This thesis describes the use of nuclear magnetic resonance techniques to determine the structures of two proteins in solution, hen egg-white lysozyme and human interleukin-4. Using 2D ¹H methods an extensive set of structural data has been collected for hen lysozyme (1158 NOE distance restraints, 68 o and 24 _?1 dihedral angle restraints) and these data have been used in distance geometry-dynamical simulated annealing calculations to determine an ensemble of NMR structures for the protein. The overall Ca RMSD from the average for a set of 16 calculated structures is 1.8 ± 0.2 A but, excluding 14 residues in exposed disordered regions, this value reduces to 1.3 ± 0.2 Å. Regions of secondary structure, and particularly the four a helices, are well defined (Ca RMSD 0.8 ± 0.3 Å for helices). Detailed comparisons of the NMR structures with crystal structures of the protein have shown the close similarity of the main chain fold and the conformation of interior side chains in solution and in crystals. ³J_aß coupling constant measurement have indicated, however, that the conformational mobility of the side chains of many surface residues is significantly more pronounced than an individual crystal structure would suggest. For human interleukin-4, a strategy involving ¹⁵N and ¹³C labelled recombinant protein together with heteronuclear 3D NMR techniques has been employed to determine the structure of the protein. The work has provided the first structure for this protein, a left-handed four helix bundle with an up-up-down-down connectivity. For an ensemble of 10 final calculated NMR structures there is a Ca RMSD from the average of 1.6 ± 0.4 Å, the definition of the helical core of the protein being particularly good (0.8 ± 0.2 Å). There is, however, some disorder in the long overhand loops of the structure; this reflects the unusually high conformational mobility of these regions. Comparison of the interleukin-4 structure with proteins with related folds, particularly members of the haemopoietic cytokine superfamily, suggests that the fold found here for interleukin-4 may be the adopted structure throughout this cytokine superfamily. In a postscript to this thesis the NMR structure of human interleukin-4 is shown to have a very similar conformation to a crystal structure of the protein which has been solved very recently.

Chalkbrood, caused by Ascosphaera apis (Maassen ex Claussen) Olive & Spiltor, is a cosmopolitan fungal disease of honey bee larvae (Apis mellifera L.) for which there is no chemotherapeutic control. Using in vitro larval rearing methods, lysozyme-HCl, a food-grade antimicrobial extracted from hen egg albumen, was found to suppress chalkbrood at levels of 0.75-1.5% (g/mL) of larval diet. In field trials, lysozyme-HCl did not affect adult bee survival or brood production and did effectively suppress the development of chalkbrood disease. Daily chalkbrood mummy production decreased by a factor of 10 in colonies treated with three treatments of 6000 mg of lysozyme-HCl when compared with infected, untreated controls and reduced disease symptoms to levels observed in uninfected colonies. Honey production was also found to be significantly negatively correlated with increased disease severity. Lysozyme-HCl is a promising safe therapeutic agent for the control of chalkbrood in honey bee colonies.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Andrew D. Ellington. Vita. Includes bibliographical references. Also available from UMI.

Universiẗat, Diss., 2003--Bonn.

Misfolding and accumulation of insoluble protein aggregates in the form of amyloid fibrils is associated with a number of prevalent and debilitating mammalian disorders. In addition, amyloid-like nanostructures exhibit robust material properties, biological compatibility and replicative properties, making them of particular interest in the development of novel nanomaterials. Understanding fibril formation is essential to the development of strategies to control, manipulate or prevent fibril growth. The amyloid hypothesis is that since amyloid-like fibrils share a common core structure, they also share common formation mechanisms. Utilising a combination of turbidity and extrinsic fluorescence techniques this thesis provides insight into the diagnostic strength of simple, inexpensive kinetic measurements of aggregate growth. These simple techniques are found to be capable of delivering a substantial amount of information about the growth mechanisms controlling aggregation, and the effect of solution and environmental conditions, forming a solid basis for further investigation. Two competing fibrillar pathways are observed for hen egg white lysozyme at low pH in the presence of salt. These two pathways, leading to the formation of either curvilinear, worm-like fibrils or to the more widely recognised rigid, straight fibrils are not particular to hen egg white lysozyme, and similar competition may affect growth curve analysis in many other protein assays, including a-synuclein. Many proteins aggregate in the presence of membranes and detergents, and the kinetics of a-synuclein aggregation in the presence of SDS are strongly influenced by SDS concentration. Most descriptions of amyloid fibril growth currently lack heterogeneous nucleation events, and these may be important for predicting aggregation of membrane-active species in vivo. It is clear that simple analytical solutions to growth models are unable in many cases to capture the complexities of filament growth. Even in relatively simple in vitro experiments different growth processes can dominate growth rate over time, competing fibrillar species can result in composite kinetic growth signals and some growth mechanisms have not yet been sufficiently incorporated into an overall description of fibril growth.

