Neste estudo, o extrato bruto obtido da cultura do fungo E. nigrum foi extraído com hexano (HEX), diclorometano (DCM) e acetato de etila (AE). Após fracionamento por HPLC, a atividade antifúngica das frações foi avaliada pelo ensaio de concentração inibitória mínima (CIM) contra Candida albicans (CA), Trychophyton rubrum (TR), Cryptococcus neoformans (CN) e Aspergillus fumigatus (AF). O extrato de AE não foi efetivo, e os extratos HEX e DCM inibiram os patógenos na faixa de 31,25 a 250 µg/mL. Das frações hexânicas, apenas HEX-F9 apresentou atividade antifúngica, com CIM entre 31,25 e 250 mg/mL, exceto sobre AF. As frações DCM-F3, F5, F6, F9 e F10 apresentaram atividade antifúngica com CIM de 31,25 a 500 mg/mL contra todos os patógenos testados. A fração DCM-F9 foi efetiva com CIM entre 62,5 e 250 mg/mL contra CA, TR e CN. As frações DCM-F9 e DCM-F11 estão em fase final de purificação para posterior caracterização físico-química. / The aim of this study was to purify the crude extract produced from E. nigrum and characterize antifungal isolated molecules. E. nigrum was cultivated and the culture supernatant extracted with hexane (HEX), dichloromethane (DCM) and ethyl acetate (AE). Fractions were obtained by HPLC. The antifungal activity were evaluated by minimal inhibitory concentration (CIM) against C. albicans (CA), T. rubrum (TR), C. neoformans (CN) and A. fumigatus (AF). The HEX extract inhibited all pathogens with CIM from 31,25 to 62,5 mg/mL while DCM from 62,5 to 250 mg/mL. From HEX fractions only HEX-F9 showed antigungal activity with CIM from 31,25 to 250 mg/mL, except AF. The DCM-F3, F5, F6, F9 and F10 fractions showed antifungal activity, mainly DCM-F6 (CIM from 31,25 to 62,5 mg/mL against all pathogens) and DCM-F9 (CIM from 62,5 to 250 mg/mL against CA, TR and CN). DCM-F6 was not produced anymore by the fungus in subsequent cultures. DCM-F9 LC/MS, CG/MS and RMN results suggested impure fraction, making difficult to determine the main compound. New approaches are being considered.

Yeung Kai-shing, Elvis. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 98-104). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgement --- p.v / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / Abbreviations --- p.xiii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review / Chapter 2.1 --- Allergy to shellfish --- p.3 / Chapter 2.2 --- Identification and characterization of crustacean allergens --- p.7 / Chapter 2.3 --- Identification and characterization of mollusk allergens --- p.10 / Chapter 2.4 --- Cross-reactivity between shellfish allergens --- p.14 / Chapter 2.5 --- Epitopes of shellfish allergens --- p.17 / Chapter Chapter 3 --- Isolation and characterization of allergens in cephalopods / Chapter 3.1 --- Introduction --- p.22 / Chapter 3.2 --- Materials and methods --- p.25 / Chapter 3.2.1 --- Animals --- p.25 / Chapter 3.2.2 --- Sera --- p.25 / Chapter 3.2.3 --- Cephalopod tissue extracts --- p.25 / Chapter 3.2.4 --- Quantitation of protein --- p.27 / Chapter 3.2.5 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.27 / Chapter 3.2.6 --- Immunoblotting --- p.29 / Chapter 3.2.7 --- Immunological detection of IgE binding protein --- p.30 / Chapter 3.2.8 --- Controlling of ribonuclease (RNase) activity --- p.31 / Chapter 3.2.9 --- Isolation of total RNA --- p.31 / Chapter 3.2.10 --- Construction of cDNA library --- p.32 / Chapter 