Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-04-24T13:13:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Ricardo M. Katak.pdf: 1544893 bytes, checksum: b7b82696df95a9ce886dc0401cb6f29c (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-04-24T13:14:03Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Ricardo M. Katak.pdf: 1544893 bytes, checksum: b7b82696df95a9ce886dc0401cb6f29c (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-04-24T13:14:36Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Ricardo M. Katak.pdf: 1544893 bytes, checksum: b7b82696df95a9ce886dc0401cb6f29c (MD5) / Made available in DSpace on 2017-04-24T13:14:36Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Ricardo M. Katak.pdf: 1544893 bytes, checksum: b7b82696df95a9ce886dc0401cb6f29c (MD5) Previous issue date: 2015-11-27 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Insect vectors of tropical diseases such as Ae. Aegypti, the main vector of dengue virus, chikungunya, zika and West Nile virus, are a major problem for public health. One of the major control measures is to control the vector. In this regard, we sought to investigate the biological control of Ae. aegypti; with the use of Bacillus spp .; Amazon isolated from different environments. From the different samples, there was obtained a total of 118 bacterial strains, and 41 strains identified by phenotypic characteristics such as bacilli. Of these, 38 were positive and gram 2 gram negative bacilli. The molecular identification of these strains allowed the identification of 29 strains were characterized in three different genres. The molecular characterization of patients bacilli of Cry4 genes, cry11 and PHAC. Cry4 genes were observed in BtAM41, 2R6.2.1LB lines with no toxicity. The Bio19LB lines, BSBioLB, Bio01LB and Bio011LB presented the cry11 genes, as the larvicidal activity were efficient in both phases of bioassays except Bio011 lineage that any result below 50% in the second stage of bioassays. The 2WISP2, K5NA lines, R8ISP2, R15ISP2, Bio16LB, BtAM125LB, BtAM49LB just presented the PhaC gene. Just BtAM49LB strain was effective in larvicidal activity. Considering the results of the first and second stage, the best results when there were interaction sterile lysed cell supernatant (SN), showing 100% in 72 h. In this sense, it is recommended to study the characterization of metabolites of these strains. The K4NA lines; 103PHAISP2, BtAM220NA; ALP2ISP2, BtAM74LB and PHA50ISP2 bear the cry11 and PHAC genes showed no larvicidal activity above 50% considering it was not observed correlation of genes associated with larvicidal activity. Only Bio19LB strain showed the cry11 gene and Cry4, with larvicidal activity above 50% in the supernatant of lysed cells with interaction with 70% mortality within 72 h. There was no correlation of the phaC gene and Cry isolated, but the best results were in the supernatant of lysed cells with consortium, possibitando the possibility of interaction of molecules with insecticidal activity. So before these results are needed more detailed studies to understand and elucidate the interaction of strains that showed higher larvicidal activity, it is concluded that the strains that showed larvicidal activity above 50% and the same carriers of cry11 genes, Cry4 and PHAC can be associated with other virulence and pathogenicity factors, becoming future potential lines for the control of Ae. Aegypti. / Insetos vetores de patógenos tropicais como Ae. Aegypti, principal vetor do vírus dengue, chikungunya, zika e vírus do Nilo Ocidental, são um grande problema para saúde pública. Uma das principais medidas de combate é o controle do vetor. Neste sentido, buscou-se investigar o controle biológico de Ae. aegypti; com uso de Bacillus spp.; isolados de diferentes ambientes Amazônicos. A partir de diferentes amostras, obteve-se o total de 118 linhagens bacterianas, sendo 41 linhagens identificadas por características fenotípicas como bacilos. Destes, 39 foram bacilos gram positivos e 2 gram negativos. A identificação molecular destas linhagens permitiu identificar 29 linhagens, sendo caracterizadas em três gêneros distintos. Quanto a caracterização molecular dos bacilos portadores dos genes Cry4, Cry11 e PhaC. Foram observados genes Cry4 nas linhagens BtAM41, 2R6.2.1LB, com ausência de toxicidade. As linhagens Bio19LB, BSBioLB, Bio01LB e Bio011LB apresentaram os genes Cry11, quanto a atividade larvicida foram eficientes nas duas etapas dos bioensaios exceto a linhagem Bio011 que apresentou resultados abaixo de 50 % na segunda etapa dos bioensaios. As linhagens 2WISP2, K5NA, R8ISP2, R15ISP2, Bio16LB, BtAM125LB, BtAM49LB apresentaram apenas o gene PhaC. Apenas a linhagem BtAM49LB foi eficiente na atividade larvicida. Considerando os resultados dos bioensaios seletivos de extratos brutos de