Os compostos antimicrobianos naturais são um tema de grande interesse devido ao aumento da demanda por alimentos seguros e de alta qualidade. A utilização de lipossomas é uma alternativa interessante para proteger antimicrobianos nos alimentos, além de fornecer compostos naturais de liberação controlada. Os lipossomas revestidos com polissacarídeos apresentam melhor estabilidade, representando uma alternativa aos lipossomas convencionais. Inicialmente, os nanolipossomas que encapsulam a nisina foram preparados com fosfatidilcolina de soja (PC) e pectina ou ácido poligalacturônico. Os lipossomas desenvolvidos apresentaram alta eficiência de encapsulação, baixo índice de polidispersão e foram estáveis durante 21 dias a 7 °C e 25 °C. A atividade antimicrobiana foi observada contra cinco cepas diferentes de Listeria em placas de ágar de leite, com uma melhor eficiência contra L. innocua 6a. Em um segundo momento, as características estruturais dos lipossomas foram estudadas por dispersão de raios-X de pequeno ângulo (SAXS) e as amostras foram submetidas a ciclos de temperatura (20-60 °C). Para isso, os lipossomas foram desenvolvidos contendo pectina ou ácido poligalacturônico pelos métodos de hidratação de filme e evaporação em fase reversa, para encapsular nisina. A análise de SAXS confirmou a presença de estruturas lamelares em todas as amostras. Além disso, parte da estrutura multilamelar tornou-se cúbica, provavelmente devido à presença de nisina nos lipossomas. A adição de polissacarídeos mostrou diferenças entre as fases cúbicas formadas. Em última análise, a mistura de lisozima e nisina foi encapsulada em lipossomas contendo polissacarídeos. O diâmetro médio dos lipossomas foi de 85,6 e variou para 77,3 e 79,9 nm com a incorporação de pectina ou ácido poligalacturônico, respectivamente. O potencial zeta dos lipossomas com polissacarídeos foi de cerca de -30 mV, mostrando alta eficiência de encapsulação. A atividade antimicrobiana foi avaliada a 37 °C, mostrando que a PC-pectina reduziu a população de L. monocytogenes em 2 log UFC/mL e 5 log UFC/mL em leite integral e desnatado, respectivamente. Em refrigeração, a PC-pectina reduziu a população de L. monocytogenes para quase zero por até 25 dias em leite desnatado. Portanto, pode dizer-se que os lipossomas que contêm polissacarídeos podem ser uma tecnologia promissora para o encapsulamento da lisozima e nisina. Além disso, a existência de estrutura cúbica nos lipossomas pode proporcionar liberação controlada de antimicrobianos. / Natural antimicrobial compounds are a topic of utmost interest due to the increased demand for safe and high-quality foods. The use of liposomes is an interesting alternative to protect antimicrobials in food, also providing controlled release natural compounds. Polysaccharides coated liposomes present better stability, representing an alternative to conventional liposomes. Initially, nanoliposomes encapsulating nisin were prepared with soy phosphatidylcholine (PC) and pectin or polygalacturonic acid. The liposomes developed presented high encapsulation efficiency, low polydispersity index, and were stable for 21 days at 7°C and 25°C. The antimicrobial activity was observed against five different strains of Listeria in milk-agar plates, with a better efficiency against L. innocua 6a. In a second moment, structural characteristics of liposomes were studied by small angle X-ray scattering (SAXS) and the samples were submitted to temperature cycles (20-60°C). For this, liposomes were developed containing pectin or polygalacturonic acid by the thin-film hydration method and reverse phase evaporation method for nisin encapsulation. The analysis of SAXS confirmed the presence of lamellar structures in all the samples. In addition, part of the multilamellar structure became cubic, probably due to the presence of nisin in the liposomes. The addition of polysaccharides showed differences between the cubic phases formed. Ultimately, the mixture of lysozyme and nisin were encapsulated in liposomes containing polysaccharides. The mean diameter of the liposomes was 85.6 and varied to 77.3 and 79.9 nm with the incorporation of pectin or polygalacturonic acid, respectively. The zeta potential of liposomes with polysaccharides were around -30 mV, showing high encapsulation efficiency. The antimicrobial activity was assessed at 37 °C, showing that PC-pectin reduced the population of L. monocytogenes to 2 log CFU/mL and 5 log CFU/mL in whole and skim milk, respectively. At under refrigeration, PC-pectin reduced the population of L. monocytogenes to almost zero for up to 25 days in skim milk. Therefore, it can say that the liposomes containing polysaccharides can be a promising technology for the encapsulation of lysozyme and nisin. In addition, the existence of cubic structure in the liposomes can provide controlled release of antimicrobials.

