Yap Hong Kin. / "April 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 102-116) / 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.

Lau, Shong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 124-129). / Abstracts in English and Chinese. / Abstract of thesis --- p.i / 碩士論文摘要 --- p.iii / Acknowledgement --- p.iv / Declaration --- p.v / Abbreviations --- p.vi / Genetic symbols --- p.viii / Table of content --- p.ix / List of figures --- p.xiii / List of tables --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Thesis outline --- p.1 / Chapter 1.2 --- A. chrysogenum --- p.3 / Chapter 1.3 --- Antibiotic industry --- p.4 / Chapter 1.4 --- Cephalosporins --- p.4 / Chapter 1.5 --- CPC biosynthetic pathway --- p.5 / Chapter 1.6 --- DAC --- p.8 / Chapter 1.7 --- Aims of study --- p.10 / Chapter Chapter 2 --- Construction of transformation cassettes --- p.11 / Chapter 2.1 --- Introduction --- p.11 / Chapter 2.2 --- Materials and Methods --- p.13 / Chapter 2.2.1 --- Construction of cassette RTCAH --- p.13 / Chapter 2.2.1.1 --- Cultivation of R. toruloides --- p.13 / Chapter 2.2.1.2 --- Preparation of R. toruloides genomic DNA --- p.13 / Chapter 2.2.1.3 --- PCR of R. toruloides CAH gene --- p.14 / Chapter 2.2.1.4 --- Construction of pRTCAHhyr --- p.16 / Chapter 2.2.2 --- Construction of cassette GHG --- p.17 / Chapter 2.3 --- Results and Discussion --- p.18 / Chapter 2.3.1 --- pGHG construction --- p.18 / Chapter 2.3.2 --- pRTCAHhyr construction --- p.18 / Chapter Chapter 3 --- Optimization of integrative transformation of A. chrysogenum --- p.22 / Chapter 3.1 --- Introduction --- p.22 / Chapter 3.2 --- Materials and Methods --- p.24 / Chapter 3.2.1 --- Strain and culture medium --- p.24 / Chapter 3.2.2 --- Reagents --- p.24 / Chapter 3.2.3 --- Standard transformation procedures --- p.25 / Chapter 3.2.3.1 --- Cell cultivation --- p.25 / Chapter 3.2.3.2 --- Protoplast preparation --- p.25 / Chapter 3.2.3.3 --- PEG mediated protoplast fusion --- p.27 / Chapter 3.2.4 --- Examination of transformation parameters --- p.28 / Chapter 3.3 --- Results and Discussion --- p.29 / Chapter 3.3.1 --- Cell growth period --- p.30 / Chapter 3.3.2 --- DNA concentration --- p.32 / Chapter 3.3.3 --- PEG molecular weight --- p.35 / Chapter 3.3.4 --- Transformation additives --- p.37 / Chapter 3.3.5 --- Modified transformation protocol --- p.39 / Chapter Chapter 4 --- Metabolic engineering of A. chrysogenum --- p.42 / Chapter 4.1 --- Introduction --- p.42 / Chapter 4.2 --- Materials and Methods --- p.43 / Chapter 4.2.1 --- Transformation of A. chrysogenum --- p.43 / Chapter 4.2.2 --- Screening of transformants --- p.43 / Chapter 4.2.3 --- HPLC analysis --- p.43 / Chapter 4.2.4 --- A. chrysogenum genomic DNA preparation --- p.44 / Chapter 4.2.5 --- Genotyping by PCR --- p.45 / Chapter 4.2.5.1 --- GHG transformants --- p.45 / Chapter 4.2.5.2 --- RTCAH transformants --- p.47 / Chapter 4.2.6 --- Genotyping of GHG transformants by Southern hybridization --- p.47 / Chapter 4.2.6.1 --- Preparation of DIG-labeled GHG probe by PCR --- p.48 / Chapter 4.2.6.2 --- PCR preparation of DIG-labeled Cef-EF probe --- p.48 / Chapter 4.2.6.3 --- Restriction digestion of genomic DNA of GHG transformants --- p.49 / Chapter 4.2.6.4 --- Agarose gel electrophoresis and DNA transfer