Submitted by (edna.saturno@ufrpe.br) on 2016-12-01T14:58:58Z No. of bitstreams: 1 Franklin Magliano da Cunha.pdf: 1121804 bytes, checksum: 66cbfdb39feb9b059c8f94887174e30c (MD5) / Made available in DSpace on 2016-12-01T14:58:58Z (GMT). No. of bitstreams: 1 Franklin Magliano da Cunha.pdf: 1121804 bytes, checksum: 66cbfdb39feb9b059c8f94887174e30c (MD5) Previous issue date: 2011-02-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Despite of the Bt formulation and transgenic Bt plants efficiency against several lepidopteran species, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) exhibits low susceptibility to the majority of them. The reasons to this low susceptibility specifically to toxins Cry1Ac and Cry1Ab are not fully clarified. Also, it is possible that species moderately or not susceptible to Cry1Ac can convey the toxin to the third trophic level. This study investigated response of S. frugiperda related to Bt with three hypotheses about its low suscepbility: regenerative cells from midgut are activated by Cry1Ac protein and renovate the epithelial cells (i) in hemolymph they increase the nitric oxide and phenoloxidases as a toxin response (ii) and the tolerance is associated to the qualitative and quantitative variations from hemocytes. We still tested the hypothesis that S. frugiperda fed with Bt-cotton can induce alterations in the ultrastructure and histochemistry of digestive cells, in humoral parameters, hemogram and ultrastructure of hemocytes of Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae), a generalist predator. It was concluded that Cry1Ac in Bt-cotton is not toxic enough to kill the armyworm, but it induce to humoral and cellular alterations as signal of response to xenobiotic. The Cry1Ac toxin ingested by S. frugiperda can expose the third trophic level (P. nigrispinus) and it causes alterations in the distribution of glycogen, lipids and calcium caused by perimicrovillar matrix disorganization. The TEM revealed the presence of spherocrystals that appear to be involved in the defense against the toxin. The humorals parameters and the hemogram of P. nigrispinus are not affected when they are reared with prey S. frugiperda fed daily with leaf Bt-cotton. However it is capable of induces little modifications in granulocytes and plasmocytes. / Apesar da eficiência de formulações Bacillus thuringiensis (Bt) e plantas transgênicas Bt contra vários lepidópteros, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) apresenta baixa suscetibilidade a maioria deles. As razões dessa baixa suscetibilidade especificamente para toxinas Cry1Ac e Cry1Ab não estão totalmente esclarecidas. É possível que espécies moderadamente ou não suscetíveis ao Cry1Ac possam adquirir essa toxina e expô-la ao terceiro nível trófico. Este estudo investigou a resposta de S. frugiperda relacionadas ao Bt baseado em três hipóteses sobre sua baixa suscetibilidade: células regenerativas do intestino médio são ativadas pela toxina Cry1Ac e renovam as células epiteliais (i), na hemolinfa aumentam a produção de óxido nítrico e fenoloxidase como resposta à toxina (ii), e a tolerância está associada a variações qualitativas e quantitativas dos hemócitos. Testamos ainda a hipotese de que o consumo de S. frugiperda alimentadas com algodão Bt pode levar a alterações na ultraestrutura e histoquímica das células digestivas, nos parâmetros humorais, hemograma e ultraestrutura dos hemócitos de Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae). Conclui-se que toxina Cry1Ac no algodão Bt não é suficientemente tóxica para matar as lagartas, mas induz alteração humoral e celular como sinal de resposta a um xenobiótico. A toxina Cry1Ac ingerida por S. frugiperda pode expor o terceiro nível trófico (P. nigrispinus) e ocasionar alterações na distribuição de glicogênio, lipídios e cálcio em decorrência da desorganização da matriz perimicrovilar. O Microscópio Eletrônico de Transmissão (MET) revelou a presença de esferocristais que parecem estar envolvidos na defesa contra toxina. Os parâmetros humorals e o hemograma de P. nigrispinus não são afetados quando são criados com presa S. frugiperda alimentadas diariamente com folhas frescas de algodão Bt. No entanto, é capaz de induzir modificações pequenas em granulócitos e plasmócitos.