Thesis (doctoral)--University of Stockholm, 1983. / Publications on which thesis is based are appended. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Roberta F. Colman, Dept. of Chemistry and Biochemistry. Includes bibliographical references.

Thesis (Ph. D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 98 pages. Includes Vita. Includes bibliographical references.

Orientadores : Carmen Silvia Passos Lima, Fernando Ferreira Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-02T07:57:10Z (GMT). No. of bitstreams: 1 Nascimento_Helvia_M.pdf: 15695574 bytes, checksum: 707cf2971721d8745f1510ed82343542 (MD5) Previous issue date: 2002 / Resumo: As enzimas do sistema da glutationa S-transferase (GST) mediam a exposição a agentes citotóxicos e genotóxicos e parecem compromissadas com a susceptibilidade ao câncer. Os genes GSTM1 e GSTT1 apresentam um genótipo variante, a deleção homozigótica, no qual o gene inteiro está ausente. A associação dos genótipos do GST com o risco de ocorrência do câncer colorretal (CC) não está completamente esclarecida. Por outro lado, uma maior freqüência do polimorfismo 0104N do gene CaL 18A1, um inibidor da angiogênese, foi observada em pacientes com adenocarcinoma prostático, quando comparados a indivíduos normais, sugerindo que sua presença possa influenciar o desenvolvimento de tumores sólidos dependentes da angiogênese, como o CC. Neste estudo, nós testamos se a deleção homozigótica do genes GSTM1 e GSTT1 e o polimorfismo 0104N alteram o risco de adenocarcinoma colorretal esporádico (ACE). Para cumprir tais objetivos, o ONA genômico de 102 pacientes com ACE e 300 controles foi analisado por meio da reação em cadeia da polimerase e digestão enzimática. As freqüências da deleção dos genes GSTM1 (49,9%) e GSTT1 (16,6%) em pacientes foram similares àquelas observadas em controles (44,6 e 17,3%, respectivamente). Não foram também observadas diferenças significativas entre as freqüências da deleção combinada dos genes em pacientes e controles (8,8% vs 8,0%). A observação de riscos de 1,03 (IC 95%: 0,96-1,10) e 1,08 (IC 95%: 0,99-1,18) associados com as deleções isoladas dos genes GSTM'/ e GSTT1, respectivamente (P=0,45 e P=0,08), e de 1,18 (IC 95%: 0,47-2,90) associado com a deleção combinada dos genes (P=0,74), sugere que a ausência hereditária desta via de detoxificação de carcinógenos não teve importância na determinação do ACE em nossos casos. Ainda, as freqüências do polimorfismo 0104N foram similares em pacientes e controles (15,7% e 14,0%, respectivamente; P=0,80). O risco de 0,98 (IC 95%: 0,89-1,08), associado com este polimorfismo do gene CaL 18A1, sugere que ele também não influenciou a susceptibilidade ao ACE em nossos casos. Entretanto, a deleção do gene GSTT1 foi mais comum em pacientes com idade menor do que 60 anos, quando comparados àqueles com idade maior do que 60 anos (28,8% vs 4,0%, respectivamente; P=0,001), sugerindo que este genótipo possa ter influenciado a idade de manifestação da doença em nossa amostra / Abstract: It has been postulated that the glutathione S-transferase (GST) enzymes mediate the exposure to cytotoxic and genotoxic agents and may be involved in susceptibility to cancer. 80th GST mu 1 (GSTM1) and GST theta 1 (GSTT1) genes have a null variant allele, in which the entire gene is absent. The association of the GST null genotype and the risk of developing colorectal cancer (CC) is not yet fully clarified. On the other hand, higher frequency of the polymorphism 0104N of the gene CaL 18A1, um inhibitor of angiogenesis, was seen in prostatic adenocarcinoma in comparison with contrais, suggesting that the gene abnormality may influence the developing of solid tumours dependent of angiogenesis, such as CC. In this study, we tested whether the null genotypes for GSTM1 and GSTT1 genes and 0104N mutation altered the risk for the sporadic colorectal adenocarcinoma (SCA). For this purpose, genomic ONA from 102 SCA patients and 300 contrais were analysed by polymerase chain reaction and restriction digestion. The frequencies of GSTM1 (49.9%) and GSTT1 (16.6%) null genotypes in patients were similar to those observed in contrais (44.6 and 17.3%, respectively). No significant differences in the null combined genotype frequencies were also found between patients and controls (8.8% vs 8.0%). The observation of a 1.03 (95%CI: 0.96-1.10) and 1.08-fold (95%CI: 0.99-1.18) risk associated with the GSTM1 and GSTT1 null genotypes, respectively (P=0.45 and P=0.08) and a 1.18-fold (95%CI: 0.47-2.90) risk associated with the combined null genotype (P=0.74), suggests that the inherited absence of this carcinogen detoxification pathway was an unimportantdeterminant of the SCA in our cases. In addition, the frequencies of 0104N polymorphism were also similar in patients and contrais (15.7% and 14.0%, respectively; P=0.80). A 0.98-fold (95%CI: 0.89-1.08) risk associated with this CaL 18A1 gene polymorphism suggests that it did not influence the susceptibility for SCA in our cases. However, GSTT1 null genotype was more common in patients who were diagnosed before the age of 60 years than in those who were diagnosed at an older age (28.8% vs 4.0%, respectively; P=0.001), suggesting that this genotype could had influenced the age of onset of the disease in our cases / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Acyl Coenzyme A: Isopenicillin N Acyl Transferase (AT) is a key enzyme in the biosynthesis of β-lactam antibiotics in penicillin producing organisms such as P. chrysogenum and A. nidulans. Its natural activity is to exchange the side chain of the low activity antibiotic IPN [18] for the phenylacetyl side chain resulting in the more active antibiotic Penicillin G [5]. The biosynthesis of β-lactams has been exploited towards producing these compounds for therapeutic use. However, increasing bacterial resistance means new analogues in this compound class are constantly sought.As well as improving current production methods of β-lactam antibiotics, AT's broad substrate specificity means it could potentially play a role in the development and production of alternative β-lactam antibiotics that are able to overcome resistance.This thesis describes the identification of an AT mutant with improved acylation activity (AAT activity) via screening of an AT library using a previously developed screening method. Approaches towards the development of a method for the identification of AT mutants with improved hydrolysis activity were also explored. The main problem to overcome in developing such a screen is the inhibitory effect of 6-APA [1], the product of hydrolysis, on AT's IAT activity. The first approach investigated the potential of increasing the sensitivity of an assay by targeting AT to the periplasm. A second approach using β-lactamases to hydrolyse 6-APA [1] thus freeing up the active site of AT was also investigated.

Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. Mutants with deletions in two PEA transferase genes were significantly more susceptible to β-defensins, and the triple mutant was significantly more susceptible to both α- and β-defensins, but not LL-37; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface, suggesting these three genes contribute to the addition of positively charged moieties on the cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdtA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin may be more important than defensin resistance to H. ducreyi pathogenesis.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

No description available.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy. / A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is widely used as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an ad titional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible for-ration of significant amounts of 160 -kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, ultraviolet melting, differential scanning micro calorimetry , and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the umolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration with a ~GO(H20) = 26 ±1.7 kcal/mol. The conformational stability was unchanged in the presence of the leading antischistosomal drug Praziquantel, which bound the protein with a Kd = 9 ±1.8 p,M. The strong relationship observed between the m-v,llue and the size of the protein indicates that the amount of protem. surface exposed to solvent upon unfolding is the major structural de.erminant for the dependence of the protein's free energy of unfolding on urea concentration. 'Ihermograms obtained by differential scanning calorimetry also fitted to a two-state irreversible unfolding transition, both in the presence and absence of Praziquantel, with values of ~Cp = 1779 cal mol-IK-I , ~HcaI = 227 kcal/mol, AHVH ::::::233 kcal/mol (r :::::~:HVHIAlIcal = 1.02) and AS = 354 cal mol''K". The low ~Cp and ~S, when compared with the theoretically determined values, implied that the thermal denaturation of Sj26GST did not result in complete unfolding of the protein, / Andrew Chakane 2018

