sensíveis da levedura Saccharomyces cerevisiae NCYC 1006. O precipitado proteico da levedura apresenta várias proteínas com diferentes tamanhos moleculares e, parte dessas proteínas é positiva para atividade de β1,3-glucanase, quitinase e protease, podendo ser uma dessas enzimas ou, o sinergismo entre elas, o responsável pelo fator killer da levedura. A funcionalidade de suas proteínas para atividade killer aumenta em pH 4.9 e em uma faixa de temperatura de 22 °C a 27 °C e, o melhor agente precipitante das proteínas da levedura foi o etanol 80%. A purificação parcial das proteínas, que constituem o precipitado proteico de S. koalae, mostrou a existência de aproximadamente quatro bandas proteicas em uma das frações de purificação, Fração 1, com tamanhos aproximados que variaram de 8 a 65 kDa. A atividade killer, presente no precipitado proteico da levedura S. koalae, não apresenta ação antagônica sobre Geotrichum citri-aurantii e Penicillium digitatum. / The killer phenomenon is characterized by yeasts that produce and excrete proteins or glycoproteins that are inhibitors of sensitive microbial cells. The stability and activity of killer toxins are highly sensitive to the factors such as pH, the temperature of incubation of yeasts, composition and physical-chemical properties of the medium and concentration of sensitive cells. Sporobolomyces koalae is a new species of yeast with potential for killer toxin production. In order to know more about the functions of this microorganism in the ecosystem, this work had as objectives: (i) to detect the killer activity of the S. koalae protein precipitate, (ii) to characterize biochemically and functionally the S. koalae protein precipitate, (iii) to partially purify the killer toxin produced by S. koalae, and, finally, (iv) to verify its antagonistic action on Geotrichum citri-aurantii and Penicillium digitatum, pathogens that occur in the postharvest of citrus. By the results obtained in this work, it was possible to detect the presence of killer activity in the protein precipitate of S. koalae against sensitive cells of Saccharomyces cerevisiae NCYC 1006. The protein precipitate from yeast has several proteins with different molecular sizes and some of these proteins are positive for β1,3-glucanase, chitinase and protease activity. It may be one of these enzymes or synergism between them, the responsible for the killer factor. The functionality of their proteins for killer activity increases at pH 4.9 and a temperature range of 22 oC to 27 oC and the best precipitant of protein from yeast was ethanol (80%). Partial purification of the proteins showed the existence of approximately 4 protein bands in one of the purification fractions, Fraction 1, with approximate sizes ranging from 8 to 65 kDa. The killer activity, present in the protein precipitate of yeast S. koalae, did not present antagonistic action on G. citri-aurantii and P. digitatum. / FAPESP: 14/25067-3

Les objectifs de cette étude sont de caractériser des altérations des qualités sensorielles et technologiques de la viande de dinde et de mettre en relation ces altérations avec les caractéristiques des protéines musculaires. Nous avons, tout d'abord, sélectionné des muscles Pectoralis major (PM) de dindes en fonction de leur couleur. Les propriétés sensorielles et technologiques de la viande ne diffèrent jamais entre le groupe ayant une couleur « normale » et le groupe ayant une couleur « pâle » (expérience couleur). Dans un deuxième temps, nous avons essayé d'analyser l'effet de la vitesse de chute du pH post mortem sur la qualité de la viande issue du PM de dinde. Cet effet a été mesuré avec différents types génétiques de dindes : des souches BUT9 (expériences BUT9.1 et BUT9.2) et BIG6 (expérience BIG6) comparées dans des conditions d'abattage commercial ou des souches BUT9 et Label Rouge (expérience Label) comparées en générant artificiellement le défaut PSE. Nos différentes expériences montrent que le pouvoir de rétention en eau et la texture de la viande sont diminués lorsque la glycolyse musculaire post mortem est accélérée. Par contre, la couleur de la viande est peu affectée. Nous avons aussi mis en évidence des altérations de protéines de structure (α-actinine), de protéines contractiles (actine et chaîne lourde de la myosine) et de protéines sarcoplasmiques (GAPDH, aldolase A, myokinase, ATP synthase et phosphorylase). Le type génétique des dindes ne semble pas être déterminant dans l'augmentation de l'apparition des défauts de qualité de viande.

