Made available in DSpace on 2016-06-02T19:02:41Z (GMT). No. of bitstreams: 1 4328.pdf: 9852557 bytes, checksum: 9a38f4661fc82092a95f76aa328b7cf9 (MD5) Previous issue date: 2012-02-16 / Financiadora de Estudos e Projetos / In recent years, the technology animal cells culture has allowed the development of many byproducts, especially those with pharmacological interest. Some of these products, the recombinant proteins, can be produced by heterologous expression systems on a commercial scale. Bacteria, yeast, mammalian cells and insects are some of the hosts used in these processes. As source of proteins with farmacological interest, the catterpillar Lonomia obliqua hemolinph was demonstrated to be a helpful organismo. Antiviral, antiapoptotic, antimicrobial and inducing growth proteins, are some of examples. Since the control of viral infections is a major interest to public health, the searching for new antiviral drugs has utmost importance. Several studies have reported the presence of active principles in the arthropods hemolymph. Recently, we demonstrated the existence of an antiviral protein in the hemolymph of the caterpillar Lonomia obliqua. This purified protein induced viral production reduction (TCID50 mL-1) over 157 times in cells infected with the measles virus, 61 times for polio and 61 times for influenza virus H1N1 infections. Thus, the present goals were building and expression of a recombinant plasmid contained coding sequences for expression of viral proteins (using baculovirus) in insect cell Sf-9 system. By this process, it was aimed to test biological activity of the protein. Further sequence analyses of this protein were performed using bioinformatics tools. The RNA of L. obliqua was extracted with Trizol reagent. RNA product was used in RT-PCR reactions with primers specific for the antiviral protein, based on the sequence of the cDNA libraries of L. obliqua tegument and spines, using all possible frame of translation for each cDNA. Restriction sites were inserted in cDNA sequence to insert it in pFastBacTM1 donor vector (Invitrogen). The sequence contained in selected clone of Escherichia coli DH5α was used for transformation into E. coli DH10Bac to obtain a bacmid by transposition process. This bacmid was used for antiviral recombinant protein expression in Sf-9 cells. This recombinant protein activity was tested in Picorna (EMC enchephalomiocardite), Rubeola and Herpes virus. In these trials, it was observed a replication reduction of 10,000, 10,000 and 1,000000 times, respectively. The bioinformatics analysis demonstrated that this protein is secreted, globular and probably belongs to a new class of proteins. / A tecnologia de cultivo de celulas animais tem permitido nos ultimos anos o desenvolvimento de inumeros bioprodutos. Principalmente com interesse farmacologico, alguns desses produtos, as proteinas recombinantes, podem ser produzidas em sistemas de expressao heterologos em escala comercial. Bacterias, leveduras, celulas de mamiferos e de insetos sao alguns dos hospedeiros utilizados nestes processos. Como fonte de proteinas de interesse farmacologico, a hemolinfa da lagarta Lonomia obliqua mostrou-se um organismo bastante promissor. Proteinas antivirais, antiapoptoticas, antimicrobianas e indutoras de crescimento sao alguns destes exemplos. Como o controle das infeccoes virais e de grande interesse pra saude publica, a busca por novos antivirais e de extrema importancia. Diversos estudos relatam a presenca de principios ativos na hemolinfa de artropodes. Recentemente nos demonstramos a existencia de uma proteina antiviral na hemolinfa da lagarta Lonomia obliqua. Esta proteina purificada mostrou-se capaz de reduzir a producao viral (TCID50 mL 1) mais de 157 vezes o virus do sarampo, 61 vezes para o virus da polio e 61 vezes para o virus influenza H1N1. Assim, este estudo objetivou a construcao e expressao de um recombinante contendo sequencias de codificacao da proteina antiviral para a expressao em sistema baculovirus/celula de inseto Sf-9 e a realizacao de testes de atividade biologica e caracterizacao por bioinformatica. Para sintetizar cDNA, o RNA de L. obliqua foi extraido com o reagente Trizol e usado nas reacoes de RT-PCR com primers especificos para a proteina antiviral, com base na sequencia das bibliotecas de cDNA de L. obliqua de tegumento e espiculas, utilizando todos os frames de traducao possiveis para cada cDNA. Sitios de restricao foram inseridos no cDNA para ligacao ao vetor doador pFastBacTM 1 (Invitrogen). O plasmideo recombinante selecionado em Escherichia coli DH5α foi utilizado na transformacao em E. coli DH10Bac para a obtencao do bacmideo pelo processo de transposicao. O bacmideo recombinante foi utilizado para a expressao da proteina antiviral em celulas SF-9. A atividade desta proteina recombinante foi testada em Picornavirus (EMC - encephalomiocardite), Rubeola e Herpes. Nestes testes foi observado que a proteina reduziu em 10.000, 10.000 e 1.000.000 a titulacao viral, respectivamente. As analises de bioinformatica demonstraram que esta proteina e secretada, globular e provavelmente pertenca a uma nova classe de proteinas.