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / A planta Calotropis procera pertencente à famÃlia Asclepiadaceae à encontrada em vasta extensÃo do Nordeste Brasileiro. à uma planta laticÃfera e sua produÃÃo endÃgena de lÃtex à extraordinÃria. Embora haja na literatura cientÃfica diversas publicaÃÃes que relatam o potencial medicinal de diversas partes da planta, principalmente de seu lÃtex, nÃo hà ainda uma abordagem bioquÃmica e funcional de seu fluido laticÃfero. Esta pesquisa foi desenvolvida no sentido de proceder a um estudo bioquÃmico parcial de atividades endÃgenas da fraÃÃo protÃica majoritÃria do lÃtex e de avaliar seus efeitos inseticidas sobre diferentes modelos de pragas agrÃcolas. A fraÃÃo protÃica majoritÃria do lÃtex foi preparada a partir de um protocolo previamente desenvolvido no laboratÃrio em que sÃo envolvidas etapas de centrifugaÃÃo e diÃlise. A fraÃÃo protÃica isenta de borracha e de molÃculas de baixa massa molecular foi testada para atividades enzimÃticas endÃgenas proteolÃticas, quitinÃsicas e a-amilÃsica e atividades inibitÃrias para proteases e a-amilase. Ensaios enzimÃticos de digestÃo das proteÃnas do lÃtex por extratos digestivos de insetos foram tambÃm realizados. Bioensaios utilizando-se dietas artificiais contendo diferentes proporÃÃes das proteÃnas do lÃtex Ãntegras, digeridas por pronase ou aquecidas a 98 ÂC foram realizados com seis insetos pertencentes a quatro ordens. Nos bioensaios foram avaliados parÃmetros tais como desenvolvimento, sobrevivÃncia, tempo de emergÃncia dos indivÃduos alÃm de ser avaliado o efeito cumulativo de proteÃnas do lÃtex na dieta por sucessivas geraÃÃes. Na fraÃÃo protÃica do lÃtex foram encontradas atividades proteolÃticas do tipo cisteÃnica e serÃnica, alÃm de atividade quitinolÃtica. NÃo foi detectada atividade a-amilÃsica. SoluÃÃes de proteÃnas do lÃtex nÃo exibiram atividade inibitÃria do tipo a-amilÃsica e tripsÃnica ou quimiotripsÃnica. ApÃs tratamento tÃrmico, proteÃnas do lÃtex ainda solÃveis foram capazes de inibir a atividade da papaÃna. Posteriormente esta atividade inibitÃria foi capaz de inibir as atividades proteolÃticas de Callosobruchus maculatus e Dysdercus peruvianus. As proteÃnas do lÃtex foram inseticidas para C. maculatus, Zabrotes subfasciatus, Anticarsia gemmatalis e Ceratitis capitata enquanto que nÃo foram para Spodoptera frugiperda e D. peruvianus. NÃo houve efeito deletÃrio acumulativo em insetos submetidos a uma dieta contendo proteÃnas do lÃtex durante seis geraÃÃes. Quando digeridas por pronase e aquecidas por 30 min a 98 ÂC, a aÃÃo inseticida das proteÃnas do lÃtex ainda foi mantida sobre o C. maculatus. As proteÃnas do lÃtex foram completamente resistentes à proteÃlise por enzimas digestivas de Dysdercus peruvianus e Callosobruchus maculatus. Entretanto, a aÃÃo proteolÃtica endÃgena do lÃtex promoveu a proteÃlise do extrato enzimÃtico de C. maculatus embora isto nÃo tenha ocorrido no extrato intestinal de D. peruvianus. O lÃtex de C. procera apresenta forte atividade proteolÃtica e resistÃncia à proteÃlise por enzimas digestivas de insetos. Atividade quitinolÃtica foi tambÃm observada. O lÃtex possui atividade inseticida para diferentes pragas agrÃcolas e esta atividade parece estar associada à presenÃa de um inibidor de atividade proteolÃtica do tipo cisteÃnica, alÃm da presenÃa de quitinases e elevada atividade proteolÃtica endÃgena. Assim, a fraÃÃo protÃica do lÃtex pode ser considera como parte constituinte da defesa da planta contra insetos. / The plant Calotropis procera belonging to Asclepiadaceae is found around a vast extension of the Northeast from Brazil. It is a lactifers plant and its endogenous production of latex is admirable. Despite continuous reports appearing in the literature give mention to the medicinal properties of different parts of the plant, including the latex, there is not scientific approaches comprising biochemical of functional aspects of the latex from C. procera. The aims this work was determine the presence of endogenous enzymatic activities and inhibitory activity against different proteases and a-amylase, to evaluate insecticidal properties against different crop pests and to correlate enzymatic content and insecticidal action with possible role of the latex in protecting the plant to insect pathogens. The major soluble protein fraction from the latex was prepared following the protocol previously defined that includes centrifugations and dialysis steps. The protein fraction devoided of rubber and low molecular mass molecules was assayed to the presence of endogenous proteolytic activities as well as a-amylase activity. Even, the presence of inhibitor of such activities was investigated. Additionally the latex proteins were submitted to proteolysis by digestive content of different insects. Bioassays base on artificial diets with different contents of native, pronase digested or heated treated (98 ÂC) latex proteins were performed against six insects belonging to 4 different orders. Increment in bodyweight of larvae, time of development and percentage of survive were the parameters considered to analyze detrimental effects of latex proteins upon insects. The effect of latex proteins upon Callosbruchus maculatus insects by continuous exposure until F-6 generation was reached was performed. It was found strong endogenous proteolytic activity and latex proteins were resistent to proteolysis by insect digestive extracts. In addition chitinolytic activity was also detected. The latex exhibited insecticidal activity against different insect groups and this effect may be correlated to the presence of a cysteine protease inhibitor associated to its proteolytic properties and chitinolytic activity. It is suggested that the protein fraction of the latex from Calotropis procera plays a relevant role in defending the plant against pathogens.