3.2.10.1 --- Synthesis of first-strand cDNA --- p.33 / Chapter 3.2.10.2 --- Amplification of cDNA by LD-PCR --- p.33 / Chapter 3.2.10.3 --- Polishing of ds cDNA --- p.34 / Chapter 3.2.10.4 --- Ligation of adaptor --- p.34 / Chapter 3.2.10.5 --- Phosphorylation of adaptor-ligated cDNA --- p.35 / Chapter 3.2.10.6 --- Size fractionation of cDNA --- p.35 / Chapter 3.2.10.7 --- Ligation of cDNA to vector --- p.36 / Chapter 3.2.10.8 --- Packaging of plasmids to bacteriophage --- p.36 / Chapter 3.2.10.9 --- Titration of the packaging reaction --- p.38 / Chapter 3.2.10.10 --- Amplification of the cDNA library --- p.39 / Chapter 3.2.11 --- Immunoscreening of octopus cDNA library --- p.40 / Chapter 3.2.12 --- Extraction of phage DNA --- p.42 / Chapter 3.2.13 --- Purification of cDNA insert from phage plasmid --- p.42 / Chapter 3.2.14 --- cDNA cloning and protein expression --- p.43 / Chapter 3.2.15 --- Nucleotide sequencing analysis of DNA --- p.45 / Chapter 3.2.16 --- Immunoblot analysis of the recombinant allergen --- p.46 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Immunoblotting of allergens in cephalopod --- p.47 / Chapter 3.3.2 --- RNA from octopus --- p.47 / Chapter 3.3.3 --- The cDNA library of octopus --- p.51 / Chapter 3.3.4 --- Immunoscreening of cDNA library from octopus --- p.56 / Chapter 3.3.5 --- Nucleotide sequencing determination and analysis --- p.61 / Chapter 3.3.6 --- Protein expression of Oct l I --- p.76 / Chapter 3.3.7 --- Immunoblot analysis of recombinant tropomyosin from octopus --- p.83 / Chapter 3.4 --- Discussion --- p.87 / Chapter Chapter 4 --- Conclusions --- p.96 / References --- p.98

Yuen, Yee Lok. / On t.p. "alpha" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 165-177). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiii / List of Figures --- p.xv / List of Tables --- p.xx / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Peroxisome proliferator-activated receptor (PPAR) --- p.1 / Chapter 1.1.1 --- PPARα isoforms --- p.1 / Chapter 1.2 --- PPARα ligands --- p.2 / Chapter 1.3 --- Biological roles of PPARα --- p.3 / Chapter 1.3.1 --- Lipid metabolism --- p.3 / Chapter 1.3.2 --- Bile acid metabolism --- p.4 / Chapter 1.3.3 --- Biotransformation --- p.6 / Chapter 1.4 --- Roles of PPARα in hepatocarcinogenesis --- p.7 / Chapter 1.4.1 --- Cell proliferation and apoptosis --- p.7 / Chapter 1.4.2 --- Oxidative stress --- p.8 / Chapter 1.5 --- Discovery of novel PPARα target genes --- p.9 / Chapter 1.5.1 --- Identification of a novel PPARα-regulated gene L5#55 by fluorescent differential mRNA display (FDD) analysis --- p.9 / Chapter 1.6 --- Sulfotransferase (SULT) --- p.15 / Chapter 1.7 --- Objective of the present study --- p.16 / Chapter Chapter 2 --- Molecular cloning and characterization of mouse liver sulfotransferase-like (mL-STL) gene --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and methods --- p.17 / Chapter 2.2.1 --- Animals --- p.17 / Chapter 2.2.2 --- Treatments --- p.18 / Chapter 2.2.3 --- Total RNA extraction --- p.18 / Chapter 2.2.3.1 --- Materials --- p.18 / Chapter 2.2.3.2 --- Methods --- p.19 / Chapter 2.2.4 --- Rapid amplification of cDNA ends (RACE) --- p.19 / Chapter 2.2.4.1 --- Materials --- p.19 / Chapter 2.2.4.2 --- Methods --- p.20 / Chapter 2.2.4.2.1 --- Primer design --- p.20 / Chapter 2.2.4.2.2 --- Rapid amplification of 5'- and 3'-cDNA ends --- p.20 / Chapter 