Os compostos antimicrobianos naturais são um tema de grande interesse devido ao aumento da demanda por alimentos seguros e de alta qualidade. A utilização de lipossomas é uma alternativa interessante para proteger antimicrobianos nos alimentos, além de fornecer compostos naturais de liberação controlada. Os lipossomas revestidos com polissacarídeos apresentam melhor estabilidade, representando uma alternativa aos lipossomas convencionais. Inicialmente, os nanolipossomas que encapsulam a nisina foram preparados com fosfatidilcolina de soja (PC) e pectina ou ácido poligalacturônico. Os lipossomas desenvolvidos apresentaram alta eficiência de encapsulação, baixo índice de polidispersão e foram estáveis durante 21 dias a 7 °C e 25 °C. A atividade antimicrobiana foi observada contra cinco cepas diferentes de Listeria em placas de ágar de leite, com uma melhor eficiência contra L. innocua 6a. Em um segundo momento, as características estruturais dos lipossomas foram estudadas por dispersão de raios-X de pequeno ângulo (SAXS) e as amostras foram submetidas a ciclos de temperatura (20-60 °C). Para isso, os lipossomas foram desenvolvidos contendo pectina ou ácido poligalacturônico pelos métodos de hidratação de filme e evaporação em fase reversa, para encapsular nisina. A análise de SAXS confirmou a presença de estruturas lamelares em todas as amostras. Além disso, parte da estrutura multilamelar tornou-se cúbica, provavelmente devido à presença de nisina nos lipossomas. A adição de polissacarídeos mostrou diferenças entre as fases cúbicas formadas. Em última análise, a mistura de lisozima e nisina foi encapsulada em lipossomas contendo polissacarídeos. O diâmetro médio dos lipossomas foi de 85,6 e variou para 77,3 e 79,9 nm com a incorporação de pectina ou ácido poligalacturônico, respectivamente. O potencial zeta dos lipossomas com polissacarídeos foi de cerca de -30 mV, mostrando alta eficiência de encapsulação. A atividade antimicrobiana foi avaliada a 37 °C, mostrando que a PC-pectina reduziu a população de L. monocytogenes em 2 log UFC/mL e 5 log UFC/mL em leite integral e desnatado, respectivamente. Em refrigeração, a PC-pectina reduziu a população de L. monocytogenes para quase zero por até 25 dias em leite desnatado. Portanto, pode dizer-se que os lipossomas que contêm polissacarídeos podem ser uma tecnologia promissora para o encapsulamento da lisozima e nisina. Além disso, a existência de estrutura cúbica nos lipossomas pode proporcionar liberação controlada de antimicrobianos. / Natural antimicrobial compounds are a topic of utmost interest due to the increased demand for safe and high-quality foods. The use of liposomes is an interesting alternative to protect antimicrobials in food, also providing controlled release natural compounds. Polysaccharides coated liposomes present better stability, representing an alternative to conventional liposomes. Initially, nanoliposomes encapsulating nisin were prepared with soy phosphatidylcholine (PC) and pectin or polygalacturonic acid. The liposomes developed presented high encapsulation efficiency, low polydispersity index, and were stable for 21 days at 7°C and 25°C. The antimicrobial activity was observed against five different strains of Listeria in milk-agar plates, with a better efficiency against L. innocua 6a. In a second moment, structural characteristics of liposomes were studied by small angle X-ray scattering (SAXS) and the samples were submitted to temperature cycles (20-60°C). For this, liposomes were developed containing pectin or polygalacturonic acid by the thin-film hydration method and reverse phase evaporation method for nisin encapsulation. The analysis of SAXS confirmed