Bacillus spp., os melhores resultados foram quando houve a interação do sobrenadante estéril com células lisadas (SN), apresentando 100 % em 72 h. Neste sentido, recomenda-se estudos da caracterização dos metabolitos destas linhagens. As linhagens K4NA; 103PHAISP2, BtAM220NA; ALP2ISP2, BtAM74LB e PHA50ISP2 portadoras dos genes Cry11 e PhaC, não apresentaram atividade larvicida acima de 50 %, considerando que não foi observado correlação dos genes associados com atividade larvicida. Somente a linhagem Bio19LB apresentou os genes Cry11 e Cry4, apresentando atividade larvicida acima de 50 % na interação de sobrenadante com células lisadas com 70 % de mortalidade em 72 h. Não foi observado correlação dos genes PhaC e Cry nos isolados, mas os melhores resultados foram no consórcio do sobrenadante + células lisadas, possibilitando a hipótese de interação de moléculas com atividade inseticida. Portanto, diante destes resultados são necessários mais estudos detalhados para compreender e elucidar a interação das linhagens que apresentaram maior atividade larvicida, conclui-se que as linhagens que apresentaram atividade larvicida acima de 50 % e as mesmas portadoras dos genes Cry11, Cry4 e PhaC podem estar associados a outros fatores de virulência e patogenicidade, tornando-se futuramente linhagens potenciais para o controle de Ae. Aegypti no Estado do Amazonas.

No abstract available / Dissertation (MMus)--University of Pretoria, 2008. / Music / unrestricted

The diptericidal $\textit{Bacillus thuringiensis}$ (Bt) ssp. $\textit{fukuokaensis}$ strains 84-I and 17A were investigated for the presence of novel Cry proteins. N-terminal amino acid, immunological and PCR analysis indicated that both strains contain a novel set of $\delta$-endotoxins. N-terminal amino acid sequence analysis indicated that the larger proteins from each strain (90 and 72-kDa of 84-I and 70 and 65-kDa of 17A) were related to the Cry proteins of Bt ssp. $\textit{israelensis}$(Bti). Immunoblotting experiments confirmed that Cry10A-type proteins were present in both strains although subsequent PCR did not give a positive reaction for either strain using $\textit{cry10A}$ specific primers indicating that the Cry10-types were indeed novel. To further investigate the 65-kDa protein of 17A, the gene encoding it was cloned from a size-enriched plasmid DNA library. Unsuccessful attempts were also made to clone the 90-kDa protein of 84-I. Sequence alignments of the deduced protein product of the 17A gene ($\textit{am1}$) showed it to represent the second identification of a natural C-terminal truncate of a Cry4-type protein, the first being Cry10A. The missing C-terminal region of AMl appears to be encoded as a complete Orf ($\textit{am2}$) immediately downstream of the first protein gene. When DNA containing both the $\textit{am1}$ and $\textit{am2}$ genes was subcloned into the pSVP27A expression vector high levels of expression of both proteins were observed in acrystalliferous Bt. The protein was deposited in inclusion bodies which were found to be toxic to $\textit{Dacus oleae}$. Extensive phylogenetic analysis was carried out to determine the relationship between, and possible evolutionary origins of, AMl, the Cry proteins of Bti and two further Cry10A-type $\delta$-endotoxins (Cry19A from Bt ssp. $\textit{jegathesan}$ and Cry20A from 84-I) identified in other laboratories during the course of this project. Based on the amino acid sequence alignment, all seven proteins appear to have evolved from a common ancestor to form three distinct groups which mirror the structural organisation of the genes. Based on these groupings and a previous hypothesis of Dervyn $\textit{et al.}$ (1995), a hypothesis was proposed as to the evolution of the 130-kDa Cry4-type proteins from a 70-kDa Cry2-type ancestor. The above hypothesis is based on the assumption that transfer of $\delta$-endotoxin genes between subspecies has occurred at some point in evolutionary history. Evidence for this transfer was found when the genetic context of the $\textit{am1}$ gene was investigated. Two novel insertion sequences (Tl) and (T2) were identified with sequence similarity to IS$\textit{240A}$ from Bti and an insertion sequence associated with the $\textit{Orf1}$ gene of 84-I. The identification of a further incomplete reading frame with similarity to integrase/recombinase proteins involved in Class II transposition raises the possibility that T1 and T2 form part of a novel Class II transposon. A novel $\alpha$/$\beta$-type small, acid soluble protein (SASP) gene was also discovered. This gene, which may be plasmid encoded, showed considerable sequence similarity to $\alpha$/$\beta$-type SASP from $\textit{Bacillus megaterium}$. The discovery of this gene raises new questions about taxonomic relations between the $\textit{Bacilli}$.