2.2.5 --- Cloning of the 5'- and 3' RACE products --- p.25 / Chapter 2.2.5.1 --- Materials --- p.25 / Chapter 2.2.5.2 --- Methods --- p.25 / Chapter 2.2.6 --- Northern blot analysis --- p.28 / Chapter 2.2.6.1 --- Materials --- p.28 / Chapter 2.2.6.2 --- Methods --- p.28 / Chapter 2.2.6.2.1 --- Formaldehyde-agarose gel electrophoresis and blotting of RNA --- p.31 / Chapter 2.2.6.2.2 --- PCR DIG-labeling --- p.31 / Chapter 2.2.6.2.3 --- Hybridization and signal detection --- p.32 / Chapter 2.2.7 --- Reverse transcription (RT)-PCR --- p.34 / Chapter 2.2.7.1 --- Materials --- p.34 / Chapter 2.2.7.2 --- Methods --- p.34 / Chapter 2.3 --- Results and discussion --- p.37 / Chapter 2.3.1 --- Cloning of the full-length mL-STL cDNA --- p.37 / Chapter 2.3.2 --- In silico analysis of the mL-STL cDNAs --- p.50 / Chapter 2.3.3 --- Genomic organization of the mL-STL gene --- p.61 / Chapter 2.3.4 --- Tissue distribution of mL-STL mRNA transcript --- p.68 / Chapter 2.3.5 --- "PPARα-dependent regulation of mL-STL mRNA expression by fasting and Wy-14,643 treatment" --- p.74 / Chapter Chapter 3 --- Identification of the native mL-STL protein in mouse liver --- p.86 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Materials and methods --- p.87 / Chapter 3.2.1 --- Animal and treatments --- p.87 / Chapter 3.2.2 --- Cloning of the mL-STL cDNA into a modified pRSET (mpRSET) expression vector --- p.88 / Chapter 3.2.2.1 --- Materials --- p.88 / Chapter 3.2.2.2 --- Methods --- p.88 / Chapter 3.2.2.2.1 --- Amplification of mL-STL cDNA fragments --- p.88 / Chapter 3.2.2.2.2 --- Preparation of mpRSET expression vector --- p.92 / Chapter 3.2.2.2.3 --- "Ligation, transformation, and screening of recombinants" --- p.92 / Chapter 3.2.3 --- Over-expression of the mL-STL recombinant proteins in E coli strains --- p.94 / Chapter 3.2.3.1 --- Materials --- p.94 / Chapter 3.2.3.2 --- Methods --- p.94 / Chapter 3.2.4 --- Mass spectrometry analysis of the mL-STL recombinant proteins --- p.95 / Chapter 3.2.4.1 --- Materials --- p.96 / Chapter 3.2.4.2 --- Methods --- p.96 / Chapter 3.2.4.2.1 --- Trypsin digestion and peptide extraction --- p.96 / Chapter 3.2.4.2.2 --- Matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometry --- p.97 / Chapter 3.2.5 --- Purification of the mL-STL recombinant proteins --- p.98 / Chapter 3.2.5.1 --- Materials --- p.98 / Chapter 3.2.5.2 --- Methods --- p.98 / Chapter 3.2.5.2.1 --- Semi-purification of the mL-STL recombinant proteins by preparative SDS-PAGE --- p.98 / Chapter 3.2.5.2.2 --- Purification of mL-STL recombinant proteins by column chromatography --- p.99 / Chapter 3.2.6 --- Rabbit immunization using purified mL-STL recombinant proteins --- p.101 / Chapter 3.2.7 --- Subcellular fractionation of mouse liver by ultracentrifugation --- p.101 / Chapter 3.2.7.1 --- Materials --- p.101 / Chapter 3.2.7.2 --- Methods --- p.102 / Chapter 3.2.8 --- Western blot analysis of the native mL-STL protein --- p.104 / Chapter 3.2.8.1 --- Materials --- p.104 / Chapter 3.2.8.2 --- Methods --- p.104 / Chapter 3.2.8.2.1 --- SDS-PAGE and electro-blotting of proteins --- p.104 / Chapter 3.2.8.2.2 --- Immunostaining and signal detection --- p.105 / Chapter 3.3 --- Results and discussion --- p.106 / Chapter 3.3.1 --- Cloning of the mL-STLl and mL-STL2 cDNAs into a modified pRSET (mpRSET) vector --- p.106 / Chapter 3.3.2 --- IPTG induction of the mpRSET-mL-STL protein