Diss. (sammanfattning) Umeå : Umeå Universitet, 2007. / Härtill 3 uppsatser. I publikationen saknas serienummer.

CARVALHO, Eunice Bobô de. Efeito da modulação da glutamina, alanil-glutamina, ß-caroteno, zinco e do leite de cabra transgênico contendo lisozima humana, em células epiteliais intestinais sob ação da Escherichia coli enteroagregativa. 2011. 167 f. Tese (Doutorado em Ciências Médicas) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2011. / Submitted by denise santos (denise.santos@ufc.br) on 2014-02-17T12:03:16Z No. of bitstreams: 1 2011_tese_ebcarvalho.pdf: 2631693 bytes, checksum: 166da1c1df6395be1be97a064c00e23e (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2014-02-17T12:03:43Z (GMT) No. of bitstreams: 1 2011_tese_ebcarvalho.pdf: 2631693 bytes, checksum: 166da1c1df6395be1be97a064c00e23e (MD5) / Made available in DSpace on 2014-02-17T12:03:43Z (GMT). No. of bitstreams: 1 2011_tese_ebcarvalho.pdf: 2631693 bytes, checksum: 166da1c1df6395be1be97a064c00e23e (MD5) Previous issue date: 2011 / The enteric infections cause 2.5 million deaths each year. The Enteroaggregative Escherichia coli (EAEC) is associated with persistent cause of diarrheal diseases. This study examined in vitro (IEC-6, Caco-2 and HEp-2 cells) the role of the micronutrients glutamine (Glu), alanyl-glutamine (Ala-Glu), beta-carotene (ß-Carot), zinc (Zn), and the milk of transgenic goats containning human lysozyme (M-Lyso) and their respective controls (Ctrle) in the following assays: proliferation, migration, viability, apoptosis, cell necrosis, and bacterial adhesion in response to infection caused by the EAEC-042 bacterial strain at a concentration of 2.5 x 105 CFU/mL. The effect of infection by EAEC-042 bacterial strain was evidenced by significant reduction in migration (p <0.001) and cellular viability (p <0.001); also increased apoptosis (p <0.001) and necrosis (p <0.001) in response to damage to the intestinal epithelium. It was observed that the micronutrients in the presence of bacteria significantly reduced apoptosis and necrosis caused by EAEC-042, as well as significantly reduced bacterial adhesion and increases cell migration. The control and transgenic milk abolished bacterial adhesion (p <0.001), independent of milk fat, and significantly reduce apoptosis (p <0.001) and necrosis (p <0.001) caused by EAEC-042. The qualitative analysis of EAEC adherence, considered as gold standard method, showed a reduction in bacterial adherence associated with intervention with micronutrients when compared with the EAEC-042 infection control. In conclusion, our study demonstrates the importance of intervention with micronutrients and milk (transgenic or not) in protecting the intestinal epithelial challenged by bacterial aggression. / As infecções entéricas causam cerca de 2,5 milhões de mortes ao ano. A EAEC está associada à causa de doenças diarréicas persistentes. Este estudo analisou in vitro (IEC-6, Caco-2 e HEp-2), o papel dos micronutrientes glutamina, alanil-glutamina, ß-caroteno, zinco, e dos leites de cabra transgênico com lisozima humana e controle nos ensaios de proliferação, migração, viabilidade, apoptose, necrose celular, adesão bacteriana em resposta à infecção causada pela cepa de EAEC-042 na concentração de 2,5 x 105 UFC/mL. A cepa bacteriana de EAEC-042 mostrou redução significativa na migração (p<0,001) e na viabilidade celular (p<0,001) e esta aumentou a apoptose (p<0,001) e necrose (p<0,001) em resposta a lesão ao epitélio intestinal. Foi observado que os micronutrientes na presença da bactéria reduziram significativamente a apoptose e necrose ocasionados por esta, bem como reduziram significativamente a adesão bacteriana, além de aumentar a migração celular. Os leites controle e transgênico apresentaram redução significativa da adesão bacteriana (p<0,001), independente da presença da camada de gordura, além de reduzirem significativamente a apoptose (p<0,001) e a necrose (p<0,001) ocasionadas pela EAEC-042. A análise qualitativa de aderência celular, considerada padrão ouro, mostrou redução na aderência bacteriana quando associados aos micronutrientes, comparados ao controle com EAEC-042. Nota-se a quase ausência de aderência em ambos os leites. Este estudo mostra a importância dos micronutrientes e leite de cabra transgênico ou não, sobre a proteção epitelial intestinal nas agressões bacterianas.