to positively charged nylon membrane --- p.50 / Chapter 4.2.6.5 --- Pre-hybridization and hybridization --- p.52 / Chapter 4.2.6.6 --- Membrane washing and blocking --- p.52 / Chapter 4.2.6.7 --- Membrane detection --- p.53 / Chapter 4.2.7 --- Genotyping of RTCAH transformants by Southern hybridization --- p.53 / Chapter 4.2.7.1 --- PCR preparation of DIG-labeled RTCAH probe --- p.54 / Chapter 4.2.7.2 --- Restriction digestion of genomic DNA of RTCAH transformants --- p.54 / Chapter 4.2.7.3 --- Agarose gel electrophoresis and Southern hybridization of RTCAH transformants --- p.55 / Chapter 4.3 --- Results and Discussion --- p.56 / Chapter 4.3.1 --- Hygromycin B screening and HPLC analysis of GHG transformants --- p.56 / Chapter 4.3.2 --- Genotyping of GHG232 by PCR --- p.56 / Chapter 4.3.3 --- Genotyping of GHG232 by Southern hybridization --- p.58 / Chapter 4.3.4 --- Hygromycin B screening and HPLC analysis of RTCAH transformant --- p.61 / Chapter 4.3.5 --- Genotyping of RTCAH transformants by PCR --- p.61 / Chapter 4.3.6 --- Genotyping of RTCAH transformants by Southern hybridization --- p.63 / Chapter Chapter 5 --- Characterization of modified strains and fermentation study --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- Materials and Methods --- p.67 / Chapter 5.2.1 --- Comparison of DAC production in GHG232 and RTCAH transformants by small-scale fermentation --- p.67 / Chapter 5.2.2 --- CPC conversion assay --- p.67 / Chapter 5.2.3 --- Evaluation of fermentation media --- p.68 / Chapter 5.2.4 --- Factorial design for medium formulation --- p.68 / Chapter 5.3 --- Results and Discussion --- p.71 / Chapter 5.3.1 --- Evaluation of DAC production profiles of GHG232 and RTCAH transformants --- p.71 / Chapter 5.3.2 --- CPC conversion assay --- p.79 / Chapter 5.3.3 --- Medium formulation --- p.81 / Chapter 5.3.4 --- Factorial design for medium formulation --- p.85 / Chapter 5.3.4.1 --- CSL and NH4OAc --- p.85 / Chapter 5.3.4.2 --- CaC03 --- p.91 / Chapter 5.3.4.3 --- Sucrose --- p.94 / Chapter 5.3.4.4 --- Starch and soy oil --- p.97 / Chapter 5.3.4.5 --- Methionine --- p.99 / Chapter Chapter 6 --- Conclusive remarks --- p.101 / Appendix: Study of reusability of commercial plasmid extraction kit --- p.103 / Chapter A1 --- Introduction --- p.103 / Chapter A2 --- Materials and Methods --- p.105 / Chapter A2.1 --- Preparation of competent E. coli DH5a cells --- p.105 / Chapter A2.2 --- Transformation and cultivation ofE. coli DH5a cells --- p.106 / Chapter A2.3 --- Column and solutions for plasmid DNA preparation --- p.107 / Chapter A2.4 --- Plasmid preparation --- p.108 / Chapter A2.5 --- Regeneration and storage of DNA extraction column --- p.109 / Chapter A2.6 --- Yield and quality assessment of the prepared plasmid DNA --- p.109 / Chapter A3 --- Results and Discussion --- p.111 / Chapter A3.1 --- Plasmid DNA yield and purity comparison --- p.111 / Chapter A3.2 --- Column regeneration and EtOH storage --- p.111 / Chapter A3.2.1 --- DNA yield after column regeneration --- p.111 / Chapter A3.2.2 --- Purity of plasmid DNA --- p.112 / Chapter A3.2.3 --- Restriction digestion --- p.117 / Chapter A3.2.4 --- E. coli DH5α cells transformation --- p.121 / Chapter A4 --- Conclusive remarks --- p.123 / Bibliography --- p.124

No description available.