expression --- p.106 / Chapter 3.3.3 --- Confirmation of mL-STL recombinant proteins by mass spectrometry --- p.118 / Chapter 3.3.4 --- Purification of mL-STL recombinant proteins for rabbit immunization and polyclonal antisera production --- p.130 / Chapter 3.3.5 --- Antigenicity of mL-STL antisera --- p.134 / Chapter 3.3.6 --- Identification of mL-STL native protein and its induction pattern in mouse liver --- p.139 / Chapter 3.3.7 --- "Time-course of fasting and Wy-14,643 treatment on the mL- STLl native protein expression" --- p.147 / Chapter Chapter 4 --- Overall discussion --- p.153 / Future study --- p.163 / References --- p.165 / "Appendix A. Alignment of nucleotide sequences of mouse chromosome 7，Riken2810007J24, mL-STLl, and mL-STL2 cDNA sequences" --- p.178 / Appendix Bl. Theoretical tryptic peptide masses of mpRSET- mL-STLl protein --- p.217 / Appendix B2. Raw data from mass spectrometry analysis of mpRSET-mL-STLl protein --- p.218 / Appendix C1. Residue molecular mass of amino acids --- p.219 / Appendix C2. Di-peptide table --- p.220 / Appendix D1. Theoretical tryptic peptide masses of mpRSET- mL-STL2 protein --- p.221 / Appendix D2. Raw data from mass spectrometry analysis of mpRSET-mL-STL2 protein --- p.222

Xu, Jian. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 238-255). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016).

Neste estudo, o extrato bruto obtido da cultura do fungo E. nigrum foi extraído com hexano (HEX), diclorometano (DCM) e acetato de etila (AE). Após fracionamento por HPLC, a atividade antifúngica das frações foi avaliada pelo ensaio de concentração inibitória mínima (CIM) contra Candida albicans (CA), Trychophyton rubrum (TR), Cryptococcus neoformans (CN) e Aspergillus fumigatus (AF). O extrato de AE não foi efetivo, e os extratos HEX e DCM inibiram os patógenos na faixa de 31,25 a 250 µg/mL. Das frações hexânicas, apenas HEX-F9 apresentou atividade antifúngica, com CIM entre 31,25 e 250 mg/mL, exceto sobre AF. As frações DCM-F3, F5, F6, F9 e F10 apresentaram atividade antifúngica com CIM de 31,25 a 500 mg/mL contra todos os patógenos testados. A fração DCM-F9 foi efetiva com CIM entre 62,5 e 250 mg/mL contra CA, TR e CN. As frações DCM-F9 e DCM-F11 estão em fase final de purificação para posterior caracterização físico-química. / The aim of this study was to purify the crude extract produced from E. nigrum and characterize antifungal isolated molecules. E. nigrum was cultivated and the culture supernatant extracted with hexane (HEX), dichloromethane (DCM) and ethyl acetate (AE). Fractions were obtained by HPLC. The antifungal activity were evaluated by minimal inhibitory concentration (CIM) against C. albicans (CA), T. rubrum (TR), C. neoformans (CN) and A. fumigatus (AF). The HEX extract inhibited all pathogens with CIM from 31,25 to 62,5 mg/mL while DCM from 62,5 to 250 mg/mL. From HEX fractions only HEX-F9 showed antigungal activity with CIM from 31,25 to 250 mg/mL, except AF. The DCM-F3, F5, F6, F9 and F10 fractions showed antifungal activity, mainly DCM-F6 (CIM from 31,25 to 62,5 mg/mL against all pathogens) and DCM-F9 (CIM from 62,5 to 250 mg/mL against CA, TR and CN). DCM-F6 was not produced anymore by the fungus in subsequent cultures. DCM-F9 LC/MS, CG/MS and RMN results suggested impure fraction, making difficult to determine the main compound. New approaches are being considered.

