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Mémoire de Maîtrise
DOI
Document
Auteur
Nom complet
Ricardo Harakava
Adresse Mail
Unité de l'USP
Domain de Connaissance
Date de Soutenance
Editeur
Piracicaba, 1993
Directeur
Titre en portugais
Estudos sorológicos sobre o fungo Hemileia vastatrix Berk. et BR. e sobre sua interação com plantas do gênero Coffea
Mots-clés en portugais
ANTICORPOS
FERRUGEM DO CAFEEIRO
FUNGOS FITOPATOGÊNICOS
GALINHAS POEDEIRAS
SEROLOGIA
Resumé en portugais
Galinhas poedeiras foram utilizadas para a produção de anticorpos, os quais foram extraídos das gemas dos ovos através de métodos baseados em POLSON et alii (1985). Uma galinha cuja produção de anticorpos foi acompanhada diariamente, atingiu título máximo (1:32) a partir do décimo oitavo dia após a última injeção, de uma série de 3. As linhas de precipitação formadas em teste de dupla difusão foram mais intensas quando utilizado tampão adicionado de NaCl a 1,4 M que a 0,14 M. Anticorpos para antígenos do fungo coletado no campo reagiram com antígeno homólogo e com antígenos de cafeeiros de diferentes espécies. Anticorpos para cafeeiros cv. Mundo Novo reagiram somente com antígenos de cafeeiros. Os antígenos comuns presentes no extrato de cafeeiro mundo novo foram precipitados pelo etanol a 80%, apresentaram afinidade pelo trocador aniônico DEAE Sephadex A-25 e foram eluídos principalmente no volume externo de coluna de Sephadex G-100. Esses antígenos mostraram-se resistentes à fervura em banho-maria por até 60 min, mas a autoclavagem à 120ºC por 30 min causou perda total da reação sorológica. Tratamentos com metaderiodato de sódio e pronase não afetaram a reação
Titre en anglais
Serologial studies on the fungus Hemileia vastatrix Berk. et BR. and on its interaction with plants of tiie genus Coffea
Resumé en anglais
The utilization of Iaying hens for the production of antibodies to be employed in serological tests has been analysed. These animals have shown easy handling, for both the daily care and the injection of the antigens. The methodology of POLSON et alii (1985), for the extraction of the antibodies present in the eggs' yolks, was successfully employed, being accessible for any laboratory' that has a centrifuge that develops between 8,000 to 12,000 g, preferentially one that bears tubes with capacity above 40 ml. The utilization of this methodology, besides rendering the bleedings unnecessary, results in Iarge ammounts of antibodies and, as the eggs can be kept in refrigerator, it is possible to extract the antibodies from many of them simultaneously. The production of antibodies by a hen immunized against antigens extracted from urediniospores of the race II of H. vastatrix, was verified for 53 days after the Iast injection. The maximun titer (1 : 32) was verified for eggs layed between the 18th and 41st days after the last injection. Antibodies produced against antigens extracted from urediniospores of the races II and III of H. vastatrix, respectively, did not show race specificity, reacting equally with homologous and heterologous antigens and loosing completely the capacity of reacting with homologous antigen after absorption with heterologous one. Precipitation lines were more intense when the agar gel was prepared with buffer conta1ning 1.4 M NaCI as compared with those in which 0.14 M was added. Antibodies produced by a hen immunized against antigen extracted from urediniospores of H. vastatrix colIected in the field, have shown reaction with both homologous antigen and antigens extracted frorn leaves of coffee pIants of the following species: C. arahica cv. Mundo Novo, C. liberica, C. eugenioides, C. congensis, C. canéfora cv. Kouillou and cv. Robusta and Sarchimor híbrid. The occurrenee of common antigens between the fungus and all the species of coffee pIants studied, suggests an involvement of these antigens in the basic compatibility between H. vastatrix and pIants ofthe genus Coffea. Antibodies produced by a hen immunized against antigen extracted from leaves of C. Arabica cv. Mundo Novo, have shown reaction only with antigens extracted from coffee plants, and did not detect common antigens in the fungus extract. Extract obtained from leaves of C. Arabica cv. Mundo Novo was submitted, sequentially, to the precipitation with ethanol at a final concentration of 80% v/v, íon exchange chromatography in a column of DEAE Sephadex A-25 and gel filtration in a column of Sephadex G-100. Common antigens were detected only in the ethanolic precipitate, showed affinity for the íon exchanger and were eluted mainly in the void volume of the Sephadex G-100 column. The behaviour of these antigens in the íon exchange chromatography and in the gel filtration, showed to be heterogeneous in relation to both the affinity for the íon exchanger and the size of their molecules. The correspondence between the elution of proteins and of common antigens in the íon exchange chromatography and in the gel filtration, suggests a protein nature for these molecules, but the maintenance of the serological reactivity even after heating in boiling water for 60 min or after pronase treatment, suggests a polysaccharide nature for the epitope to which the antibodies bind. Therefore, the common antigens seem to be complex protein-polysaccharide macrornolecules in which the antigenic sites are located in the polysaccharide moiety
 
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Date de Publication
2019-11-08
 
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