No description available.

Les parasporines sont des toxines Cry du bacille de Thuringe actives contre des cellules tumorales. Ce travail montre que la parasporine PS1Aa2 (Cry31Aa2) forme des pores dans des membranes artificielles, comme de nombreuses toxines Cry. Ceux-ci ont plusieurs niveaux de conductance dont les plus fréquents étaient de 11, 16 et 21 pS dans une solution de 150 mM KCl. Nos résultats de microspectrofluorométrie avec la sonde Fura-2 montrent que la présence de la PS1Aa2 peut produire des augmentations du calcium intracellulaire, la plupart du temps sous la forme d’oscillations calciques et parfois des augmentations soutenues. Ces réponses ont été observées en présence et en absence de calcium extracellulaire, dans les lignées tumorales HeLa et HepG2 et dans la lignée non tumorale HEK 293. Bien que quelques études aient montré que le calcium semble intervenir dans leur mode d’action, de telles oscillations calciques n’ont jamais été décrites auparavant pour des toxines Cry. Les expériences ont dû être faites à des concentrations beaucoup plus élevées de toxine que prévues sur la base des résultats publiés de cytotoxicité. Malgré la présence des fragments identifiés auparavant comme actifs, sa faible efficacité semble liée à la présence d’ADN dans les préparations qui entraîne la précipitation de la protéine. Les travaux futurs sur cette toxine seraient donc grandement facilités par une amélioration de sa méthode de préparation. / Parasporins are Cry toxins from Bacillus thuringiensis that are active against tumor cells. This work shows that parasporin PS1Aa2 (Cry31Aa2) forms pores in artificial membranes like many Cry toxins. These pores have several levels of conductance; the most frequently seen in 150 mM KCl solutions were of 11, 16 and 21 pS. Our microspectrofluorometric results with the Fura-2 probe showed that the presence of PS1Aa2 can induce changes in intracellular calcium levels, most often in the form of calcium oscillations and sometimes as sustained increases. Such responses were observed in the presence and absence of extracellular calcium, with the tumor cell lines HeLa and HepG2, and with the non-tumorous cell line HEK 293. Calcium oscillations have not been described previously for Cry toxins even though some studies have shown that calcium appears to be involved in their mode of action. Our experiments required the use of much higher concentrations of toxin than suggested from the published cytotoxicity results. Despite the presence of fragments previously identified as active, its low efficacy appears to be related to the presence of DNA in the preparations causing the protein to precipitate. Future work on this toxin would therefore be greatly facilitated by an improvement in its method of preparation.