the presence of lamellar structures in all the samples. In addition, part of the multilamellar structure became cubic, probably due to the presence of nisin in the liposomes. The addition of polysaccharides showed differences between the cubic phases formed. Ultimately, the mixture of lysozyme and nisin were encapsulated in liposomes containing polysaccharides. The mean diameter of the liposomes was 85.6 and varied to 77.3 and 79.9 nm with the incorporation of pectin or polygalacturonic acid, respectively. The zeta potential of liposomes with polysaccharides were around -30 mV, showing high encapsulation efficiency. The antimicrobial activity was assessed at 37 °C, showing that PC-pectin reduced the population of L. monocytogenes to 2 log CFU/mL and 5 log CFU/mL in whole and skim milk, respectively. At under refrigeration, PC-pectin reduced the population of L. monocytogenes to almost zero for up to 25 days in skim milk. Therefore, it can say that the liposomes containing polysaccharides can be a promising technology for the encapsulation of lysozyme and nisin. In addition, the existence of cubic structure in the liposomes can provide controlled release of antimicrobials.

A three-column ion-exchange system was designed, fabricated and computer-automated to accommodate a novel 'elution looping' process developed by Dr. Tim Durance (U.B.C. Department of Food Science) during his doctoral studies on the recovery of lysozyme and avidin. This processing technique enhances the simultaneous recovery of these two pharmaceutically important proteins from chicken egg albumen. The processing system prototype was sized to handle throughput rates between approximately five and 300 liters per day of albumen to facilitate both laboratory and small commercial scale work. Very efficient use is made of the ion-exchange resin due to a two-column cascaded feed arrangement. The processing control software was designed to provide flexibility and ease of operation in setting up new and existing method files, allowing for the selection of any column or group of columns to use and providing a 'staged-shutdown' approach toward handling columns fouled with congealed albumen during unattended operation. This approach attempts to maximize the productivity of the system even when one or two of the columns has become fouled with congealed albumen. / Applied Science, Faculty of / Chemical and Biological Engineering, Department of / Graduate

The possibility to orient a protein in space using an external electrical field was studied bymeans of molecular dynamics simulations. To model the possible conditions of an electrospray ionization (ESI) the protein Lysozyme in vacuum was considered under the influence ofdifferent field strengths. The simulations showed three distinct patterns: (1) the protein wasdenaturated when exposed to too strong electrical fields, above 1.5 V/nm; (2) the proteinoriented without being denaturated at field strengths between 0.5 V/nm and 1.5 V/nm (3) theprotein did not orient and did not denaturate if the strength of the field became to low, below0.5 V/nm. Our simulations show that the orientation of the protein in the fields correspondingto the second pattern takes place within time intervals from about 100 ps at 1.5 V/nm to about1 ns at 0.5 V/nm. We therefore predict, that there exists a window of field strengths, which issuitable for orientation of proteins in experimental studies without affecting their structure.The orientation of proteins potentially increases the amount of information that can beobtained from experiments such as single particle imaging. This study will therefore bebeneficial for the development of such modern techniques.

