Le latex de Carica papaya est un milieu riche en cystéine protéinases. Celles-ci ont été régulièrement utilisées en cosmétique ou pour l’attendrissement de la viande. Mais ces protéines ont aussi un intérêt pharmaceutique. En effet, le latex est bien connu pour posséder une activité antifongique mais aussi une activité anthelminthique. Ces effets sont régulièrement attribués aux cystéine protéinases qui se trouvent en concentration importante dans le latex. Malgré ces concentrations importantes en protéinases, d’autres protéines restent actives dans ce milieu. C’est le cas de la glutamine cyclase, qui a été extraite intacte de ce milieu et cristallisée. Sa structure nous a révélé une architecture particulière en ‘’&61538;-propeller‘’ à cinq pales avec double fermeture. Cette structure lui confère sa très grande stabilité. <p>Les industries pharmaceutiques sont aussi à la recherche de protéines très stables et résistantes aux protéinases endogènes. Nous avons donc entrepris l’étude du protéome de Carica papaya afin de mettre en évidence d’autres protéines minoritaires relativement stables pouvant conférer au latex son activité anthelminthique. Cette analyse a permis la mise en évidence de différentes protéines appartenant à diverses familles des « pathogenesis related protéins » (PR-proteins): une &61538;-1,3 glucosidase, une analogue à la barwin, une thaumatine et deux chitinases.<p>Nous nous sommes particulièrement intéressés à ces deux dernières au cours de cette thèse. Une caractérisation de ces deux protéines a permis de montrer que celles-ci étaient bien deux protéines distinctes, identifiées comme chitinases majeure et mineure selon leur abondance dans le latex. Elles sont relativement stables et résistantes à la protéolyse. Une analyse de la séquence de la chitinase majeure a montré que celle-ci était homologue à la chitinase issue de l’orge et une analyse de sa structure révèle la présence d’une grande concentration en prolines localisées principalement dans les neuf boucles de sa structure. Cela pourrait expliquer sa grande résistance vis à vis des cystéine protéinases.<p>La cristallisation de cette même chitinase en présence de N-acétyl-glucosamine comme additif, a conduit à une structure contenant trois molécules de GlcNac, deux dans le centre actif de notre protéine et une participant au réseau cristallin. Aucune structure de chitinase n’avait encore pu être obtenue en co-cristallisation avec un substrat. A partir des deux GlcNac observés dans le centre actif, nous avons reconstruit un complexe chitinase/(GlcNac)4. L’analyse de ce complexe a permis de mettre en évidence de nouvelles interactions entre (GlcNac)4 et les acides aminés du centre actif ainsi que de confirmer le mécanisme de la famille GH 19.<p> Des tests préliminaires sur nématodes ont finalement confirmé l’activité anthelminthique du latex et montré que la chitinase pouvait aussi être un bon nématocide<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The American horseshoe crab (HSC), Limulus polyphemus, is one of four species of horseshoe crabs found throughout the world, and the only one found in North America. It is an economically and ecologically important species throughout its native range from Maine to the Yucatan Peninsula. Harvested for fertilizer and livestock feed in the 19th century, the species is now harvested as bait for whelk and eel fisheries, and for their blood by the biomedical industry. The Atlantic States Marine Fisheries Commission (ASMFC) started to formally manage HSC in 1998 with its Interstate Fisheries Management Plan (IFMP). Unique emphasis and harvest limits have been placed on the Delaware Bay stock, as it is commercially exploited and a critical food source for the threatened red knot, Calidris canutus rufa. Previously, estimates of relative and total abundance of HSC in the Delaware Bay area were based on a design-based approach using a stratified random sampling design. In Chapter 1 of this work, I developed hurdle models for each of the six HSC demographic groups to standardize catch-per-unit-effort (CPUE) and estimate relative abundance using a model-based approach. It was determined that while the two approaches resulted in mostly convergent estimates of relative abundance, external factors such as month, time-of-day, and average depth have major effects on the observed CPUE of all demographic groups. Chapter 2 involved the development of hurdle models for the three species of bycatch frequently caught in our trawls, channeled whelk (Busysotypus canaliculatus), knobbed whelk (Busycon carica), and summer flounder (Paralichthys dentatus). It was found that channeled whelk relative abundance has been at a historical low since 2016, while summer flounder has been at a consistent high. Recent estimates of knobbed whelk relative abundance have been less variable than previously seen, with estimates since 2016 being similar to those seen before 2012. These results provide the first estimates for whelk population trends in the mid-Atlantic region and add to the growing knowledge of summer flounder relative abundance in the area. In Chapter 3, I applied the hurdle models developed in Chapter 1 to estimate the total abundance of HSC in the Delaware Bay area. For this work, I developed two spatio-temporal variograms to estimate bottom temperature and bottom salinity at unmeasured cells per month in the time series. The results showed that night estimates of total abundance were consistently higher than daytime estimates, and estimates from September or