Spodoptera frugiperda (J.E. Smith) tem sido uma das pragas mais importantes da cultura do milho no Paraguai e Brasil. Este trabalho foi realizado para subsidiar programas de manejo de resistência no Paraguai, reconhecendo-se a proximidade das fronteiras agrícolas desse país com Brasil e a alta capacidade de dispersão de S. frugiperda. Para tanto, foram realizados estudos de caracterização da suscetibilidade aos inseticidas lufenuron e flubendiamide e de diversidade genética e o fluxo gênico em populações de S. frugiperda coletadas nas principais regiões produtoras de milho do Paraguai e de dois Estados fronteiriços do Brasil (Mato Grosso do Sul e Paraná). O método de bioensaio para a caracterização das linhas-básica de suscetibilidade foi o de tratamento superficial da dieta artificial com o inseticida. As variações na suscetibilidade a lufenuron e flubendiamide entre as populações de S. frugiperda testadas foram baixas (< 4 vezes), comparando-se as populações das regiões Oriental e Ocidental do Paraguai que apresentam caraterísticas climáticas distintas ou entre as populações do Paraguai e do Brasil. Para o monitoramento da suscetibilidade de populações de S. frugiperda no Paraguai foram definidas as doses diagnósticas, baseada na DL99, de 0,16 &mu;g de lufenuron.cm-2 (equivalente a 10 &mu;g de lufenuron.mL-1 de água) e 2,84 &mu;g de flubendiamide.cm-2 (180 &mu;g de flubendiamide.mL-1 de água). Mediante o uso de 12 loci microssatélites, foram verificadas maior diversidade genética dentro das populações (87,70%) quando comparada entre as populações de cada país (9,91%) e entre países (2,39%). Sendo assim, não foi constatada alta estruturação genética entre as populações de S. frugiperda coletadas no Paraguai e Brasil. As populações do Paraguai mostraram-se mais distintas entre si quando comparadas com as populações do Brasil. Nossos resultados apontam baixa estruturação genética e moderado/alto fluxo gênico entre as localidades de coleta de populações de S. frugiperda e entre as populações do Paraguai e Brasil. Os resultados obtidos na presente pesquisa servirão para a implementação de um programa de manejo da resistência de S. frugiperda a inseticidas no Paraguai, com ênfase na necessidade de ações no âmbito regional considerando os estados fronteiriços do Brasil. / Spodoptera frugiperda (J.E. Smith) has been one of the most important corn pests in Paraguay and Brazil. This research was conducted to collect information to insect resistance management programs in Paraguay considering the proximity of agricultural borders of this country with Brazil and the high dispersal capacity of S. frugiperda. We conducted studies to characterize the susceptibility to the insecticides lufenuron e flubendiamide and to evaluate the genetic diversity and gene flow among S. frugiperda populations collected from major corn-producing regions in Paraguay and two frontier States of Brazil (Mato Grosso do Sul and Paraná). The bioassay method to characterize the baseline susceptibility was the diet surface treatment with the insecticide. The variation in the susceptibility to lufenuron and flubendiamide among S. frugiperda populations tested was low (< 4-fold) by comparing populations from Estearn and Western regions of Paraguay with distinct climatic conditions or populations from Paraguay and Brazil. For susceptibility monitoring to lufenuron and flubendiamide in S. frugiperda populations in Paraguay, we defined the diagnostic doses based on LD99 of 0.16 &mu;g of lufenuron.cm-2 (equivalent to 10 &mu;g of lufenuron.mL-1 of water) and 2,84 &mu;g of flubendiamide.cm-2 (180 &mu;g of flubendiamide.mL-1 of water). Using 12 microsatellite loci, higher genetic diversity within populations (87.70%) than among populations within each country (9.91%) and between countries (2.39%). Thus, there was no high genetic structure between S. frugiperda populations collected in Paraguay and Brazil. The populations of S. frugiperda from Paraguay were more distinct among themselves when compared with the populations from Brazil. Our results suggest low genetic structure and moderate/high gene flow among different sampling locations of S. frugiperda populations as well as between populations from Paraguay and Brazil. The results obtained in this research will support the implementation of resistance management programs of S. frugiperda to inseticides in Paraguay with focus on the need of plan of actions at regional level considering the border States of Brazil.

