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The 55K product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation. Both biochemical and genetic approaches have been used to show that this 496-residue (496R) protein of adenovirus (Ad5) is phosphorylated at both serine and threonine residues at sites near the carboxy terminus within sequences characteristic of a casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased virus replication and reduced the efficiency of transformation of primary baby rat kidney cells, suggesting that phosphorylation at these sites is of importance in the regulation of 55K biological activity. / Thesis / Master of Science (MS)

In this study the structural characteristics and antibacterial activities of two peptides derived from the carboxy-terminal of a tick defensin were investigated. Two defensin isoforms (OsDef1 and OsDef2) were previously identified in the midgut of the tick, Ornithodoros savignyi. Both OsDef1 and OsDef2 were found to be active against Gram-positive bacteria but showed no antibacterial activity against Gram-negative bacteria. OsDef2 was found to be slightly more active than OsDef1 and was, therefore, used as the template for the design of smaller antimicrobial peptides. Two peptide analogues were synthesised using the carboxy-terminal sequence of OsDef2 and differed in that in the one peptide the cysteine residues were present (Os) and in the other the cysteine residues were omitted (Os-C). Structurally, Os contained more α-helical properties than Os-C, whereas Os-C was more β-sheeted when prepared in 25 mM SDS. Both Os and Os-C showed no antibacterial activity when tested in Luria-Bertani broth or Mueller-Hinton broth indicating that the activities of Os and Os-C were influenced by the presence of broth salts and proteins. When tested in sodium phosphate buffer, both Os and Os-C exhibited Gram-positive and Gram-negative antibacterial activity. Os was slightly more active than Os-C against 3 of the 4 tested strains, with minimum bactericidal concentrations (MBCs) ranging from 0.94 μg/ml to 3.75 μg/ml. Os retained bactericidal activity against both Staphylococcus aureus and Escherichia coli when tested in the presence of 100 mM NaCl or 30% human serum. Os-C retained activity against E. coli in the presence of NaCl but became inactive in 30% human serum against both bacterial strains. At the MBCs, Os exhibited faster killing kinetics than Os-C killing both Bacillus subtilis and E. coli within 5 min, whereas Os-C took up to 120 min and 60 min, respectively. SYTOX Green permeabilization assays showed that both Os and Os-C caused permeabilization of E. coli membranes after 30 min exposure. At high peptide concentrations, both Os and Os-C were shown to interact with plasmid DNA. Both Os and Os-C exhibited no cytotoxic effects against SC-1 and Caco-2 cell lines, even at peptide concentrations 32 times higher than the highest MBC. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Biochemistry / unrestricted

Tsang Wai Kai, Paul. / "July 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 133-157). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

In order to facilitate a biophysical analysis of the carboxy terminal acidic transactivation domain (AAD) of Vmw65 from Herpes Simplex Virus Type 1 (HSV-1), an overexpression system in Escherichia coli was constructed and optimized to produce milligram quantities of this polypeptide. Purification of the polypeptide was facilitated by creating a fusion protein to glutathione S-transferase (GST) from Schizosoma japonicum using a commercially available vector. Upon thrombin digestion of the fusion protein, the carrier and AAD products were resolved by anion-exchange chromatography. With typically 15 mg of AAD available from a 12 litre culture, several biophysical studies were initiated. Circular dichroism and fluorescence spectroscopy both described a polypeptide with an extended structure reminicent of a random-coil; that is, it did not possess substantial quantities of known elements of secondary structure such as a-helicies and β-sheets under physiological conditions. A new structure high in α-helical content was induced upon addition of trifluoroethanol to mimic a hydrophobic milieu. Ultracentrifugation data supported the spectroscopic observations by describing an extended, monomeric polypeptide. The ultimate goal of the study, a teritiary structure, was sought by attempting to crystallize AAD with popular salts and organic solvents. Biologically, the described random-coil structure of AAD could be relevant to its role as a promoter and stablizer of the transcriptional pre-initation complex, the determining step in gene expression. A structurally labile domain would support AAD’s ability to interact with several targets including TFIID and TFIIB, though not necessarily by similar mechanisms. The requirement for a drastic conformational change such as a random-coil to α-helical transition currently remains unclear though observations made in this study of AAD in trifluoroethanol have shown that a conformational change is indeed possible. With a means of producing large quantities of AAD, the opportunity now arises to study its interaction with available cloned targets. The ensuing biophysical studies will then provide a greater understanding of AAD’s important role in gene expression. / Thesis / Master of Science (MSc)