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Little is understood about off-host behavior and ecology of immature Amblyomma maculatum Koch (Gulf Coast tick). A more complete understanding of this tick is essential to protect human and animal health. My research focused on seasonality and distribution of immatures in Mississippi, potential suitability of some insect and human hosts to larvae, and aspects of nymphal questing behavior. A single larva was collected (third off-host collection reported) when sampling A. maculatum habitat using a novel device. Collection of this larva in November expands the stage’s known seasonality and confirmed a prediction concerning seasonality of larval A. maculatum. Low frequency of immatures (8.3%) confirmed that they’re incredibly difficult to collect off-host. Nymphal collections peaked in March, and known seasonality was extended for both nymphs and adults. I examined known records, elucidating seasonality and distribution of A. maculatum in Mississippi. Either multiple generations per year or diapause are responsible for observed bi-modal distribution of immature collections. Additionally, I compiled the most extensive host record of immature A. maculatum in Mississippi and investigated seasonality patterns using USDA plant hardiness zones. I compiled the most complete record of ticks found on arthropods. Amblyomma americanum and A. maculatum were both confirmed to crawl onto arthropods, giving support to occasional, unintentional dispersal by phoresy. There was no conclusive evidence that larval A. maculatum feed on arthropods, however data supported feeding by larval A. americanum. These results have interesting implications regarding evolution of pathogens/endosymbionts. I provided the first evidence that larval A. maculatum can attach to humans. Rickettsia parkeri, a human pathogen transmitted by this species has recently been shown to be capable of transovarial transmission. Therefore, larval A. maculatum may provide another avenue of transmission. I have demonstrated that A. maculatum are difficult to collect off-host in part because they prefer to quest low to the ground. In choice studies, 5-cm-tall stems were most likely to be occupied by nymphs released into an array of stems. Low vapor pressure deficit encouraged questing, while higher VPD and warmer temperature increased questing height. These results may have implications in understanding host-seeking behavior in other tick species as well.

L’abeille domestique (Apis mellifera) est un insecte qui contribue à l’agriculture par sa pollinisation. Le taux élevé des mortalités hivernales des colonies est préoccupant depuis des décennies au Canada. Plusieurs facteurs sont impliqués, particulièrement Varroa destructor; un parasite qui se nourrit du corps gras de l’abeille. Le développement d’outils adaptés permettrait un meilleur suivi des colonies. Le projet consiste à corréler l’infestation de varroa avec les concentrations en sucres sériques et les co-infections (virales et bactériennes). Cette étude compare dans le temps six ruches fortement infestées et six ruches traitées (témoins). Un prélèvement d’hémolymphe a été effectué pour mesurer les concentrations en sucres en utilisant un glucomètre humain préalablement validé. Les concentrations en sucres (glucose et tréhalose) dans l’hémolymphe étaient significativement plus faibles (p<0.001) dans les ruches fortement infestées que les témoins en septembre. L’analyse RT-PCR multiplexe de six virus (DWV A/B, BCQV, KBV, IAPV et ABPV) a démontré que les ruches fortement infestées présentent une infection simultanée virale avec des charges plus élevées que chez les ruches témoins (p<0.05) pour la majorité des virus, sauf pour ABPV. Chez les ruches fortement parasitées, les charges virales pour DWVA et BQCV sont plus élevées en septembre qu’en juillet (p≤0.0001). Serratia marcescens a été seulement détectée dans une ruche infestée et une ruche témoin. Une exposition continue et élevée à varroa occasionne, en automne, une augmentation des charges virales et une diminution des sucres, suggérant une altération de l’immunité, du métabolisme et des réserves. Ces paramètres provoquent une faiblesse et une mortalité des colonies. / The European honeybee (Apis mellifera) contributes to the agriculture by its pollination; however, the mean overwintering loss rate of colonies over the last decades in Canada is worrisome. Varroa destructor, which feeds on the fat bodies of honeybees, is considered one of the most important causes of bee colony declines. The development of adapted diagnostic tools would improve the monitoring of honeybee health. This project aims to correlate the infestation by varroa to the hemolymph sugar concentrations (trehalose and glucose) and bacterial and viral coinfections. Six highly infested and six treated hives were compared over time. Pooled hemolymph of honeybees was collected for sugar concentration measurements using a previously validated portable glucometer. The hemolymph samples were also submitted for bacteriology. Multiplex RT-PCR analyses were performed on pooled honeybees for six viruses: Deformed wing virus A and B (DWV-A/B), Bee Queen Cell Virus (BQCV), Acute Bee Paralysis Virus (ABPV), Kashmere Bee Virus (KBV), Israeli Acute Paralysis Virus (IAPV). The results show that, in September, sugar concentrations in hemolymph were significantly lower in highly infested hives (p<0.001). Infested hives showed markedly higher viral loads (p<0.05), except for ABPV. Viral loads were significantly higher (p≤0.0001) in September than in July for DWV-A and BQCV. Serratia marcescens was only detected in one infested hive and one control. Overall, a continued and severe exposure to varroa leads to increased viral charges and decreased sugar concentrations, suggesting alterations in immunity, metabolism and reserve mobilization. All these parameters contribute to the weakening and mortality of the colonies.