Maize (Zea mays) is an important agricultural crop frequently targeted by pests that pose a threat to plant development and survival. To deal with this problem, maize generates a wide variety of responses to attack by pests, from activation of woundresponse pathways to the release of volatile compounds. Several maize lines have been developed that show resistance to one common pest, the larvae of the fall armyworm (Spodoptera frugiperda). Analysis of the volatiles released by the resistant and susceptible lines in the presence and absence of the fall armyworm was conducted using SPME coupled to GC/MS. Caryophyllene, a commonly released plant volatile, was identified in the resistant line. In the susceptible line, caryophyllene was detected in smaller quantities or not at all. The results of a preference study demonstrated that fall armyworm larvae show a statistically significant preference for yellow-green whorl tissue from the susceptible over the resistant line.

Organic insecticides play a big role in reducing the usage of chemical insecticides and their negative impact on the environment. Bacillus thuringiensis (Bt) spays are the only tool that organic farmers are allowed to use for the control of pests. Genetic engineering and modification of crops have been made possible with scientific advances in cell and molecular biology. These advances are used to transfer some of the Bt Cry toxins into crops for control of target species to reduce yield loss. Bt maize were commercialised for the first time in South Africa in 1998 and the economic important stem borers, Busseola fusca (Fuller) (Lepidoptera: Noctuidae), Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) and Sesamia calamistis (Hampson) (Lepidoptera: Noctuidae) were exposed to the Cry1Ab toxin that is found in Bt maize. Busseola fusca developed resistance to Cry1Ab under field conditions within eight years after it had been released. Eldana saccharina (Walker) (Lepidoptera: Pyralidae) is a major pest on sugarcane in South Africa and although it has not been recorded on maize in this country, is it known as a major pest of maize in other African countries. African armyworm, Spodoptera exempta (Walker) (Lepidoptera: Noctuidae) has a very wide distribution in Africa and is known to be an occasional pest on maize. The aims of this dissertation were to determine the efficacy of Bt spray applications for control of four lepidopteran pests and whether development of Cry1Ab resistance by B. fusca caused a loss in susceptibility to other Bt toxins (i.e. cross-resistance). Susceptibility bioassays with 10 day old larvae were conducted under laboratory conditions. Treatments included application of various dosage rates of Dipel® and deltamethrin as well as exposure to MON810 (maize leaves). Stemborer populations of C. partellus, E. saccharina, and B. fusca (Venda) as well as the S. exempta were effectively controlled by the Bt spray, Dipel®. Care should be taken not be interpret the percentage C. partellus, E. saccharina and S. exempta larvae that survived after exposure to MON810 and Bt spray treatments as development of resistance without verification of these experiments with earlier instars that are known to be more susceptible. Spodoptera exempta is active throughout a year in temperate zones of Africa. If S. exempta develop resistance to Cry toxins and Bt maize events would be released for commercial planting in these areas, S. exempta pose a threat added to their injuriousness. Busseola fusca larvae were sampled from Venda (susceptible population), Ventersdorp and the Vaalharts Irrigation Scheme (resistant population). The Ventersdorp B. fusca population was controlled by MON810 and MON89034 and Bt sprays, but the percentage larvae that survived showed reduced susceptibility within the population. Dipel® treatments, MON810 and MON89034 did not provide effective control of the Vaalharts B. fusca population reported to be resistant to Cry1Ab, in two experiments. The high survival rates indicate a reduction in susceptibility to Cry toxins other than Cry1Ab and therefore development of cross resistance in the Vaalharts B. fusca population. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Organic insecticides play a big role in reducing the usage of chemical insecticides and their negative impact on the environment. Bacillus thuringiensis (Bt) spays are the only tool that organic farmers are allowed to use for the control of pests. Genetic engineering and modification of crops have been made possible with scientific advances in cell and molecular biology. These advances are used to transfer some of the Bt Cry toxins into crops for control of target species to reduce yield loss. Bt maize were commercialised for the first time in South Africa in 1998 and the economic important stem borers, Busseola fusca (Fuller) (Lepidoptera: Noctuidae), Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) and Sesamia calamistis (Hampson) (Lepidoptera: Noctuidae) were exposed to the Cry1Ab toxin that is found in Bt maize. Busseola fusca developed resistance to Cry1Ab under field conditions within eight years after it had been released. Eldana saccharina (Walker) (Lepidoptera: Pyralidae) is a major pest on sugarcane in South Africa and although it has not been recorded on maize in this country, is it known as a major pest of maize in other African countries. African armyworm, Spodoptera exempta (Walker) (Lepidoptera: Noctuidae) has a very wide distribution in Africa and is known to be an occasional pest on maize. The aims of this dissertation were to determine the efficacy of Bt spray applications for control of four lepidopteran pests and whether development of Cry1Ab resistance by B. fusca caused a loss in susceptibility to other Bt toxins (i.e. cross-resistance). Susceptibility bioassays with 10 day old larvae were conducted under laboratory conditions. Treatments included application of various dosage rates of Dipel® and deltamethrin as well as exposure to MON810 (maize leaves). Stemborer populations of C. partellus, E. saccharina, and B. fusca (Venda) as well as the S. exempta were effectively controlled by the Bt spray, Dipel®. Care should be taken not be interpret the percentage C. partellus, E. saccharina and S. exempta larvae that survived after exposure to MON810 and Bt spray treatments as development of resistance without verification of these experiments with earlier instars that are known to be more susceptible. Spodoptera exempta is active throughout a year in temperate zones of Africa. If S. exempta develop resistance to Cry toxins and Bt maize events would be released for commercial planting in these areas, S. exempta pose a threat added to their injuriousness. Busseola fusca larvae were sampled from Venda (susceptible population), Ventersdorp and the Vaalharts Irrigation Scheme (resistant population). The Ventersdorp B. fusca population was controlled by MON810 and MON89034 and Bt sprays, but the percentage larvae that survived showed reduced susceptibility within the population. Dipel® treatments, MON810 and MON89034 did not provide effective control of the Vaalharts B. fusca population reported to be resistant to Cry1Ab, in two experiments. The high survival rates indicate a reduction in susceptibility to Cry toxins other than Cry1Ab and therefore development of cross resistance in the Vaalharts B. fusca population. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

MgAl layered double hydroxides nanoparticles (LDHs) are known as the useful materials in agrochemsitry. LDHs can be used as a bio-insecticide carrier to enhance insecticide’s activity efficiency. In our study, to improve the insecticide activity of anacardic acid, an extract from cashew nut shell liquid, we intercalated it MgAl layered double hydroxides nanoparticles. Different hybridization between anacardic acid and LDHs (37, 74, 148, and 296μg/mL) (L-As) were made and tested on the survivals of cutworms (Spodoptera litura). L-As or free anacardic acid was sprayed directly on the leaves mustard to feed cutworms or directly on the skin of cutworms. Our results showed that in all L-As treatments, the worm killing efficiency was higher than the free anacardic acid treatment. / Hạt nano lớp đôi hydroxides MgAl (LDHs) được biết đến như là những vật liệu hữu ích trong nông ngành hóa học nông nghiệp. LDHs có thể được dùng như là một loại chất mang cho thuốc trừ sâu sinh học để tăng cường hiệu lực diệt sâu. Trong nghiên cứu này, để tăng cường hiệu lực diệt sâu của anacardic acid, một loại hoạt chất được chiết từ dầu vỏ hạt điều, chúng tôi đã gắn chèn nó lên hạt nano lớp đôi hydroxides MgAl. Các nồng độ khác nhau của dạng lai của anacardic và LDHs (37, 74, 148 và 296μg/mL) (L-As) đã được kiểm tra tỷ lệ sống của ấu trùng sâu khoang (Spodoptera litura). Các nghiệm thức L-As và dạng anacardic acid tự do đã được phun lên lá rau cải ngọt cho ấu trùng sâu ăn hoặc phun trực tiếp lên da ấu trùng sâu. Kết quả cho thấy, tất cả các công thức có xử lý bằng L-As, hiệu lực diệt ấu trùng sâu đều cao hơn so với dạng anacardic acid ở trạng thái tự do.