Syftet med denna studie var att utforska nautiska befäls attityder till larmen de kan ställas inför på sin brygga och den eventuella kausaliteten. Tidigare forskning inom larm och forskning inom optimering av brygglayouter presenterade ett problematiskt fenomen vilken av Papastavrou och Lehto kallas för cry-wolf. Detta fenomen beskriver att operatörer av system med larm, till exempel sjöfartsofficerare, tenderar att ignorera larmen om de utsätts för en hög frekvens av larm, denna höga frekvens kallar Al-Dabbagh och Tongwen för alarm-floods. Denna studie kommer fram till att cry-wolf fenomenet existerar ombord det fartyg studien utfördes på. Datainsamlingen utfördes med den kvalitativa forskningsmetodiken, främst genom semi-strukturerade intervjuer. Dessa intervjuer kompletterades sedan med ostrukturerade observationer vilka utfördes under en nio dagar lång fältstudie. Studiens primärdata analyserades genom innehållsanalyser och detta gjordes ur det hermeneutiska perspektivet. Studien fann att befälen tenderar att ignorera larmen om larmen inte klassas som nödlarm. Vidare presenteras två potentiella lösningar som ämnar ge tillbaka den tyngd larmen borde associeras med. Utöver detta presenteras också en hypotes vilken syftar till att ta reda på om ett givet fartyg påverkas av cry-wolf fenomenet. Denna hypotes måste valideras innan den kan godtas som gångbar för sjöfarten som helhet. / The main purpose of this thesis was to explore nautical officer’s attitudes towards the alarms they may encounter on the bridge and the eventual causality between the officers and their actions concerning the alarms. Earlier research into alarms and research regarding optimization of bridge-layouts presented a problematic theory which Papastavrou and Lehto call cry-wolf. Wherein the operators of systems, or ships, with advanced alarms tend to ignore the alarms if they are subjected to a high frequency of alarms, or alarm-floods as Al-Dabbagh and Tongwen calls it. This study shows that, on board the vessel which the study was conducted, the cry-wolf phenomena exist. The study was conducted on board a Swedish vessel from the hermeneutical perspective. The data collection was done with the qualitative method, primarily through semi-structured interviews and these were then enriched with data from unstructured observations which were made during a nine-day stay aboard said vessel. The collected data has been analysed through content analysis from the hermeneutical perspective and the study reveals that the officers tend to ignore the alarms, unless they are classified as emergency alarms. This study proposes two potential fixes for how to make the alarms less overwhelming and how to give back the importance of the alarms. Furthermore, the study presents a hypothesis which aims to find whether or not a given vessel is subject to the cry-wolf phenomena. This hypothesis needs to be proven in future research before it can be proclaimed as a valid hypothesis.

In der vorliegenden Arbeit werden die Schreientwicklung von Säuglingen und die dabei auftretenden Regularitäten und Gesetzmäßigkeiten untersucht. Es wurde dazu eine Analyse von Grundfrequenzverläufen (Melodien) in Säuglingsschreien der ersten 5 Lebensmonate von 30 medizinisch unauffälligen monozygoten Zwillingen durchgeführt. Diese Studie belegt, daß der Säuglingsschrei weit mehr ist, als nur ein angeborenes biologisches Alarmsignal, das Ausnahmesituationen in der Kommunikation vorbehalten bleibt. Die gefundenen Gesetzmäßigkeiten in der Entwicklungen der Schreimelodie berechtigen, den Säuglingsschrei in eine Reihe mit frühen Nichtschrei-Vokalisationen als essentielle Wegbereiter für den Spracherwerb zu stellen. Den durch umfangreiche Studien anderer Autoren bereits nachgewiesenen Perzeptionsleistungen junger Säuglinge können durch die hier gefundenen, komplexen Leistungen bezüglich der Melodieentwicklung im Schrei nunmehr auch ebenbürtige Produktionsleistungen gegenübergestellt werden. Die analysierten 1700 Schreimelodien wurden durch einen Vergleich in Ähnlichkeitsklassen eingeteilt. Es wird ein Klassifizierungssystem vorgeschlagen, das sechs Ähnlichkeitsklassen (Melodietypen) definiert, die auf prinzipiellen Formeigenschaften (langsamen Modulationseigenschaften) der