by Yuen Wai Keung. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 99-112). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Abbreviations --- p.vi / Table of Contents --- p.viii / Chapter chapter one --- introduction --- p.1 / Chapter 1.1 --- Glutathione S-transferase --- p.2 / Chapter 1.1.1 --- Introduction --- p.2 / Chapter 1.1.2 --- Classification of mammalian GST --- p.2 / Chapter 1.1.3 --- Classification of insect GST --- p.7 / Chapter 1.1.4 --- Substrate specificity --- p.11 / Chapter 1.2 --- The chironomidae --- p.13 / Chapter 1.2.1 --- Biology and life history of chironomidae --- p.13 / Chapter 1.3 --- Chironomidae larvae --- p.16 / Chapter 1.3.1 --- Bloodworm t --- p.6 / Chapter 1.3.2 --- Sources of chironomidae larvae --- p.17 / Chapter 1.4 --- Aim of research --- p.18 / Chapter chapter two --- materials and methods --- p.20 / Chapter 2.1 --- Screening of GST in different subcellular fractions --- p.21 / Chapter 2.1.1 --- Preparation of mitochondria --- p.21 / Chapter 2.1.2 --- Preparation of microsomes --- p.22 / Chapter 2.1.3 --- Preparation of cytosol --- p.22 / Chapter 2.2 --- Assay for GST activity --- p.23 / Chapter 2.2.1 --- Activity Units --- p.23 / Chapter 2.3 --- Protein assay --- p.23 / Chapter 2.4 --- Preparation of glutathione-affinity column --- p.25 / Chapter 2.5 --- Purification of cytosolic GSTs --- p.26 / Chapter 2.5.1 --- Preparation of cytosol --- p.26 / Chapter 2.5.2 --- Chromatography on Sephadex G25 --- p.26 / Chapter 2.5.3 --- Affinity Chromatography --- p.26 / Chapter 2.5.3.1 --- Specific elution of GSTs --- p.26 / Chapter 2.5.3.2 --- Non-specific elution of GSTs --- p.27 / Chapter 2.5.4 --- Fast Protein Liquid Chromatography with Mono Q --- p.27 / Chapter 2.6 --- Determination of molecular mass --- p.29 / Chapter 2.6.1 --- Subunit molecular mass --- p.29 / Chapter 2.6.2 --- Native molecular mass --- p.31 / Chapter 2.7 --- Isoelectric focusing PAGE --- p.31 / Chapter 2.8 --- Enzyme activities and kinetic studies --- p.34 / Chapter 2.8.1 --- Optimum pH --- p.34 / Chapter 2.8.2 --- Heat inactivation assay --- p.34 / Chapter 2.8.3 --- Km and Vmax --- p.34 / Chapter 2.8.4 --- Substrate specificity --- p.35 / Chapter 2.8.5 --- Glutathione peroxidase activity --- p.38 / Chapter 2.9 --- N-terminal amino acid sequence analysis --- p.39 / Chapter 2.9.1 --- Semidry electroblotting --- p.39 / Chapter 2.9.2 --- Staining of proteins on PVDF membrane --- p.40 / Chapter 2.9.3 --- N-terminal amino acid sequence analysis --- p.40 / Chapter 2.9.4 --- On-membrane deblocking of protein --- p.40 / Chapter 2.9.5 --- BLAST search --- p.41 / Chapter chapter three --- results --- p.42 / Chapter 3.1 --- Screening of GST in different subcellular fractions --- p.43 / Chapter 3.2 --- Purification of cytosolic GSTs by chromatography --- p.45 / Chapter 3.2.1 --- Sephadex G25 column --- p.45 / Chapter 3.2.2 --- GSH affinity column --- p.45 / Chapter 3.2.3 --- Mono-Q column --- p.45 / Chapter 3.3 --- Determination of molecular mass --- p.53 / Chapter 3.3.1 --- Subunit molecular mass --- p.53 / Chapter 3.3.2 --- Native molecular mass --- p.53 / Chapter 3.4 --- Isoelectric point determination --- p.53 / Chapter 3.5 --- Enzymes activities and kinetic studies --- p.57 / Chapter 3.5.1 --- Optimum pH --- p.57 / Chapter 3.5.2 --- Thermostability --- p.57 / Chapter 3.5.3 --- Km and Vmax --- p.57 / Chapter 3.5.4 --- Substrate specificity --- p.76 / Chapter 3.5.5 --- Glutathione peroxidase Activity --- p.76 / Chapter 3.6 --- N-terminal amino acid sequence analysis --- p.83 / Chapter chapter four --- discussion --- p.89 / Chapter 4.1 --- GST in different subcellular fractions --- p.90 / Chapter 4.2 --- Purification of cytosolic GST --- p.91 / Chapter 4.3 --- Physical properties --- p.93 / Chapter 4.3.1 --- Subunit molecular mass --- p.93 / Chapter 4.3.2 --- Native molecular mass --- p.93 / Chapter 4.3.3 --- Isoelectric point --- p.95 / Chapter 4.4 --- Kinetic properties --- p.94 / Chapter 4.4.1 --- Optimum pH --- p.94 / Chapter 4.4.2 --- Thermostability --- p.95 / Chapter 4.4.3 --- Km and Vmax --- p.95 / Chapter 4.4.4 --- Substrate specificity --- p.96 / Chapter 4.4.5 --- Glutathione peroxidase activity --- p.96 / Chapter 4.5 --- N-terminal amino acid sequence data --- p.97 / Chapter 4.6 --- Conclusion --- p.98 / references --- p.99