This thesis describes an investigation of the mechanism of the bifunctional, a-ketoglutarate dependent dioxygenase, deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS), which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of this to deacetylcephalosporin C (DAC). The conversion of the unnatural substrate 3-exomethylene cephalosporin C by DAOC/DACS has been investigated in detail. A new metabolite was isolated from incubations of the deuterated [4-²H]-3-exomethylene cephalosporin C, and was identified as the 3β-spiroepoxide cepham, (2Ṟ,3Ṟ,6Ṟ,7Ṟ)-l-aza-[2-²H]-3-spiroepoxy-7-[(5Ṟ)-5-amino- 5-carboxypentanamido]-8-oxo-5-thiabicyclo[4.2.0]octane-2-carboxylic acid. The results obtained indicate that this metabolite is a shunt product whose formation is enhanced by the operation of a deuterium kinetic isotope effect on an enzyme-bound intermediate. It has also been found that this 3β-spiroepoxide cepham is further converted by DAOC/DACS to 3-formyl cephalosporoate products. The mechanism of oxygenation of DAOC/DACS was investigated through ¹⁸O-labelling studies. Incubations of [2-¹³C,3-²H]penicillin N and [4-²H]-3-exomethylene cephalosporin C with DAOC/DACS were carried out under ¹⁸O₂ or in H₂¹⁸O. Incorporation of ¹⁸O-label into the products [3-¹³C]DAC, [3-¹³C,4-²H]-3β-hydroxycepham and 3β-spiroepoxide cepham was observed from both sources. The results suggest that intermediates capable of oxygen-exchange are formed during the enzymatic reactions. Two substrate analogues, the 5-epipenicillin N and the 2β-difluoromethyl penicillin N, have been synthesised in order to probe the substrate specificity of DAOC/DACS with respect to the ring-expansion activity. The 5-epipenicillin N was not accepted as a substrate by DAOC/DACS, and the observations made indicate that it was unstable under the incubation conditions. No product was either observed from incubations of the 2β-difluoromethyl penicillin N with DAOC/DACS, although bioassay tests suggested a cephem product had been formed in very small amounts. Finally, the results of a substrate specificity comparison between the soluble recombinant enzymes deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS) from Cephalosporium acremonium and deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus are described.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2007. / Härtill 4 uppsatser.

Made available in DSpace on 2014-06-11T19:25:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-04Bitstream added on 2014-06-13T20:33:02Z : No. of bitstreams: 1 pedroso_tm_me_arafcf_parcial.pdf: 91662 bytes, checksum: f5c6c2b00880819f56fc5a7a78654f9d (MD5) Bitstreams deleted on 2015-04-13T18:26:27Z: pedroso_tm_me_arafcf_parcial.pdf,Bitstream added on 2015-04-13T18:27:06Z : No. of bitstreams: 1 000712772.pdf: 1022206 bytes, checksum: 44846063b688caab618840158e9fc515 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os antimicrobianos têm um papel fundamental no controle de doenças infecciosas. As cefalosporinas se mantêm como uma classe de antimicrobianos que se sobressaem pela sua importância terapêutica, elevada frequência de utilização, segurança e efetividade contra um amplo espectro microbiano. A cefazolina sódica (CFZ) é um agente antimicrobiano β-lactâmico, classificada como cefalosporina de primeira geração, muito utilizado como agente terapêutico e na profilaxia perioperatória. Neste trabalho foram desenvolvidos novos métodos analíticos para análise qualitativa e para a quantificação de cefazolina sódica, visando a obtenção de métodos mais rápidos, mais seguros para os operadores, mais econômicos e de fácil execução, que são essenciais para a análise desta cefalosporina na indústria farmacêutica. Na análise qualitativa, a cefazolina foi estudada quanto a sua solubilidade, ponto de fusão, pH, espectrofotometria na região do ultravioleta (UV) e na região de infravermelho (IV), cromatografia em camada delgada e cromatografia líquida de alta eficiência (CLAE), possibilitando a identificação da amostra. Para quantificação do fármaco três métodos foram validados. O método espectrofotométrico na região do ultravioleta foi validado utilizando o comprimento de onda de 270 nm, com faixa linear de concentração de 8 a 28 μg/mL utilizando água purificada como solvente, o teor foi de 99,10% e a exatidão foi de 100,56%. Ensaio microbiológico por método turbidimétrico utilizou o micro-organismo Staphylococcus aureus ATCC 26923, com faixa linear de 6 a 11,46 μg/mL e apresentou potência de 100,07% e exatidão foi 99,92%. A validação do método por CLAE foi realizada empregando fase móvel composta por água purificada e acetonitrila (60:40 v/v), com pH 8 ajustado com trietilamina (TEA), no comprimento de onda... / The antimicrobials have a crucial role in the control of infectious diseases. Cephalosporins remain one class of antimicrobials that stand out for their therapeutic importance, high frequency of use, safety and effectiveness against a broad microbial spectrum. Cefazolin sodium (CFZ) is a