Conformationally constrained bicyclic amino acids are invaluable in the synthesis of natural products and peptidomimetics. Aeruginosins contain novel bicyclic amino acid, 2--‐carboxy--‐6--‐hydroxyl octahydrindole (Choi) as the core structure. Aeruginosins are tetra peptide serine protease inhibitors isolated from marine sponges and cyanobacterial water blooms. Rigid bicyclic amino acid(Choi)is an essential core structure, which strongly influences biological activity of aeruginosin family members. Aeruginosins showed promising inhibitory activity against thrombin, trypsin, and factor VIIa. Thrombin and factor VIIa play a major role in blood clotting cascade; excessive coagulation leads to thrombosis and other cardiovascular diseases. Several research groups have reported a number of synthetic aeruginosin analogs. In this thesis, some of the synthetic methodologies of bicyclic amino acid core of aeruginosins are presented. Importance of bicyclic amino acids in peptidomimetic synthesis and drug designing is presented. Mainly, syntheses of ring oxygenated Choi analogs starting from glucose and mannose are presented.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A Cannabis sativa L. é a droga de abuso de uso proscrito mais consumida no mundo. O consumo desta droga por condutores de veículo automotor está associado com o aumento do risco de acidentes de trânsito, com o aumento da gravidade e com o aumento das taxas de mortalidade. O objetivo deste trabalho foi propor métodos analíticos aplicáveis à rotina laboratorial forense para monitoramento do consumo de canábis por condutores. Para tanto, foi utilizada como metodologia a extração líquido-líquido seguida de análise por cromatografia em fase gasosa acoplada a detector de massas para pesquisa de canabinoides em matrizes biológicas como sangue total (ST) e fluido oral (FO). A análise de 11-nor-9-carbóxi-delta9-THC (THC-COOH) em amostras de ST por cromatografia gasosa envolve a derivatização desta molécula e, consequentemente, do THC extraído desta matriz (quando presente), uma vez que ambos estão presentes no mesmo extrato, constituindo mais uma etapa analítica e que requer maior controle das condições de reação. Por outro lado, a detecção de THC em FO pode ser realizada sem a realização desta etapa, o que constitui uma vantagem analítica. A determinação de THC-COOH e THC em ST não demonstrou repetibilidade, o que inviabilizou as análises qualitativas e quantitativas nesta matriz. A detecção de THC em FO se mostrou uma análise simples e passível de validação, porém com limite de detecção (200ng/mL) acima do recomendado pelas guias forenses internacionais (2 ng/mL). A metodologia analítica desenvolvida se mostrou compatível com aplicação na análise confirmatória em casos de intoxicação aguda pelo consumo de canábis, demonstrando a necessidade de utilização de técnicas de concentração de amostras como extração em fase sólida ou microextração em fase sólida, para obtenção de menores limites de detecção, podendo assim ser aplicado na rotina laboratorial para o monitoramento do uso frequente de canábis, e não apenas em casos de intoxicação aguda. Além disso, foi realizada uma abordagem sobre a interpretação da detecção de THC em diferentes matrizes biológicas. / Cannabis sativa L. is the illegal drug of abuse most consumed in the world. The consumption of this drug by motor vehicle drivers is associated with an increased risk of traffic accidents, increased severity and increased mortality rates. The objective of this work was to propose analytical methods applicable to forensic laboratories to verify the consumption of cannabis by drivers. Liquid-liquid extraction followed by gas chromatography (GC) coupled to mass spectrometry (MS) detector has been applied to cannabinoid analysis in biological samples such as whole blood (WB) and oral fluid (OF). The analysis of 11-nor-9-carboxy-delta9-THC (THC-COOH) in WB by GC required the derivatization of this molecule and also involved the derivatization of THC since both were extracted from the same sample. The derivatization constitutes another analytical step, which requires greater control of the reaction conditions. Fortunately, the detection of only THC in OF can be done without performing this step. The determination of THC-COOH and THC in WB did not demonstrate repeatability, which impaired the qualitative and quantitative analyzes in this matrix. The detection of THC in OF proved to be a simple analysis, that could be validated, but the limit of detection (200ng/mL) was higher than the recommended by the international forensic guides (2 ng/mL). The chromatographic method developed was compatible with the application of a confirmatory analysis in acute cannabis intoxication, demonstrating the need to use techniques of samples concentration such as SPE or SPME, in order to obtain lower limits of detection, thus being able to be applied in the laboratory routine for the monitoring of the frequent use of cannabis, and not only in cases of acute intoxication. In addition, it was made an approach of THC detection in different biological matrices.