Schreimelodien beruhen. Die Formeigenschaften der Melodietypen sind also bis auf lineare Transformationen der Zeitachse und einem konstanten Shiftwert in der Frequenzachse (bei ungeändertem Frequenzhub) invariant. Alle Schreimelodien wurden eindeutig den gebildeten Melodietyp-Klassen zugeordnet und ohne irgendwelche Skalen-Transformationen in Frequenz oder Zeit dargestellt. Die Konsistenz aller im Rahmen der vorliegenden Studie durchgeführten Untersuchungen hat die vorgeschlagene Klassifizierung gerechtfertigt. Es konnte gezeigt werden, daß die Synchronizität und die Konkordanz in der Melodieentwicklung für die Zwillinge eines Paares deutlich größer ist als für nichtverwandte Zwillinge gleichen Geschlechts und Alters. Die Monozygotie der Zwillinge und die damit erhaltene höhere Synchronizität der Entwicklung ließen die Universalität der Entwicklung wesentlich prägnanter hervortreten, als dies bei einer durchschnittlichen Population der Fall gewesen wäre. Es konnte eine kontinuierliche, gesetzmäßige Melodieentwicklung bei allen Zwillingen nachgewiesen und in Form eines Entwicklungsdiagramms dargestellt werden. Es wurde eine höhere Konkordanz und Synchronizität zwischen den monozygoten Zwillingen der gleichen Paare bezüglich der Art der erzeugten Melodietypen und deren absoluten Häufigkeit in einem bestimmten Alter gefunden. Dies und die ebenfalls nachgewiesene Fähigkeit der Zwillinge zur hohen formähnlichen Reproduktion der Melodien sind ein starkes Argument für die Beteiligung genetischer Faktoren an der Erzeugung der Melodietypen nach einem internen zerebralen Programm. / The cry development of babies and the regularly occurring phenomena and principles are discussed in the present paper. The time variations of the fundamental frequency (melodies) in babies cries in the first five months of life was analyzed in 30 medically normal monozygotic twins. This study documents that the baby s cry is far more than only an innate biological alarm signal which is reserved to exceptional situations of communication. The developmental principles of the cry melody found justify the categorization of the baby s cry on the same level as other early noncry vocalizations as essential precursors for the acquisition of speech. The perceptual capacity of young infants as already demonstrated by extensive studies of other authors can now also be compared to equivalent cry production on the basis of the complex phenomena found with regard to cry melody development of melody in the cry. The 1,700 cry melodies analyzed were classified into similarity classes by means of a comparison. A system of classification defining six similarity classes (melody types) which are based on fundamental form characteristics (slow modulation properties) of the cry melodies was suggested. The form characteristics of the melody types are invariant apart from linear transformations of the time axis and a constant shift value in the frequency axis (with unchanged frequency hub). All cry melodies were unequivocally assigned to the classes of melody type formed and visualized in frequency or time without any scale transformations. The consistency of all the investigations conducted in the present study justify the suggested classification. It could be shown that the synchronicity and the concordance in the melody development for monozygotic twins is very much greater than that for unrelated twins of the same sex and age. The monozygosity of the twins and the greater synchronicity of the development thereby attained resulted in the universality in the development being manifested very much more distinctly than would have been the case in an average population. A continuous regular development of melody could be demonstrated in all twins and depicted in the form of a development diagram. A greater concordance and synchronicity between the monozygotic twins of the same pair was found with regard to the characteristics of the melody types produced and their absolute frequency at a certain age. This and the likewise demonstrable ability of the twins to reproduce the melodies in a highly similar form are very strong arguments corroborating a role of genetic factors in the production of the melody types according to an internal cerebral program.