A single column cation exchange method was developed which allowed simultaneous recovery of lysozyme and avidin from undiluted egg white using a unique elution sequence which involved accumulation of avidin on the column through several cycles of egg white application and lysozyme elution. Lysozyme was recovered with higher yields than reported for the isoelectric precipitation methods often used in the industry (86% vs 60 - 80%) and in high purity. Avidin recovery was also as good or better than that of previously reported ion exchange methods (74 - 80% vs 17 -80%). The purity of the avidin fraction (up to 40.9%) was superior to other reported primary avidin fractions. Avidin was shown to have a greater potential for both electrostatic and hydrophobic interactions with Duolite C-464 than lysozyme but under the conditions of this separation, electrostatic interactions were dominant. Secondary purification of avidin by carboxymethyl celluose cation exchange (CMC), gel filtration, metal chelate interaction chromatography (MCIC), aliphatic hydrophobic interaction chromatography (HIC), and Phenyl-Sepharose interaction chromatography (PSIC) each resulted in considerable increase in avidin purity. In terms of resin capacity, yields, and avidin purity however, CMC ion exchange was superior. A comparison of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andnative protein electrophoresis profiles gave clear evidence of protein - protein interaction between avidin and lysozyme in partially purified avidin preparations. This interaction may also occur between the native proteins in the egg white, but has not been demonstrated with certainty. The molar ratio of avidin to available biotin binding sites was estimated by 5 methods. For highly purified avidin samples the hydroxy azo benzoic acid (HABA) method proposed by Green (1965) was superior. A new method, utilizing an immobilized biotin column, which did not require extensive purification of avidin was found to give similar results. Finally, a highly sensitive assay of proteins bound to nitrocellulose membranes was developed which was capable of quantifying as little as 0.12 µg of protein. Membrane bound proteins were labeled with peroxidase via a biotin - avidin linkage as previously reported and bound peroxidase activity was related to initial protein content. The method was applicable to determination of the relative concentrations of different protein bands on Western blots of electrophoresis gels. / Land and Food Systems, Faculty of / Graduate

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1985. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1985. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Diss. (sammanfattning) Umeå : Umeå universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 98-111).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 196-228).

P22 lysozyme, encoded by gene 19, is an essential phage protein responsible for hydrolyzing the bacterial cell wall during lytic infection. P22 lysozyme is related to T4 lysozymein its mode of action, substrate specificities, and in its structure. Gene 19 was located on the phage genome, subcloned, and then sequenced. lysozyme was produced in large quantities and purified for biochemical characterization and for crystallograpic studies. Gene 19consists of 146 codons, and encodes a protein with a molecular weight of 16,117. Amber mutations were created in gene 19 by in vitro primer-directed mutagenesis. The mutations were crossed by homologous recombination onto the phage genome. The phages bearing the amber mutations in gene 19 were screened for the ability to grow on six different amber suppressor strains. Amino acid substitutions that resulted in nonfunctional or less functional lysozyme were determined. Of 60 possible amino acid substitutions at 11 different sites in P22 lysozyme, 20 are deleterious. The phage bearing amber mutations in gene 19that failed to grow on given suppressor strains were reverted and second site intragenic revertants were obtained. The mutations were sequenced. A substitution of serine for glutamine at residue 82 is compensated for by changing residue 46 from serine to leucine. This single change enables the phage to form a plaque at 300C but not at 400C. When the triple change asn42->lys; ser46->leu; and ser43->pro is present the lysozyme produced is no longer temperature sensitive. The crystal structure of P22 lysozyme is not yet solved. Assuming that the structures of T4 lysozyme and P22 lysozyme are similar, one can examine the positions of equivalent residues in the T4 lysozyme structure. The spatial arrangement of the residues changed by the secondary site mutations and the original substitution can then be visualized. The mutations discussed above all map far from the original mutation on the T4 three dimensional model. A substitution of leucine for tyrosine at position 22 is compensated for by the double mutation of arg18->ser and ser23->lys. When the equivalent residues are mapped on the T4 three dimensional model the changes map in close proximity to the original mutation.

In many vertebrate and invertebrate species mediators of innate immunity include antimicrobial peptides (AMPs) such as peptide fragments of histones and other proteins with previously ascribed different functions. Shark AMPs have not been described and this research examines the antibacterial activity of nurse shark (Ginglymostoma cirratum) peripheral blood leukocyte lysates. Screening of lysates prepared by homogenizing unstimulated peripheral blood leukocytes identified muramidase (lysozyme-like) and non-muramidase antibacterial activity. Lysates were tested for lysozyme using the lysoplate assays, and antibacterial (AB) activity was assayed for by a microdilution growth assay that was developed using Planococcus citreus as the target bacterium. Fractionation of crude lysates by ion exchange and affinity chromatography was followed by a combination of SDS-PAGE with LC/MS-MS and/or N-terminal sequence analysis of low molecular weight protein bands (kDa). This yielded several peptides with amino acid sequence similarity to lysozyme, ubiquitin, hemoglobin, human histones H2A, H2B and H4 and to antibacterial histone fragments of the catfish and the Asian toad. Not all peptide sequences corresponded to peptides potentially antibacterial. The correlation of a specific protein band in active lysate fractions was accomplished by employing the acid-urea gel overlay assays in which AB activity was seen as zones of growth inhibition on a lawn of P. citreus at a position corresponding to that of the putative AB protein band. This study is the first to describe putative AMPs in the shark and their potential role in innate immunity.