A Cannabis sativa L. é a droga de abuso de uso proscrito mais consumida no mundo. O consumo desta droga por condutores de veículo automotor está associado com o aumento do risco de acidentes de trânsito, com o aumento da gravidade e com o aumento das taxas de mortalidade. O objetivo deste trabalho foi propor métodos analíticos aplicáveis à rotina laboratorial forense para monitoramento do consumo de canábis por condutores. Para tanto, foi utilizada como metodologia a extração líquido-líquido seguida de análise por cromatografia em fase gasosa acoplada a detector de massas para pesquisa de canabinoides em matrizes biológicas como sangue total (ST) e fluido oral (FO). A análise de 11-nor-9-carbóxi-delta9-THC (THC-COOH) em amostras de ST por cromatografia gasosa envolve a derivatização desta molécula e, consequentemente, do THC extraído desta matriz (quando presente), uma vez que ambos estão presentes no mesmo extrato, constituindo mais uma etapa analítica e que requer maior controle das condições de reação. Por outro lado, a detecção de THC em FO pode ser realizada sem a realização desta etapa, o que constitui uma vantagem analítica. A determinação de THC-COOH e THC em ST não demonstrou repetibilidade, o que inviabilizou as análises qualitativas e quantitativas nesta matriz. A detecção de THC em FO se mostrou uma análise simples e passível de validação, porém com limite de detecção (200ng/mL) acima do recomendado pelas guias forenses internacionais (2 ng/mL). A metodologia analítica desenvolvida se mostrou compatível com aplicação na análise confirmatória em casos de intoxicação aguda pelo consumo de canábis, demonstrando a necessidade de utilização de técnicas de concentração de amostras como extração em fase sólida ou microextração em fase sólida, para obtenção de menores limites de detecção, podendo assim ser aplicado na rotina laboratorial para o monitoramento do uso frequente de canábis, e não apenas em casos de intoxicação aguda. Além disso, foi realizada uma abordagem sobre a interpretação da detecção de THC em diferentes matrizes biológicas. / Cannabis sativa L. is the illegal drug of abuse most consumed in the world. The consumption of this drug by motor vehicle drivers is associated with an increased risk of traffic accidents, increased severity and increased mortality rates. The objective of this work was to propose analytical methods applicable to forensic laboratories to verify the consumption of cannabis by drivers. Liquid-liquid extraction followed by gas chromatography (GC) coupled to mass spectrometry (MS) detector has been applied to cannabinoid analysis in biological samples such as whole blood (WB) and oral fluid (OF). The analysis of 11-nor-9-carboxy-delta9-THC (THC-COOH) in WB by GC required the derivatization of this molecule and also involved the derivatization of THC since both were extracted from the same sample. The derivatization constitutes another analytical step, which requires greater control of the reaction conditions. Fortunately, the detection of only THC in OF can be done without performing this step. The determination of THC-COOH and THC in WB did not demonstrate repeatability, which impaired the qualitative and quantitative analyzes in this matrix. The detection of THC in OF proved to be a simple analysis, that could be validated, but the limit of detection (200ng/mL) was higher than the recommended by the international forensic guides (2 ng/mL). The chromatographic method developed was compatible with the application of a confirmatory analysis in acute cannabis intoxication, demonstrating the need to use techniques of samples concentration such as SPE or SPME, in order to obtain lower limits of detection, thus being able to be applied in the laboratory routine for the monitoring of the frequent use of cannabis, and not only in cases of acute intoxication. In addition, it was made an approach of THC detection in different biological matrices.