November resulted in the highest estimated catch for all demographic groups. The results suggest that when comparing September model-based estimates at night to those of the design-based approach, nearly a third of all previous design-based estimates significantly underestimated the total abundance of HSC in the Delaware Bay area. This result suggests that the ASMFC can recommend increased harvest limits for mature individuals if that action aligns with the goals of their adaptive resource management (ARM) framework. / Master of Science / The American horseshoe crab (HSC), Limulus polyphemus, is one of four species of horseshoe crabs found throughout the world, and the only one found in North America. It is an economically and ecologically important species throughout its native range from Maine to the Yucatan Peninsula. Harvested by the millions in the 19th century, the species is still harvested as bait for whelk and eel fisheries, and for their blood by the biomedical industry, on the order of hundreds of thousands. Formal management of HSC by the Atlantic States Marine Fisheries Commission (ASMFC) began in 1998, and allowable catch and landings have decreased since 1999. A strong focus has been placed on the Delaware Bay population, as it is the center of HSC abundance and provides a critical food source for the threatened red knot, Calidris canutus rufa, while also being commercially exploited. To effectively manage the species, it is important to accurately estimate relative and total abundance so that proper harvest limits can be set. Previously, estimates of relative and total abundance of HSC in the Delaware Bay area were based on a design-based approach using a stratified random sampling design. In Chapter 1 of this work, I developed hurdle models (a generalized linear model that models the probability of observations and the observed positive counts using two separate regression models that are then combined) for each of the six HSC demographic groups to remove the effect of external factors (year, latitude, longitude, depth [inshore/offshore], topography, average trawl depth, time-of-day, month, bottom temperature, bottom salinity, and distance from shore) on our observed catch-per-unit-effort (CPUE) and estimate relative abundance using a model-based approach. It was determined that while the two approaches resulted in mostly convergent estimates of relative abundance, factors like month, time-of-day, and average depth had major effects on the observed CPUE of all demographic groups. Chapter 2 involved developing similar hurdle models for three species of bycatch frequently caught in our trawls, i.e., channeled whelk (Busysotypus canaliculatus), knobbed whelk (Busycon carica), and summer flounder (Paralichthys dentatus). It was found that channeled whelk relative abundance has been at a historical low since 2016, while summer flounder has been at a consistent high. Recent estimates of knobbed whelk relative abundance have been less variable than previously seen, with estimates since 2016 being similar to those seen before 2012. These results provide the first estimates for whelk population trends in the mid-Atlantic region and add to the growing knowledge of summer flounder relative abundance in the area. In Chapter 3, I applied the hurdle models developed in Chapter 1 to estimate the total abundance of HSC in the Delaware Bay area. To do this, the bottom temperature and salinity had to be estimated for each geographic cell. This was accomplished by developing two spatio-temporal variograms which allowed me to estimate either variable at an unmeasured point and time based on its spatial and temporal distance from a measured value in a process known as spatio-temporal kriging. The results showed that night estimates of total abundance were consistently higher than daytime estimates and that estimates from September or November resulted in the highest estimated total abundance for all demographic groups. The results suggest that when using September model-based estimates at night to compare against the design-based approach, nearly a third of all previous design-based estimates significantly underestimated the total abundance of HSC in the Delaware Bay area. This outcome could justify ASMFC increasing recommended harvest limits for mature individuals if that action aligns with the goals of their adaptive resource management (ARM) framework.

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Submitted by Deyse Queiroz (deysequeirozz@hotmail.com) on 2018-05-18T13:02:41Z No. of bitstreams: 1 FRANCISCA AMANDA ABREU MARTINS - DISSERTAÇÃO PPGHT 2016.pdf: 1114179 bytes, checksum: 1804a3728b4b1b900e48cfd4d2008a1d (MD5) / Made available in DSpace on 2018-05-18T13:02:42Z (GMT). No. of bitstreams: 1 FRANCISCA AMANDA ABREU MARTINS - DISSERTAÇÃO PPGHT 2016.pdf: 1114179 bytes, checksum: 1804a3728b4b1b900e48cfd4d2008a1d (MD5) Previous issue date: 2016-08-29 / O ácaro rajado, Tetranychus urticae Koch, é considerado uma das principais pragas de frutíferas no Brasil, dentre elas, a cultura do Carica papaya tem sido intensamente danificada por esse artrópode. Na produção de mamão são inúmeros os desafios para o controle desta praga. Este trabalho foi elaborado visando contribuir para o fomento de programas de manejo de T. urticae na cultura do mamão, com seguintes objetivos: a) prospectar isolamentos de insetos moribundos linhagens fúngicas de Beauveria bassiana, visando seu uso no controle do T. urticae; b) avaliar a eficiência na mortalidade do T. urticae nos estágios