Orientadora: Janete Apparecida Desidério Sena / Banca: Manoel Victor Franco Lemos / Banca: Cristina Lacerda Soares Petrarolha Silva / Banca: Gislayne Fernandes Lemes Trindade Vilas-Bôas / Banca: Odair Aparecido Fernandes / Resumo: Aedes aegypti (Linnaeus), principal vetor da dengue no Brasil, tem sido combatido com o uso maciço de produtos químicos, contribuindo com o desenvolvimento de resistência e inviabilizando o controle do inseto. Bioinseticidas à base de Bacillus thuringiensis vêm apresentando resultados satisfatórios no controle de dípteros, devido à produção de proteínas bioinseticidas denominadas Cry (cristal), Cyt (citolíticas) e Chi (quitinase) e os efeitos sinergísticos existentes entre elas. O presente trabalho objetivou a seleção de isolados de B. thuringiensis, portadores de genes cry, cyt e chi com alta eficiência no controle de A. aegypti. Uma coleção de 1073 isolados de B. thuringiensis, provenientes de diversas regiões brasileiras, foi submetida à Reação em Cadeia da Polimerase (PCR), com base nos iniciadores específicos para os genes cry4Aa, cry4Ba, cry10Aa, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa e chi. Os isolados dípteros-específico foram avaliados quanto à estruturação genética e através de bioensaios seletivos e quantitativos, para determinação da CL50 e CL90. Apenas 45 (4,19%) apresentaram amplificação para os genes cry e cyt e destes, 25 (54,34%) eram quitinolíticos. Foram definidos 21 haplótipos entre os 45 isolados de B. thuringiensis, os quais foram submetidos aos bioensaios seletivos, indicando 13 isolados que causaram 100% de mortalidade às larvas de A. aegypti. Os bioensaios quantitativos e análise de agrupamento permitiram selecionar quatro isolados altamente tóxicos às larvas de A. aegypti. A identificação dos genes cry, cyt e chi de B. thuringiensis e análise dos efeitos sinergísticos entre as toxinas, associadas à análise da toxicidade a insetos vetores permitiram a seleção de isolados que poderão ser utilizados em formulações de novos bioinseticidas brasileiros, podendo contornar possíveis problemas de resistência. / Abstract: Aedes aegypti (Linnaeus), the main vector of dengue fever in Brazil, has been controlled with massive use of chemical products wich, contributed to the development of resistance decreasing the insect control efficacy. Bioinsecticides based on Bacillus thuringiensis is currently presenting satisfactory results on controlling dipterans, due to the production of Cry proteins (crystal proteins), Cyt (citolitic) and Chi (chitinase) with synergistic effects between them. The present work aimed to select B. thuringiensis isolates with cry, cyt and chi genes exhibiting high efficiency on A. aegypti larvae. A bacterial collection containing 1073 isolates of B. thuringiensis, obtained from different Brazilian locations, had their DNA isolated and submitted to PCR amplifications using specific primers for the genes cry4Aa, cry4Ba, cry10Aa, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa e chi. The dipterans-especific isolates were then evaluated for the genetic structure and through selective and latter to quantitative bioassays for the determination of LC50 and LC90. Forty five out of 1073 isolates (4.19%) presented amplicons for the cry and cyt genes and from these 25 (54.34%) were detected as chitinolitic. Twenty one haplotypes were identified among the 45 isolates of B. thuringiensis, which were submitted to the selective bioassays, indicating 13 isolates that caused 100% mortality of A. aegypti larvae. The quantitative bioassays and the statistical analysis have pointed four toxic isolates to A. aegypti larvae. The identification of the cry, cyt and chi genes of B. thuringiensis and the analysis of the synergistic effects among the toxins, along with the toxicity analysis towards the vector insects allowed the selection of a set of isolates that can be used for the formulation of new bioinsecticides from Brazilian origin, that would avoid or at least delay the appearance of resistance problems. / Doutor