Philosophiae Doctor - PhD / One of the most common HIV-associated opportunistic infections is candidiasis, caused by Candida albicans or other Candida species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of Candida species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant Candida species. The mechanisms of Candida drug resistance in the African continent have also not been described. In this study, 255 Candida species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. Candida cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in Candida species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of Candida infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant Candida strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of Candida colonization mechanisms in HIV-infected African patients.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22291号 / 理博第4605号 / 新制||理||1660(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 和俊, 教授 高田 彰二, 教授 平野 丈夫 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

The master's thesis is focused on study of specific protective proteins from animal products. Two different types of antimicrobial egg white proteins were studied in detail - antimicrobial protein ovotransferrin (conalbumin) and enzyme lysozyme. Ovotransferrin belongs to transferrin group of proteins and exhibits activities similar to milk protective protein lactoferrin. The main effects of ovotransferrin are antiviral, anticancer and immunomodulatory. Antimicrobial activity of ovotransferrin based on the possibility to bind iron is still a subject of interest. For comparison the second egg protein lysozyme (N-acetyl muramidglycan hydrolase) was used. Lysozyme is a hydrolytic enzyme which primary attack cell wall of bacteria. In the theoretical part of the thesis an overview of the specific antimicrobial proteins in selected animal products was introduced mainly focused on ovotransferrin and lysozyme. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial activity, protein concentration and purity. For quantitative analysis of total proteins, optimized Hartree – Lowry spectrophotometric method was used. For the determination of molecular weight and purity SDS-PAGE was used and stained by Coomassie Brilliant Blue G250 and silver. In experimental part the real sample of egg white was compared with samples of lyophilized antimicrobial proteins and therapeutical pills supplied by industrial partner. Protein composition and purity of these preparative has been determined. Antimicrobial activity of ovotransferrin was studied on cultures of G+ bacterium Bacillus subtilis and for comparison on G– E. coli. Ovotransferrin showed antimicrobial effect only at very high concentrations of about 75 mg/ml (Bacillus subtilis) and 50 mg/ml (E coli) even with addition of high amount (100 mM) of hydrogen carbonate ions. The inhibitory effect was most evident in liquid media. On the other hand, lysozyme exhibited significant inhibitory activity from 0.3 mg/ml on gram positive bacteria. Inhibitory effect on E. coli was not observed. Another part of study was focused on isolation of ovotransferrin from egg white using gel permeation chromatography on Sephadex G100. As mobile phases 0.1 M phosphate buffer and 0.05 M Tris-HCl buffer were tested. By SDS-PAGE the purity of ovotransferin comparing to standard was evaluated. Finally, the encapsulation of ovotransferrin and lysozyme was tested. Ovotransferrin and lysozyme was encapsulated into liposome and chitosan particles. Particles stability, distribution and average size distribution were studied by dynamic light scattering and zeta potential measurement. The stability of particles in the model physiological conditions was studied too.

Introduction: Cellular membrane proteins, such as the cytochrome P450 enzyme superfamily (P450), have important roles in the physiology of the cell. P450s are important in metabolising endogenous molecules, as well as metabolising xenobiotic substances for detoxification and excretion. P450s are also implicated in cancer as they can act to ¿negatively¿ de-activate or ¿positively¿ activate cancer therapeutics. Identifying specific P450s that are highly up-regulated at the tumour site could be used to predict drug response and formulate targeted cancer therapy to help diminish systemic side-effects. Methods: Previous enrichment strategies have been unable to isolate the full complement of the P450 superfamily. To develop enrichment procedures for the P450s, a proteomic strategy was developed so that compounds could be screened for their effectiveness as general P450 probes. A standardised work-flow was created, encompassing affinity chromatography, protein concentration/desalting, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-mass spectrometry (HPLC-MS). A ketoconazole analogue and a 2-EN analogue, with known P450 inhibition, were immobilised on a solid support for comparison to immobilised histamine. Co-factor removal, competitive elution and DTT cleavage of disulfide bonds of probes were utilised to elute bound proteins. Results/Discussion: Inhibitor-beads bound a large range of proteins, including P450¿s, of which some were eluted by co-factor removal, some by competitive elution. Specificity of binding was improved by optimising buffer conditions and solid supports, however non-specific binding was not totally eradicated. All human P450s from spiked samples and 18 P450s from more complex mouse liver samples were recovered using one or more ligands. / Bruker Daltonics

No description available.