A Cannabis sativa L. é a droga de abuso de uso proscrito mais consumida no mundo. O consumo desta droga por condutores de veículo automotor está associado com o aumento do risco de acidentes de trânsito, com o aumento da gravidade e com o aumento das taxas de mortalidade. O objetivo deste trabalho foi propor métodos analíticos aplicáveis à rotina laboratorial forense para monitoramento do consumo de canábis por condutores. Para tanto, foi utilizada como metodologia a extração líquido-líquido seguida de análise por cromatografia em fase gasosa acoplada a detector de massas para pesquisa de canabinoides em matrizes biológicas como sangue total (ST) e fluido oral (FO). A análise de 11-nor-9-carbóxi-delta9-THC (THC-COOH) em amostras de ST por cromatografia gasosa envolve a derivatização desta molécula e, consequentemente, do THC extraído desta matriz (quando presente), uma vez que ambos estão presentes no mesmo extrato, constituindo mais uma etapa analítica e que requer maior controle das condições de reação. Por outro lado, a detecção de THC em FO pode ser realizada sem a realização desta etapa, o que constitui uma vantagem analítica. A determinação de THC-COOH e THC em ST não demonstrou repetibilidade, o que inviabilizou as análises qualitativas e quantitativas nesta matriz. A detecção de THC em FO se mostrou uma análise simples e passível de validação, porém com limite de detecção (200ng/mL) acima do recomendado pelas guias forenses internacionais (2 ng/mL). A metodologia analítica desenvolvida se mostrou compatível com aplicação na análise confirmatória em casos de intoxicação aguda pelo consumo de canábis, demonstrando a necessidade de utilização de técnicas de concentração de amostras como extração em fase sólida ou microextração em fase sólida, para obtenção de menores limites de detecção, podendo assim ser aplicado na rotina laboratorial para o monitoramento do uso frequente de canábis, e não apenas em casos de intoxicação aguda. Além disso, foi realizada uma abordagem sobre a interpretação da detecção de THC em diferentes matrizes biológicas. / Cannabis sativa L. is the illegal drug of abuse most consumed in the world. The consumption of this drug by motor vehicle drivers is associated with an increased risk of traffic accidents, increased severity and increased mortality rates. The objective of this work was to propose analytical methods applicable to forensic laboratories to verify the consumption of cannabis by drivers. Liquid-liquid extraction followed by gas chromatography (GC) coupled to mass spectrometry (MS) detector has been applied to cannabinoid analysis in biological samples such as whole blood (WB) and oral fluid (OF). The analysis of 11-nor-9-carboxy-delta9-THC (THC-COOH) in WB by GC required the derivatization of this molecule and also involved the derivatization of THC since both were extracted from the same sample. The derivatization constitutes another analytical step, which requires greater control of the reaction conditions. Fortunately, the detection of only THC in OF can be done without performing this step. The determination of THC-COOH and THC in WB did not demonstrate repeatability, which impaired the qualitative and quantitative analyzes in this matrix. The detection of THC in OF proved to be a simple analysis, that could be validated, but the limit of detection (200ng/mL) was higher than the recommended by the international forensic guides (2 ng/mL). The chromatographic method developed was compatible with the application of a confirmatory analysis in acute cannabis intoxication, demonstrating the need to use techniques of samples concentration such as SPE or SPME, in order to obtain lower limits of detection, thus being able to be applied in the laboratory routine for the monitoring of the frequent use of cannabis, and not only in cases of acute intoxication. In addition, it was made an approach of THC detection in different biological matrices.