We studied the functional significance of marked differences in the DNA content of somatic cells and germ line nuclei by static Feulgen-DNA cytophotometry for several species of microcrustaceans that exhibit chromatin diminution during very early stages of embryogenesis. Mature females and males showed many gonadal nuclei with elevated amounts of DNA that persist until dispersal of this "extra" DNA throughout the cytoplasm as fragments and coalescing droplets of chromatin during anaphase of the diminution division.

INTRODUÇÃO: Onicomicose, infecção das unhas por fungo é a mais freqüente das doenças ungueais, constituindo aproximadamente metade de todas as alterações ungueais. Pode ser causada por dermatófitos, leveduras ou fungos filamentosos não dermatófitos. OBJETIVO: Caracterizar as onicomicoses causadas por fungos filamentosos não dermatófitos. (1) Verificar, dentre as suspeitas clínicas de onicomicose, qual a freqüência da recuperação de fungos,(2) Verificar, dentre as suspeitas clínicas de onicomicose, quais as espécies de fungos recuperadas, (3) Verificar, dentre o total das espécies identificadas, qual a freqüência das espécies de fungos filamentosos não dermatófitos. MÉTODOS: Duzentos e cinco indivíduos com suspeita clínica de onicomicose foram estudados no período de dezembro de 2003 a novembro de 2004, por meio de exames micológicos diretos e cultura para fungos. RESULTADOS: O diagnóstico de onicomicose foi estabelecido, pelo exame micológico direto, em 170 indivíduos. O diagnóstico etiológico foi estabelecido pela cultura para fungos. Dentre os 107 agentes identificados, os dermatófitos foram identificados em 78,52% (N=84), as leveduras em 13,08% (N=14) e os FFND em 8,40% (N=9) das vezes. CONCLUSÃO: É necessário que se estabeleça o diagnóstico etiológico dos casos de onicomicoses, já que os fungos filamentosos não dermatófitos ocorrem com freqüência considerável e são indistinguíveis daquelas por dermatófitos. / INTRODUCTION: Onychomycosis, a nail fungus infection is the most frequent nail disease, constituting about half of all nail disorder. It can be caused by dermatophytes, yeasts and non- dermatophytes filamentous fungi. OBJECTIVE: Characterize the onychomycosis caused by filamentous fungi non- dermatophytes. (1) Verify after a clinical suspicion of onychomycosis, which are the frequency of fungi recovery, (2) examine, after a clinical suspicion of onychomycosis, which species of fungi are recovered, (3) Checking, among the total of identified species , which are the frequency of the species of filamentous fungi not dermatophytes. METHODS: Two hundred and five patients with clinical suspicion of onychomycosis were studied in the period from December 2003 to November 2004, through direct mycological examination and culture for fungus. RESULTS: The diagnosis of onychomycosis has been established by direct mycological examination, in 170 individuals. The etiological diagnosis was established by the culture for fungus. Among the 107 persons identified, the dermatophytes were recovered in 78.52% (N = 84), the yeast in 13.08% (N = 14) and filamentous fungi non- dermatophytes in 8,40% (N = 9). CONCLUSION: It is necessary to establish the etiological diagnosis of the cases of onychomycosis, as filamentous fungi non- dermatophytes occur often than ever considered and are its clinic features are indistinguishable from those of the dermatophytes.