β-lactam antimicrobial agent, classified as first-generation cephalosporin, often used as a therapeutic agent and for perioperative prophylaxis. In this work, new analytical methods were developed for qualitative and quantitative analysis of cefazolin sodium in lyophilized powder, seeking obtain method faster, safer for operators, more economical and easiness of implementation, which are essential for the analysis of this cephalosporin in pharmaceutical industry. For qualitative analysis, several methods were performed, including analyzes of solubility, melting point and pH; ultraviolet (UV) and infrared (IR) spectrophotometry; thin layer chromatography and high performance liquid chromatography (HPLC) allowing the identification of samples. For quantification of the drug, three analytical methods were validated. The spectrophotometric method in the ultraviolet region was validated at 270 nm, with a linear range of concentration from 8 to 28 mg/mL, using purified water as solvent, the content of 99.10% accuracy of 100.56%. In the microbiological assay by turbidimetric method, Staphylococcus aureus ATCC 26923 was used as microrganism test, with linear range from 6 to 11.46 mg/mL, the method presented potency of 100.07% and accuracy of 99,92%. Validation of the HPLC method consisted of mobile phase composed of purified water and acetonitrile (60:40 v/v), adjusted to pH 8 with triethylamine (TEA), and detection wavelength of 270 nm, yielding a retention time of 3.6 minutes in the linear range evaluated from 30 to 80 μg/mL, content... (Complete abstract click electronic access below)

Orientador: Hérida Regina Nunes Salgado / Banca: Armando da Silva Cunha Junior / Banca: Marlus Chorilli / Resumo: Os antimicrobianos têm um papel fundamental no controle de doenças infecciosas. As cefalosporinas se mantêm como uma classe de antimicrobianos que se sobressaem pela sua importância terapêutica, elevada frequência de utilização, segurança e efetividade contra um amplo espectro microbiano. A cefazolina sódica (CFZ) é um agente antimicrobiano β-lactâmico, classificada como cefalosporina de primeira geração, muito utilizado como agente terapêutico e na profilaxia perioperatória. Neste trabalho foram desenvolvidos novos métodos analíticos para análise qualitativa e para a quantificação de cefazolina sódica, visando a obtenção de métodos mais rápidos, mais seguros para os operadores, mais econômicos e de fácil execução, que são essenciais para a análise desta cefalosporina na indústria farmacêutica. Na análise qualitativa, a cefazolina foi estudada quanto a sua solubilidade, ponto de fusão, pH, espectrofotometria na região do ultravioleta (UV) e na região de infravermelho (IV), cromatografia em camada delgada e cromatografia líquida de alta eficiência (CLAE), possibilitando a identificação da amostra. Para quantificação do fármaco três métodos foram validados. O método espectrofotométrico na região do ultravioleta foi validado utilizando o comprimento de onda de 270 nm, com faixa linear de concentração de 8 a 28 μg/mL utilizando água purificada como solvente, o teor foi de 99,10% e a exatidão foi de 100,56%. Ensaio microbiológico por método turbidimétrico utilizou o micro-organismo Staphylococcus aureus ATCC 26923, com faixa linear de 6 a 11,46 μg/mL e apresentou potência de 100,07% e exatidão foi 99,92%. A validação do método por CLAE foi realizada empregando fase móvel composta por água purificada e acetonitrila (60:40 v/v), com pH 8 ajustado com trietilamina (TEA), no comprimento de onda... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The antimicrobials have a crucial role in the control of infectious diseases. Cephalosporins remain one class of antimicrobials that stand out for their therapeutic importance, high frequency of use, safety and effectiveness against a broad microbial spectrum. Cefazolin sodium (CFZ) is a β-lactam antimicrobial agent, classified as first-generation cephalosporin, often used as a therapeutic agent and for perioperative prophylaxis. In this work, new analytical methods were developed for qualitative and quantitative analysis of cefazolin sodium in lyophilized powder, seeking obtain method faster, safer for operators, more economical and easiness of implementation, which are essential for the analysis of this cephalosporin in pharmaceutical industry. For qualitative analysis, several methods were performed, including analyzes of solubility, melting point and pH; ultraviolet (UV) and infrared (IR) spectrophotometry; thin layer chromatography and high performance liquid chromatography (HPLC) allowing the identification of samples. For quantification of the drug, three analytical methods were validated. The spectrophotometric method in the ultraviolet region was validated at 270 nm, with a linear range of concentration from 8 to 28 mg/mL, using purified water as solvent, the content of 99.10% accuracy of 100.56%. In the microbiological assay by turbidimetric method, Staphylococcus aureus ATCC 26923 was used as microrganism test, with linear range from 6 to 11.46 mg/mL, the method presented potency of 100.07% and accuracy of 99,92%. Validation of the HPLC method consisted of mobile phase composed of purified water and acetonitrile (60:40 v/v), adjusted to pH 8 with triethylamine (TEA), and detection wavelength of 270 nm, yielding a retention time of 3.6 minutes in the linear range evaluated from 30 to 80 μg/mL, content... (Complete abstract click electronic access below) / Mestre