Copper in drinking water elicits a persisting bitter, metallic, or astringent taste. Characteristics and perception mechanisms of copper sensation have not been fully understood. Saliva is assumed to influence copper sensations via binding of salivary electrolytes or proteins with copper. The interaction between salivary components and copper is thought to influence sensory perception by affecting volatility of aroma compounds, de-lubricating salivary proteins, and by controlling solubility of copper. A recent study suggested that intensity of copper taste may be dependent on the amount of solubilized copper, which increases at lower pH. This research was performed to identify 1) the temporal sensory characteristics of copper; 2) the effect of pH on perception of copper sensation; 3) the nature of copper-protein interaction and its impact on sensory perception. The effect of copper on the volatility of aroma compounds and the role of copper-protein interaction in volatile chemistry were investigated using a model mouth system containing artificial saliva at different pH levels. Headspace concentration of each volatile was measured using SPME-GC analysis. Copper (2.5 mg/L) in the model system increased headspace concentration of volatiles (hexanal, butyl acetate, 2-heptanone, and ethyl hexanoate, 0.5 microL/L each) at pH 6.5, but no change in volatility was observed at pH 7.0. At pH 7.5, presence of copper in the artificial saliva decreased headspace volatile concentration. Effect of copper on volatiles at pH 6.5 may be due to increased solubility of copper at lower pH. Copper seems to facilitate hydrophobic binding between mucin and aroma compounds at pH 7.5, possibly by exposing hydrophobic sites of mucin. A time-intensity (TI) test was performed to identify the effect of pH on temporal characteristics of copper sensation. Metallic taste, bitterness, and astringency were major attributes of drinking water containing 2.5 mg/L and 5 mg/L Cu. All three attributes were responsible for the lingering aftertaste of copper. TI test results of copper solutions did not show a common TI pattern of astringency that is characterized with slow onset and longer duration time. Increase in pH of water from 5.5 to 7.5 inhibited metallic taste of copper, but did not reduce bitterness and astringency. The level of soluble copper at pH 7.5 decreased by 50 % compared to that at pH 5.5. Soluble copper concentration and temporal profile of sensory attributes of copper solutions at different pH levels suggest that soluble copper species decide the perception of copper sensation by controlling metallic taste. The nature of copper-protein interaction and its implication on mechanisms of sensory perception were studied by investigating binding of copper to high molecular weight fractions of human saliva. At the copper concentration < 10 mg/L, most copper exists as unbound copper form while about 60 % of copper was found in protein fractions or with precipitated salivary debris. This result suggests that copper is in a soluble unbound form in saliva at low concentration (<10 mg/L) and assumed to be available for taste receptors. At higher concentration, copper either becomes insoluble or binds with proteins. Insoluble copper species are thought to cause astringency. When copper was added at the concentration equal to or greater than 10 mg/L, two salivary proteins of molecular weight 29 kDa and 33 kDa formed insoluble complexes with copper. Low molecular weight mucin (MG2), alpha-amylase, basic proline-rich proteins (PRPs), and a protein of MW 45 kDa also bound with copper. In summary, sensations elicited or influenced by copper are thought to be determined by what copper species are dominant in the mouth. Soluble copper species and insoluble copper species are assumed to interact with different sensory receptors, resulting in metallic taste or astringency. This speciation process is influenced by pH conditions, composition of other electrolytes, and organic chelators such as proteins. / Ph. D.