INTRODUÇÃO: Onicomicose, infecção das unhas por fungo é a mais freqüente das doenças ungueais, constituindo aproximadamente metade de todas as alterações ungueais. Pode ser causada por dermatófitos, leveduras ou fungos filamentosos não dermatófitos. OBJETIVO: Caracterizar as onicomicoses causadas por fungos filamentosos não dermatófitos. (1) Verificar, dentre as suspeitas clínicas de onicomicose, qual a freqüência da recuperação de fungos,(2) Verificar, dentre as suspeitas clínicas de onicomicose, quais as espécies de fungos recuperadas, (3) Verificar, dentre o total das espécies identificadas, qual a freqüência das espécies de fungos filamentosos não dermatófitos. MÉTODOS: Duzentos e cinco indivíduos com suspeita clínica de onicomicose foram estudados no período de dezembro de 2003 a novembro de 2004, por meio de exames micológicos diretos e cultura para fungos. RESULTADOS: O diagnóstico de onicomicose foi estabelecido, pelo exame micológico direto, em 170 indivíduos. O diagnóstico etiológico foi estabelecido pela cultura para fungos. Dentre os 107 agentes identificados, os dermatófitos foram identificados em 78,52% (N=84), as leveduras em 13,08% (N=14) e os FFND em 8,40% (N=9) das vezes. CONCLUSÃO: É necessário que se estabeleça o diagnóstico etiológico dos casos de onicomicoses, já que os fungos filamentosos não dermatófitos ocorrem com freqüência considerável e são indistinguíveis daquelas por dermatófitos. / INTRODUCTION: Onychomycosis, a nail fungus infection is the most frequent nail disease, constituting about half of all nail disorder. It can be caused by dermatophytes, yeasts and non- dermatophytes filamentous fungi. OBJECTIVE: Characterize the onychomycosis caused by filamentous fungi non- dermatophytes. (1) Verify after a clinical suspicion of onychomycosis, which are the frequency of fungi recovery, (2) examine, after a clinical suspicion of onychomycosis, which species of fungi are recovered, (3) Checking, among the total of identified species , which are the frequency of the species of filamentous fungi not dermatophytes. METHODS: Two hundred and five patients with clinical suspicion of onychomycosis were studied in the period from December 2003 to November 2004, through direct mycological examination and culture for fungus. RESULTS: The diagnosis of onychomycosis has been established by direct mycological examination, in 170 individuals. The etiological diagnosis was established by the culture for fungus. Among the 107 persons identified, the dermatophytes were recovered in 78.52% (N = 84), the yeast in 13.08% (N = 14) and filamentous fungi non- dermatophytes in 8,40% (N = 9). CONCLUSION: It is necessary to establish the etiological diagnosis of the cases of onychomycosis, as filamentous fungi non- dermatophytes occur often than ever considered and are its clinic features are indistinguishable from those of the dermatophytes.

No description available.

A novel probe was developed from genomic DNA of Helicobacter pylori ATCC type strain 43629. It hybridized with all 73 H. pylori clinical isolates tested but not with any of 183 non-H. pylori DNAs in dot blot hybridization. Typing tests revealed 41 different HaeIII-digestion patterns from 57 H. pylori strains tested. Based on the sequence of the probe, a nested PCR was developed that detected as little as 2 fg of H. pylori DNA or approximately equivalent to one cell. No PCR products were amplified from any of 21 non-H. pylori strains tested. Using this nested PCR, H. pylori DNA was detected in 33 of 45 (73%) saliva samples collected from patients with gastric H. pylori infection. These data suggest that the probe is useful for typing H. pylori and that the nested PCR is a valuable tool for detecting H. pylori DNA in saliva.