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-07-06T20:16:17Z No. of bitstreams: 1 VanessaOlintoDosSantosEvangelista_TESE.pdf: 5760623 bytes, checksum: 180f27b8cf175c05011adf038ebd2e0b (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-07-07T21:00:30Z (GMT) No. of bitstreams: 1 VanessaOlintoDosSantosEvangelista_TESE.pdf: 5760623 bytes, checksum: 180f27b8cf175c05011adf038ebd2e0b (MD5) / Made available in DSpace on 2016-07-07T21:00:30Z (GMT). No. of bitstreams: 1 VanessaOlintoDosSantosEvangelista_TESE.pdf: 5760623 bytes, checksum: 180f27b8cf175c05011adf038ebd2e0b (MD5) Previous issue date: 2015-12-03 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A expans?o agr?cola utilizando cultivares geneticamente modificados (GM) ? fen?meno mundial. Esse fato tem impulsionado a implementa??o de legisla??es regulat?rias para monitorar ? presen?a de variedades GM em culturas agr?colas. Outra importante demanda mundial est? relacionada ao consumo de alimentos contaminados por aflatoxinas. Essas mol?culas constituem um classe de compostos extremamente t?xicos que causam efeitos danosos para a sa?de humana e animal. Por isso, a seguran?a e qualidade dos produtos agr?colas s?o essenciais para os consumidores. Os testes de fluxo lateral s?o m?todos alternativos promissores que podem ser utilizados tanto para a detec??o de prote?nas transg?nicas expressas em culturas GM quanto para a detec??o de compostos t?xicos em alimentos. Essa t?cnica apresenta vantagens adicionais quando comparado aos m?todos convencionais, como: simplicidade, rapidez e baixo custo. Nesse estudo foram desenvolvidos dois testes de fluxo lateral, um para a identifica??o das prote?nas Cry1Ac e Cry8 Ka5 expressas em cultivares de algod?o GM e o outro, para a detec??o de aflatoxinas em produtos agr?colas. O teste, para a detec??o dos cultivares transg?nicos, foi desenvolvido no formato sandwhich. Esse teste foi desenvolvido utilizando os anticorpos monoclonais 1B1 e 5H4, produzidos contra a prote?na Cry1Ac, mas que apresentaram rea??o cruzada para a prote?na Cry8Ka5. O monoclonal anti-Cry1B1 foi conjugado com nanopart?culas de ouro coloidal (40 nm) e utilizados como reagente de detec??o. O monoclonal 5H4 foi adsorvido na membrana de nitrocelulose, na regi?o denominada de linha teste e utilizado como reagente de captura do teste. Na linha controle, foi adsorvido o anticorpo anti-mouse IgG. Esses testes foram validados utilizando amostras de folhas de plantas de algod?o GM ( Bollgard I ? e Planta 50- produzida em nosso laborat?rio) e folhas provenientes de cultivares n?o GM ( Cooker 312). Os resultados demonstraram que esse teste foi capaz de distinguir eficientemente amostras GM de n?o GM. Al?m disso, tamb?m apresentou elevada sensibilidade, sendo capaz de detectar 0,06 ?g das prote?nas respectivas nos cultivares transg?nicos. O teste para a detec??o de aflatoxinas foi desenvolvido no formato competitivo. O anticorpo ?-AFLA 3B6, produzido contra AFB1, apresentou reatividade cruzada contra as aflatoxinas AFB2, AFG1, AFG2 e AFM1 e por isso, foi utilizado para o desenvolvimento das fitas-testes. Nesse teste o anticorpo 3B6 foi conjugado com ouro coloidal (40 nm) e utilizado como reagente de detec??o. O ant?geno AFB1 foi adsorvido na linha teste e utilizado como reagente de captura e o anti-mouse IgG foi imobilizado na linha controle do teste. Para a valida??o, gr?os de soja contaminados com o fungo Aspergillus flavus, foram utilizados. Esses testes tamb?m foram avaliados quanto ? habilidade de detec??o de aflatoxinas em amostras alimentares, incluindo leite e prote?na texturizada de soja. Os resultados demonstraram que a fita eficientemente identificou amostras contendo aflatoxinas. Al?m disso, apresentou sensibilidade de 0,5 ng/mL ou 0,5 ?g/Kg. Os resultados obtidos sugerem que as fitas testes desenvolvidas podem ser utilizadas como m?todo r?pido e de baixo custo para o screening de cultivares GM, expressando as prote?nas Cry1Ac e Cry8Ka5, quanto para a detec??o de aflatoxinas em amostras alimentares. / The expansion of cultivated areas with genetically modified crops (GM) is a worldwide phenomenon, stimulating regulatory authorities to implement strict procedures to monitor and verify the presence of GM varieties in agricultural crops. With the constant growing of plant cultivating areas all over the world, consumption of aflatoxin-contaminated food also increased. Aflatoxins correspond to a class of highly toxic