Made available in DSpace on 2015-03-26T13:42:15Z (GMT). No. of bitstreams: 1 texto completo.pdf: 503594 bytes, checksum: 9404a8d4266a856855a02a401fdb8a03 (MD5) Previous issue date: 2010-07-06 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Wheat is one of the principal cereals in the world and is processed into a wide range of products. In recent decades researchers have shown concern over acquisition of cultivars with good baking quality, a characteristic which initially refers to the protein composition of the endosperm, composed principally of the proteins making up gluten. Various methodologies have been employed in the evaluation of this characteristic, including SDS-PAGE (polyacrylamide gel electrophoresis), SE-HPLC (size exclusion high performance liquid chromatography), baking tests and employment of genetic markers. In an attempt to compare the different existing methodologies and propose a strategy for wheat genetic improvement programs based on industrial quality, four different techniques were tests with 45 national cultivars. Six allele-specific markers were used for verification of the presence of the HMW subunits: Glu-D1-1d (5+10) and Glu-B1-2a (7+8), one LMW subunit: Glu-A3d, the presence or absence of 1B/1R translocation: by the presence of Glu-B1 (long arm of the wheat chromosome) or &#969;- secalin (gene of the short arm of rye) and puroindoline (Pinb-D1b). Also employed where SDS-PAGE, SE-HPLC and baking test methods. As a result of applying the markers, the presence in the genotypes observed was: 71% for subunit Glu-D1-1d (5+10), 16% for Glu-B1-2a (7+8), 60% for Glu-B1 (absence of translocation) and 8% for Pinb-D1b and Glu-A3d. The results of SDS-PAGE suggested a prevalence of the subunits 2* and 1 in chromosome 1A; 7+8, 7+9 and 17+18 in 1B; and 5+10 in 1D. The average values of SE-HPLC were 35.99% and 44.99% for the polymeric protein in protein (PPP) and unextractable polymeric protein (UPP), respectively, and 1.29 for the ratio between gliadins and glutenins (GLI/GLU), with significant variation among the genotypes (p&#8804;0.05). The baking test also showed a significant difference (p&#8804;0.05) between the cultivars under the same conditions. The UPP data were independent of the PPP percentage data, signifying that a portion of the unextractable polymeric protein (UPP) does not depend on the greater percentage of total polymeric proteins. However there was a positive correlation between the score of HMW subunits, evaluated by SDSPAGE, and the UPP values; this indicates that scoring has apparently been efficient in classifying the genotypes of greatest quality. The cultivars without translocation with rye also showed better results for UPP, PPP and GLI/GLU in relation to those possessing translocation. The GLI/LGU ratio is correlated with the specific volume of the bread, but apparently does not guarantee quality of the qualitative characteristics, which may indicate the action of gliadins in extensibility, but with a lack of structure in the gluten network. These results corroborate for the selection of HMW subunits 5+10, cultivars without translocation with rye, with high values of UPP and an appropriate GLI/GLU ratio with the objective of obtaining greater wheat baking quality. / O trigo é um dos principais cereais em todo o mundo, sendo processado para uma gama de produtos. Surgiu nas últimas décadas a preocupação dos pesquisadores com a obtenção de cultivares com boa qualidade de panificação, característica que refere-se primordialmente a constituição protéica do endosperma, composta principalmente pelas proteínas formadoras de glúten. Várias metodologias vêm sendo empregadas na avaliação dessa característica, dentre as quais, estão o SDS-PAGE (eletroforese em gel de poliacrilamida), SE-HPLC (cromatografia líquida de alta eficiência por exclusão molecular), teste de panificação e o emprego de marcadores genéticos. Buscando comparar as diferentes metodologias existentes e propor uma estratégia a programas de melhoramento de trigo voltados à qualidade industrial, testaram-se quatro diferentes técnicas em 45 cultivares nacionais. Usaram-se seis marcadores alelo-específicos direcionados à verificação da presença das subunidades de HMW: Glu-D1-1d (5+10) e Glu-B1-2a (7+8), uma subunidade de LMW: Glu-A3d, a presença ou ausência de translocação 1B/1R: pela presença de Glu-B1 (braço longo do cromossomo de