Orientadores: Albina Messias de Almeida Milani Altemani, Fernanda Viviane Mariano / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-26T21:33:07Z (GMT). No. of bitstreams: 1 ConchaGomez_CamilaAndrea_M.pdf: 7905371 bytes, checksum: d30a270e5a2b856cfa2e57fa9e5a3522 (MD5) Previous issue date: 2015 / Resumo: Introdução: Vários tumores de glândulas salivares imitam o sistema ductal, principalmente o ducto intercalado, tais como o adenoma pleomórfico, adenoma de células basais, carcinoma adenóide-cístico e carcinoma epitelial-mioepitelial. Contudo o adenocarcinoma polimorfo de baixo grau não apresenta ductos com dupla população celular como os outros descritos, mas é acreditado que este tumor é originado do ducto intercalado terminal. Em relação aos marcadores imunoistoquímicos, existem aqueles que detectam a expressão de enzimas e podem ser usados como marcadores funcionais de diferenciação celular, como lisozima e DOG1. Na glândula salivar normal, a lisozima é expressa no citoplasma das células luminais do ducto intercalado e em pequena quantidade nas células acinares, enquanto que o DOG1 é expresso na porção membranosa apical das células acinares e luminais do ducto intercalado. Objetivo: investigar a utilidade de lisozima e DOG1 na avaliação dos tumores salivares que se acredita sejam originados do ducto intercalado. Material e métodos: A expressão imunoistoquímica destes marcadores foi analisada em: 24 casos de Adenomas pleomórfico (AP), 10 Mioepiteliomas (M), 17 Tumores de Warthin (TW), 21 Adenocarcinomas polimorfo de baixo grau (APBG), 14 Carcinomas epitelial-mioepitelial CEME), 17 Carcinomas adenóide cístico (CAC), 14 Carcinomas secretor análogo da mama (MASC), 22 Carcinomas de células acinares (CCA), 11 Carcinomas mucoepidermóide (CM), 4 Carcinomas de ducto salivar (CDS), 7 Carcinomas mioepitelial (CMi). Resultados: a expressão luminal apical de DOG1 foi observada somente em: CCA, AP, APBG, CEME e CAC. Este padrão de DOG1 foi mais extenso e frequente no CCA; nos outros tumores foi mais variável, porém com maior expressão no CEME. Lisozima foi positiva em: MASC, CEME, AP, APBG e CDS. Somente nos 2 primeiros tumores, a expressão de lisozima foi encontrada em mais de 50% dos casos. Conclusões: As expressões apical luminal de DOG1 e de lisozima no AP, APBG, CEME e CAC possivelmente indicam diferenciação do tipo ducto intercalado, que é variável nestas neoplasias. Dentre elas, o CEME é que apresenta tal diferenciação com maior frequência e extensão. As expressões de DOG1 tipo apical luminal e de lisozima podem ser utilizadas no diagnóstico diferencial entre MASC e CCA, nos casos que apresentam dificuldade diagnóstica, uma vez que são fortemente expressas no primeiro e ausente no segundo / Abstract: Introduction: Several salivary tumors present a cellular composition that is similar to that of the intercalated duct, such as: Pleomorphic adenoma (PA), Basal cell adenoma (BCA), Adenoid cystic carcinoma (AdCC) and Epithelial-myoepithelial carcinoma (EMC). Regarding Polymorphic low grade adenocarcinoma (PLGA), although it does not present a dual cell composition as the other tumors, it is believed to be originated from the terminal intercalated duct. In relation to immunohistochemical markers, there are those that detect enzyme expression and can be used as functional marker of cellular differentiation, such as DOG1 and lysozyme. In normal salivary gland, lysozyme is expressed in the cytoplasm of luminal cells of intercalated duct and in small amount of acinar cells whereas DOG1 is expressed in the apical membrane of both acinar and luminal cells of intercalated ducts. Objetive: investigate the utility of DOG1 and lysozyme in the evaluation of salivary tumors. Material and methods: The immunohistochemical expression of these markers was analyzed in 24 cases of PA, 10 Myoepitheliomas (M), 17 Warthin tumors (WT), 21 PLGA, 14 EMC, 17 AdCC, 14 Mammary analogue secretory carcinoma (MASC), 22 Acinic cell carcinoma (ACC), 11 Mucoepidermoid carcinoma (MEC), 4 Salivary duct carcinoma (SDC) and 7 Myoepithelial carcinoma (MC). Results: Luminal apical expression of DOG1 was observed only in ACC, PA, PLGA, EMC and AdCC. This DOG1 pattern of expression was more frequent and extensive in ACC; in the other tumors this expression was variable but more marked in EMC. Lysozyme was detected in MASC, EMC, PA, PLGA and SDC. Only in MASC and EMC, lysozyme expression was found in more than 50% of the cases. Conclusions: Luminal apical expression of DOG1 and of lysozyme in PA, PLGA, EMC and AdCC probably represent intercalated duct differentiation, which is variable in these tumors. Among these tumors, EMC is the lesion where such differentiation occurs more frequently and extensively. Luminal apical expression of DOG1 and of lysozyme can be useful for distinguishing MASC from ACC because both protein are markedly expressed only in the former tumor / Mestrado / Patologia / Mestra em Estomatopatologia