de ovo, larva e protoninfa, in vitro,quando contaminados com fenpropatrina (0,5 g/L), abamectina (0,6 g/L), Milbemectina (0,3 g/L), calda Sulfocálcica (7,0 g/L), três isolados de B. bassiana e a testemunha água destilada. Na 1ª etapa do trabalho obteve-se 10 linhagens de B. bassiana, reisolados de insetos moribundos encontrados no alto sertão Paraibano, que estão mantidos em meios de cultura na UFCG. Destes, três (P21CZ, P22CZ e P29CZ), analisando-se os resultados obtidos in vitro, pode-se imputar como os que apresentaram maior potencial de germinação para utilização de bioenaios in vitro, tendo em vista o controle de T. urticae, e foram enviados ao Laboratório de Seletividade de Produtos Químicos, UFERSA. Na 2ª etapa do trabalho, para se iniciar os bioensaios foram transferidas fêmeas fertilizadas de T. urticae para discos de folhas de Canavallia ensiformis. Quando os ácaros atingiram, em cada bioensaio, o estágio desejado, de ovo, larva ou protoninfa, iniciava-se o bioensaio. Cada disco foi pulverizado contaminado com os tratamentos por meio de pulverizador pressurizado manualmente, em seguida deixados por um período de 1 hora para a secagem, eliminando o excesso dos resíduos. Logo após a secagem foram transferidos para câmaras climatizadas tipo BOD, onde permaneceram em condições controladas à temperatura de 25 ± 1 ºC, fotofase de 12 horas e umidade relativa de 60 ± 10%, para obtenção dos dados biológicos. Os parâmetros biológicos avaliados foram: duração e mortalidade de ovos, larvas, protoninfas e deutoninfas. Os resultados dos três bioensaios foram submetidos à análise de variância e as médias comparadas pelo teste de Tukey a 5% de probabilidade. Apesar da fase contaminada nos três bioensaios não ter sido afetada, houve mortalidade acumulada nas fases subsequentes. O inseticida fenpropatrina ocasionou 100% de mortalidade nos bioensaios com o estágio de ovo e larva. Os acaricidas químicos fenpropatrina, milbemectina e abamectina foram mais eficientes nos três bioensaios, com valores médios de taxa de mortalidade semelhantes. Para as larvas diretamente tratada o abamectina, ocasionou maior tempo de duração contato das larvas. O isolado B. bassiana P22CZ, na concentração (2,5 x 106 ), apresentou maior taxa de mortalidade 63,8%. Observou-se, uma relação positiva entre o aumento do tempo da exposição dos ácaros às concentrações do isolado P29CZ e a maior concentração aplicada. As porcentagens de mortalidade acumulada pela calda sulfocálcica nos três bioensaios, ovo, larva e protoninfa foram baixas, com valores respectivos de 8,8%, 7,5% e 25%. / The spider mite, Tetranychus urticae Koch, is considered one of the main pests of fruit in Brazil, among them, the Carica papaya culture has been heavily damaged by this arthropod. In papaya production are numerous challenges to the control of this pest. This work was prepared to contribute to the development of T. urticae management programs in papaya crop, with the following objectives: a) insulations prospect of dying insects fungal strains of Beauveria bassiana, aiming its use in the control of T. urticae; b) evaluate the efficiency in mortality of T. urticae in the egg, larva and protonymph in vitro when contaminated with fenpropathrin (0.5 g / L), abamectin (0.6 g / L), Milbemectin (0, 3 g / L), lime sulfur (7.0 g / L), three isolates of B. bassiana and distilled water control. In the 1st stage of the work was obtained 10 strains of B. bassiana, reisolated of dying insects found in high backlands of Paraiba, which are kept in culture media in UFCG. Of these, three (P21CZ, P22CZ and P29CZ), analyzing the results obtained in vitro, can be attributed to those with the highest germination potential for use in vitro bioenaios, in view of the control of T. urticae, and They were sent to the Selectivity of Laboratory Chemicals, UFERSA. In the 2nd stage of the work, to start bioassays fertilized females of T. urticae for sheet Canavallia ensiformis discs were transferred. When the mites reached in each bioassay, the desired stage, egg, larva or protonymph prefaced the bioassay. Each disc was sprayed with the treatments contaminated by manually pressurized spray then left for a period of 1 hour for drying, removing excess waste. Soon after drying were transferred to air-conditioned BOD chambers, where they were kept under controlled temperature conditions of 25 ± 1 ° C, photoperiod 12 hours and relative humidity of 60 ± 10%, to obtain biological data. The parameters evaluated were: duration and mortality of eggs, larvae, and protonymphs deutonymphs. The results of the three bioassays were subjected to analysis of variance and the means compared by Tukey test at 5% probability. Although the contaminated phase in the three bioassays have not been affected, there was cumulative mortality in the subsequent phases. The fenpropathrin insecticide caused 100% mortality in bioassays with egg and larva stage. The fenpropathrin chemical acaricides, milbemectin and abamectin were more efficient in all three bioassays with average values similar mortality. For directly treated larvae the abamectin, caused longer-term contact of the larvae. The isolated B. bassiana P22CZ, the concentration (2.5 x 106), had the highest mortality rate 63.8%. We observed a positive relation between increased exposure time of the mites to concentrations of isolated P29CZ and the highest concentration applied. The percentage of cumulative mortality by lime sulfur in the three bioassays, egg, larva and protonymph were low, with values of 8.8%, 7.5% and 25%.