trigo) ou de &#969;-secalin (gene do braço curto do centeio) e puroindolina (Pinb-D1b). Também foram empregadas as metodologias de SDS-PAGE, SE-HPLC e teste de panificação. Como resultado da aplicação de marcadores obteve-se presença nos genótipos de: 71% para subunidade Glu-D1-1d (5+10), 16% para Glu-B1-2a (7+8), 60% para Glu-B1 (ausência de translocação) e 8% para Pinb-D1b e Glu-A3d. Os resultados de SDS-PAGE apontam uma prevalência das subunidades 2* e 1 no cromossomo 1A; 7+8, 7+9 e 17+18 no 1B; e 5+10 no 1D. Enquanto, os valores médios de SE-HPLC foram de 35,99% e 44,99% para as frações de proteínas poliméricas totais (PPP) e não-extraíveis (UPP), respectivamente, e 1,29 para a relação entre gliadinas e gluteninas (GLI/GLU), com variação significativa entre os genótipos (p&#8804;0,05). O teste de panificação também apontou diferença significativa (p&#8804;0,05) entre as cultivares nas mesmas condições. Os dados de UPP foram independentes dos dados da proporção de PPP, significando que a porção de proteínas poliméricas não-extraíveis (UPP) não depende de maior proporção de proteínas poliméricas totais. Enquanto houve correlação positiva entre o escore dado às subunidades de HMW, avaliadas por SDS-PAGE, e os valores de UPP; isto indica que a pontuação aparentemente vem sendo eficaz em classificar os genótipos de melhor qualidade. As cultivares sem translocação com centeio também obtiveram melhores resultados para UPP, PPP e GLI/GLU em relação as que a possuem. A relação GLI/GLU está correlacionada ao volume específico dos pães, mas aparentemente não garantiu boa qualidade nas características qualitativas, o que pode indicar a ação de gliadinas na extensibilidade, mas com falha na estruturação da rede de glúten. Esses resultados corroboram para uma seleção a favor das subunidades 5+10 de HMW, de cultivares sem translocação com centeio, com alto valor de UPP e razão apropriada de GLI/GLU, quando se objetiva melhorar a qualidade de panificação do trigo.

Nach den Ergebnissen der zweiten Nationalen Verzehrsstudie sind in Deutschland bereits 66 % der Männer und 51 % der Frauen übergewichtig (BMI > 25) oder adipös (BMI > 30) [BMELV, 2008]. Bisher auf dem Markt befindliche „Light-Lebensmittel“ mit Fettaustausch- bzw. Fettersatzstoffen weisen jedoch häufig sensorische Mängel auf. Im Kooperationsprojekt „Pflanzliche Fettaustauschstoffe aus sphärischen Proteinmizellen“ (Universität Leipzig: Institut für Lebensmittelhygiene; Freising: Fraunhofer IVV) wurde ein Lupinenproteinisolat entwickelt, welches micellare Strukturen mit hydrophober Oberfläche ausbilden kann und sich aufgrund seiner fettähnlichen Eigenschaften als neuer proteinbasierter Fettaustauschstoff in Lebensmitteln eignet. Aufgrund der geringen mikrobiologischen Stabilität und einer hohen Belastung mit sporenbildenden Bakterien, z. T. Bacillus cereus, waren jedoch Maßnahmen zur Entkeimung der Rohstoffe sowie des Proteinisolats notwendig. Die Arbeit stellt diese Maßnahmen und deren Einfluss auf die mikrobiologische Beschaffenheit sowie sensorische, technofunktionelle und ausgewählte ernährungsphysiologische Eigenschaften dar. In der vorliegenden Arbeit wurde eine physikalische Methode der Saatgutentkeimung etabliert (130 °C/60 min), welche die mikrobielle Stabilisierung des lupinenproteinbasierten Fettaustauschstoffes sicherstellte, wobei die sensorische Qualität (Geschmack, Cremigkeit, Farbe) nur minimal, die ernährungsphysiologische (in-vitro-Verdaubarkeit, Maillard-Produkte, Polyphenolgehalt) jedoch nicht beeinflusst wurde. Starke Veränderungen der technofunktionellen Eigenschaften (z. B. Gelbildung, Wasserbindung, Emulgierbarkeit, Schaumbildung etc.) konnten sowohl im positiven als auch im negativen Sinne nicht beschrieben werden. Lichtmikroskopische Aufnahmen und Untersuchungen der Proteine mittels SDS-PAGE und DSC bestätigten eine nur geringfügige Beeinflussung der micellaren Struktur und Proteinzusammensetzung. Die Anwendung als Fettaustauschstoff in Lebensmitteln würde somit nicht beeinträchtigt. Der Einfluss der Saatgutbehandlung auf das Protein war wesentlich geringer als eine direkte thermische Behandlung des Proteinisolats. Im Hinblick auf den Gesamtprozess sollte eine Pasteurisierung der feuchten Proteinisolate im nichtproteinschädigenden Temperaturbereich (75 °C/5 min) dennoch durchgeführt werden, um während des Prozesses eingetragene Mikroorganismen zu inaktivieren.