Hepatocellular carcinoma (HCC) is the most common malignant tumor in Taiwan with more than one million new cases annually in the world. Growth factors play important roles in liver carcinogenesis. Hepatoma-derived growth factor (HDGF), originally isolated from the cultured media of human hepatoma HuH-7 cells, stimulate the growth of fibroblast cells, endothelial cells, and hepatoma cells. Overexpression of HDGF is related to the transformation of human hepatoma, lung cancer and melanoma. Besides, HDGF also exerts strong influence on the prognosis of patients with hepatoma. In this study, recombinant HDGF was expressed and purified with higher purity than 90%. The recombinant protein was used to raise polyclonal HDGF antibodies in rabbits and to generate three lines of HDGF monoclonal antibodies in mice. After antibodies characterization, an in-house, sandwich HDGF ELISA system was established using the purified polyclonal anti-HDGF IgG as the capture antibodies and the monoclonal anti-HDGF IgG as the detection antibodies. By using recombinant HDGF as standard, this ELISA system accurately evaluate the changes of HDGF release in SK-Hep-1 cells after gene delivery. In addition, we also evaluated the effect of anti-HDGF on the growth of hepatoma cells. Application of either polyclonal or monoclonal HDGF antibodies, but not preimmune antibodies, inhibited the proliferation of SK-Hep-1 hepatoma cells in a dose-dependent manner. In summary, the present study generated HDGF monoclonal antibodies for development of HDGF ELISA and application on suppressing HCC progression. Future studies should be carried out to enhance the sensitivity of HDGF ELISA and to evaluate the therapeutic potential of HDGF antibodies for treatment of HCC.

Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2007. / Titre provenant de l'écran titre. Bibliogr. f. 48-50.

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Listeria monocytogenes is a bacterial pathogen that is typically transmitted to humans through consumption of contaminated foods. Infection with this organism can lead to a severe and life-threatening illness referred to as listeriosis. The goal of this study was to develop monoclonal antibodies (MAbs) with high specificity and affinity to proteins found on the surface of all strains of L. monocytogenes while not cross-reacting with non-pathogenic Listeria spp. or other major bacterial pathogens commonly found in foods. A literature search was conducted to identify ten candidate surface proteins involved or putatively involved in the virulence of L. monocytogenes. Bioinformatics analyses using BLAST on the NCBI website showed that five of the ten candidate proteins were potentially present in L. monocytogenes strains but absent from strains of other Listeria spp. Genes encoding for these five proteins, ActA, InlA, InlC2, InlJ and LapB, were cloned and expressed in Escherichia coli. MAbs were raised against recombinant LapB, InlJ and InlC2 proteins using hybridoma technology. A total of 48 anti-LapB, 33 anti-InlJ and 37 anti-InlC2 MAbs were developed. Based on the comparison of IFM signal of each MAb against L. monocytogenes cells, seven anti-LapB MAbs and six anti-InlC2 MAbs were selected for further characterization. All of the anti-InlJ MAbs showed weak IFM signals and negative reactivity in ELISA against L. monocytogenes cells. The selected anti-LapB and anti-InlC2 MAbs were further characterized by assessing their ability to bind to cells of 51 strains representing 11 L. monocytogenes serotypes using ELISA. Six anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, M3519) reacted strongly with 44 of 51 strains representing 9 of the 11 L. monocytogenes serotypes tested. Five anti-InlC2 MAbs (M3607, M3618, M3630, M3633, M3636) reacted strongly with 47 strains representing 10 of the 11 L. monocytogenes serotypes tested. These results indicate that anti-LapB and anti-InlC2 MAbs could potentially be used as diagnostic reagents for isolation and detection of almost all L. monocytogenes strains in contaminated foods.

This thesis deals with the issue of mycotoxins, which are important pathogens of many agricultural crops and may pose a high health risk to humans and livestock. It contains an overview of the most important mycotoxins their producers and mycotoxicoses. The main focus of this thesis is the occurrence of mycotoxins in cereals and the effect of processing it on their content. The experimental part focuses on monitoring the content and the penetration of deoxynivalenol and zearalenone in winter wheat from grain to bread. We examined a total of 10 samples of treated and 10 samples of untreated variants of winter wheat. For each sample grain, flour and bread were examined for DON and ZEA content and all tested samples were positive for DON and ZEA. None of the examined samples exceeded or even approached the legislative limits.