On December 7, 1972, a hearing was held in Washington, D.C. on the use and abuse of antimicrobials. Senator Gaylor Nelson of the Sub-committee on the Monopoly of the Select Committee on Small Business stated, "Antibiotics are among the most frequently prescribed drugs in this country exceeded only by the psychoactive drugs." Dr. Charles C. Edwards, former Commissioner of the Food and Drug Administration, recognized that a problem existed and recommended the establishment of a National Task Force on the clinical use of antimicrobials (Kunin, et al., 1973). The following examples of illustrate problems the medical profession faces with antimicrobials. Of the 33 million patients discharged from general hospitals in 1972, 27% received one or more antibiotics during their hospital stay. The totals almost 9 million patients receiving antibiotics during the course of the year (McGowan, 1976). Dr. John Porterfield of JCAH recognized the Pharmacy-Therapeutics and Infection Control Committees as the formal organizational elements with the ultimate responsibility for formulating drug usage studies and overall administration of a quality assurance program. It was the opinion of Zeman et al., (1974), that in a large hospital (more than 500 beds) a separate Antibiotic Utilization Committee may more efficiently handle the volume of data and work. To implement this monitoring system, Brodie and Smith, (1976), recommended five drug utilization review principles: (1) authority, (2) operational and demographic characteristics of the delivery setting and service population, (3) knowledge of the existing pattern of utilization, (4) comparison of the later with local standards, and (5) evaluation of the impact of review on utilization patterns. Pierpaoli, et al., (1976), suggested that, conceptually a monitoring program could include utilization of retrospective and prospective chart review processes, complimented by a formal education program, and in-house controls on the use of certain antibiotics. A monitoring system could consist of evaluating antibiotics in three possible ways: (1) evaluate the usage of an antibiotic, or a family of antibiotics, in all medical-surgical cases, (2) evaluate one type of medical or surgical problem and review prophylactic and therapeutic use of all antibiotics, or (3) evaluate the usage of one antibiotic in one type of medical or surgical problem. The method of studying one antibiotic in one type of clinical condition might have some advantages since the number of variables is much smaller than either of the other two systems. I decided to use this approach and concentrate an open-heart surgery in which the cephalosporins have been used prophylactically.

Objectives: There is debate on whether the use of third-generation cephalosporins (3GC) increases the risk of clinical failure in bloodstream infections (BSIs) caused by chromosomally-mediated AmpC-producing Enterobacterales (CAE). This study evaluates the impact of definitive 3GC therapy versus other antibiotics on clinical outcomes in BSIs due to Enterobacter, Serratia, or Citrobacter species. Methods: This multicenter, retrospective cohort study evaluated adult hospitalized patients with BSIs secondary to Enterobacter, Serratia, or Citrobacter species from 1 January 2006 to 1 September 2014. Definitive 3GC therapy was compared to definitive therapy with other non-3GC antibiotics. Multivariable Cox proportional hazards regression evaluated the impact of definitive 3GC on overall treatment failure (OTF) as a composite of in-hospital mortality, 30-day hospital readmission, or 90-day reinfection. Results: A total of 381 patients from 18 institutions in the southeastern United States were enrolled. Common sources of BSIs were the urinary tract and central venous catheters (78 (20.5%) patients each). Definitive 3GC therapy was utilized in 65 (17.1%) patients. OTF occurred in 22/65 patients (33.9%) in the definitive 3GC group vs. 94/316 (29.8%) in the non-3GC group (p = 0.51). Individual components of OTF were comparable between groups. Risk of OTF was comparable with definitive 3GC therapy vs. definitive non-3GC therapy (aHR 0.93, 95% CI 0.51–1.72) in multivariable Cox proportional hazards regression analysis. Conclusions: These outcomes suggest definitive 3GC therapy does not significantly alter the risk of poor clinical outcomes in the treatment of BSIs secondary to Enterobacter, Serratia, or Citrobacter species compared to other antimicrobial agents.

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