contaminants found in agricultural products that can have harmful effects on human and animal health. Therefore, the safety and quality evaluation of agricultural products are important issues for consumers. Lateral flow tests (strip tests) is a promising method for the detection both proteins expressed in GM crops and aflatoxins-contaminated food samples. The advantages of this technique include its simplicity, rapidity and cost-effective when compared to the conventional methods. In this study, two novel and sensitive strip tests assay were developed for the identification of: (i) Cry1Ac and Cry8Ka5 proteins expressed in GM cotton crops and; (ii) aflatoxins from agricultural products. The first strip test was developed using a sandwhich format, while the second one was developed using a competitive format. Gold colloidal nanoparticles were used as detector reagent when coated with monoclonal antibodies. An anti-species specific antibody was sprayed at the nitrocellulose membrane to be used as a control line. To validate the first strip test, GM (Bollgard I? e Planta 50- EMBRAPA) and non-GM cotton leaf (Cooker 312) were used. The results showed that the strip containing antibodies for the identification of Cry1Ac and Cry8Ka5 proteins was capable of correctly distinguishing between GM samples (positive result) and non-GM samples (negative result), in a high sensitivity manner. To validate the second strip test, artificially contaminated soybean with Aspergillus flavus (aflatoxin-producing fungus) was employed. Food samples, such as milk and soybean, were also evaluated for the presence of aflatoxins. The strip test was capable to distinguish between samples with and without aflatoxins samples, at a sensitivity concentration of 0,5 ?g/Kg. Therefore, these results suggest that the strip tests developed in this study can be a potential tool as a rapid and cost-effective method for detection of insect resistant GM crops expressing Cry1Ac and Cry8Ka5 and aflatoxins from food samples.

La mosca mediterránea de la fruta, Ceratitis capitata, es la principal plaga de la fruticultura en el mundo. El desarrollo de métodos ambientalmente seguros de control de plagas, como Bacillus thuringiensis (Bt), adquiere cada vez mayor importancia. Esta tesis doctoral evalúa la validez de Bt y sus delta-endotoxinas como agentes de control de C. capitata. El análisis de la biodiversidad de Bt ha reflejado la gran riqueza presente en el agroecosistema de los cítricos. Sin embargo, ninguna de las cepas ensayadas (905) muestra alta toxicidad sobre C. capitata cuando esporas/cristales o sobrenadantes de su cultivo son ensayados. En cambio, la solubilización de los cristales de una selección de 42 cepas de Bacillus sp. ha demostrado que, para las cepas de la subespecie israelensis (Bti), este tratamiento produce una ganancia de función biológica. Adicionalmente, la predigestión de dichas protoxinas con proteasas de otro díptero, Culex pipiens, aumenta su actividad larvicida (CL50 31.26 µg/cm2). Por otro lado, se ha demostrado que, entre las delta-endotoxinas producidas por Bti, la protoxina Cyt1Aa es el factor determinante, con una CL50 sobre larvas de 4.93 µg/cm2. Sobre adultos, Cyt1Aa produce efectos subletales. Complementariamente, esta tesis propone un nuevo método de control basado en el desarrollo de toxinas recombinantes de fusión Cry-anticuerpo. Se ha puesto en práctica un sistema modelo para evaluar esta estrategia: se han desarrollado 4 variantes proteicas por la fusión entre partes de la protoxina Cry1Ab y un anticuerpo específico contra GFP (VHH anti-GFP) y éstas se han ensayado sobre larvas transgénicas de Drosophila melanogaster que expresan GFP en su intestino. Deficiencias en la unión de las toxinas de fusión a GFP, han impedido demostrar, por el momento, la estrategia propuesta. Por último, se han detectado al menos 160 proteínas distintas en las membranas intestinales de C. capitata y se han identificado las siguientes: V-ATPasa subunidades A y B, y alfa-tubulina. / Vidal Quist, JC. (2010). Estrategias para la utilización de la bacteria entomopatógena Bacillus thuringiensis (Berliner) en el control de Ceratitis capitata (Wiedemann) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8336 / Palancia