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Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-07-05Bitstream added on 2014-06-13T19:53:39Z : No. of bitstreams: 1 candido_tc_me_araca.pdf: 695625 bytes, checksum: 2f5b5c66c41e1d07fc364672891e34d8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A Leishmaniose visceral canina (LVC), conhecida como calazar, é uma antropozoonose endêmica no Brasil, causada pela Leishmania L. chagasi. O teste sorológico de ELISA tem sido empregado na rotina de inquéritos epidemiológicos e no auxílio diagnóstico clínico de cães suspeitos. O método de ensaio imunoenzimático em fase sólida (ELISA) usando o antígeno total de Leishmania L. chagasi (ELISA-AgT), assim como, o ELISA - Ligante de Fucose e Manose (ELISA-FML), tem demonstrado boa sensibilidade e especificidade para detectar a doença em cães assintomáticos e oligossintomáticos. O presente trabalho teve como objetivo comparar dois antígenos pelo método de ELISA no sorodiagnóstico de cães naturalmente infectados pela LVC, positivos no exame parasitológico, e agrupados em: grupo sintomático e, grupo oligossintomáticos, tendo como grupo controle cães de área não endêmica. Nos animais oligossintomáticos, a sensibilidade observada para ELISA-AgT foi de 86,7%, já para o método ELISA-FML apresentou valor de 90%. A especificidade foi de 100% no método de ELISA-AgT e 96,7 % para o ELISA-FML. Nos animais sintomáticos, a sensibilidade e especificidade para o ELISA-AgT foram de 90% e 93,3%, respectivamente; já o método de ELISA-FML apresentou sensibilidade e especificidade de 86,7% e 96,7%. No teste ELISA-AgT o valor preditivo positivo foi de 93,1% nos sintomáticos e 100% nos oligossintomáticos, enquanto que nos animais submetidos ao teste ELISA-FML, foi observado 96,3% para os sintomáticos e 96,4% para os oligossintomáticos. O índice Kappa foi utilizado para medir o grau de concordância real entre os dois métodos imunoenzimáticos empregados e o exame parasitológico direto, mostrando boa concordância entre os métodos realizados. / Visceral canine leishmaniasis or calazar is Brazilian an endemic antropozoonosis caused by Leishmania L. chagasi. ELISA sorologycal test has been used in routine of epidemiological studies and in diagnosis of clinical suspect dogs. The immunoenzymatic assay in solid phase (ELISA) using Leishmania L. chagasi total antigens AgT-ELISA and Fucose Manose ligant-ELISA (FML-ELISA) has been showed good sensibility and specificity for detection disease in asymptomatic and oligosymptomatic dogs. The present paper has the goal of compare the efficiency of two antigens by ELISA methods in dogs naturally affect by leishmaniasis with positive parasitological exam and grouped by symptoms. Group composed by symptomatic dogs, and Group by oligosymptomatic dogs. Control group was constituted of dogs from Leishmania free areas. Dogs of Group oligosymptomatics presented 86,7% of sensibility in AgT-ELISA and 90% at FML-ELISA. The specificity was 100% at AgT-ELISA and 96.7% for FML-ELISA. At Group symptomatics the sensibility and specificity for AgTELISA and FML-ELISA were respectively 90% and 93.3% and, the FMLELISA showed 86.7% and 96.7% corresponding to sensibility and specificity. On ELISA-AgT the positive predictive valor was 93.1% at symptomatic and 100% at oligosymptomatic, moreover in the dogs tested by FML-ELISA the valor was 96.3% for the symptomatic and 96.4% for oligosymptomatics. The Kappa was used and showed a good concordance between both methods tested. Our results had shown that in oligosymptomatics animals the FML-ELISA presented greater sensitivity, while that, in the symptomatic animals, the AgT-ELISA showed more sensible in the detention of positive.

Listeria monocytogenes is a bacterial pathogen that is typically transmitted to humans through consumption of contaminated foods. Infection with this organism can lead to a severe and life-threatening illness referred to as listeriosis. The goal of this study was to develop monoclonal antibodies (MAbs) with high specificity and affinity to proteins found on the surface of all strains of L. monocytogenes while not cross-reacting with non-pathogenic Listeria spp. or other major bacterial pathogens commonly found in foods. A literature search was conducted to identify ten candidate surface proteins involved or putatively involved in the virulence of L. monocytogenes. Bioinformatics analyses using BLAST on the NCBI website showed that five of the ten candidate proteins were potentially present in L. monocytogenes strains but absent from strains of other Listeria spp. Genes encoding for these five proteins, ActA, InlA, InlC2, InlJ and LapB, were cloned and expressed in Escherichia coli. MAbs were raised against recombinant LapB, InlJ and InlC2 proteins using hybridoma technology. A total of 48 anti-LapB, 33 anti-InlJ and 37 anti-InlC2 MAbs were developed. Based on the comparison of IFM signal of each MAb against L. monocytogenes cells, seven anti-LapB MAbs and six anti-InlC2 MAbs were selected for further characterization. All of the anti-InlJ MAbs showed weak IFM signals and negative reactivity in ELISA against L. monocytogenes cells. The selected anti-LapB and anti-InlC2 MAbs were further characterized by assessing their ability to bind to cells of 51 strains representing 11 L. monocytogenes serotypes using ELISA. Six anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, M3519) reacted strongly with 44 of 51 strains representing 9 of the 11 L. monocytogenes serotypes tested. Five anti-InlC2 MAbs (M3607, M3618, M3630, M3633, M3636) reacted strongly with 47 strains representing 10 of the 11 L. monocytogenes serotypes tested. These results indicate that anti-LapB and anti-InlC2 MAbs could potentially be used as diagnostic reagents for isolation and detection of almost all L. monocytogenes strains in contaminated foods.

This diploma thesis is focused on current problematics of monitoring of selected mycotoxins, DON and ZEA, in feeds in Czech Republic. The objective of my thesis was to elaborate a literature search from available books, electronic and periodical sources and service materials. Literature search is aimed at overall overview of mycotoxins, describes their characteristics, biological effects, methods of detection as well as summarizes recent legislation requirements concerning occurrence of these substances in feeds for animals. The aim of experimental part was a determination of selected mycotoxins in feed (DON and ZEA) by ELISA method and their evaluation according to the maximum limits. The diploma thesis was prepared in diagnostic laboratory SEVARON, s. r. o. in Brno.