<p>The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium.</p><p>Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex.</p>

<p>The aim of this study was to better understand the role of conditioned medium (CM) in Spodoptera frugiperda Sf9 insect cell proliferation and recombinant protein production using the baculovirus expression system.</p><p>CM was found to stimulate cell proliferation. Addition of CM and 10 kDa CM filtrate to an Sf9 culture decreased the lagphase and the maximum cell density was reached earlier than for cultures in fresh medium. The positive effect of 10 kDa CM filtrate showed that CM contains at least one small growth promoting factor. The effect was not eliminated by trypsin treatment. Addition of CM or 10 kDa CM filtrate to Sf9 cultures was found to have a negative effect on the recombinant protein production. The effect was thought to be indirect and most probably via the impact of CM on cell physiology. CM was also found to contain proteinase activity. The proteinase was identified as Sf9 cathepsin L. A proform with a molecular mass about 49 kDa and two active forms at about 39 and 22 kDa were found. The role of cathepsin L in Sf9 cultures is not yet clear. However, the knowledge of the presence of this proteinase in CM can be of great value for improving product quality and yield. Further, CM was found to have other properties as well: a concentrated fraction of CM exhibited strong antibacterial activity towards Bacillus megaterium and a weaker activity towards Escherichia coli. B. megaterium lysed rapidly after incubation in the CM fraction.</p><p>Repeated subculturing of Sf9 cells provoked a switch in growth kinetics. After 30-45 passages the cells started to proliferate earlier after inoculation and addition of CM had no longer a growth stimulating effect. However, CM still stimulated growth of a culture with low passage (LP) number (up to 45 passages). High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. This synchronisation was more pronounced for HP cells than for LP cells, which correlated to a higher recombinant protein production in baculovirus infected HP cells than in LP cells. Synchronisation of cells in G2/M by yeastolate-limitation before infection with baculoviruses suggested that the degree of synchronisation is connected to the cell density dependent decrease in recombinant protein production of Sf9 cultures.</p>

Le lépidoptère Spodoptera frugiperda est un ravageur important des cultures mais aussi l'organisme source de la lignée cellulaire Sf9, utilisée pour la production de protéines recombinantes dans le système d'expression baculovirus/cellule d'insecte. Dans la première partie de ce travail, nous avons étudié la transcription chez le baculovirus AcMNPV aux temps précoces de l'infection. Nous montrons tout d'abord que les régions homologues (hrs) portées par le génome de ce virus contiennent de nombreux motifs de reconnaissance pour des facteurs de transcription de type AP1/CREB, et que ces sites fixent spécifiquement des protéines de la cellule-hôte, Sf9. Par ailleurs, dans le contexte de l'infection, ces sites sont nécessaires à la transactivation médiée par le facteur viral très précoce IE1 qui se fixe lui aussi sur les hrs. Cette étude montre pour la première fois l'implication de facteurs cellulaires dans ce mécanisme de transactivation virale. La progression de ces travaux a cependant été freinée par le peu de données disponibles sur les gènes exprimés par Sf9. Pour en faciliter l'accès, nous avons donc construit une banque d'ADNc, dont l'exploitation fait l'objet de la deuxième partie de ce mémoire. Son criblage a permis d'obtenir la séquence complète de l'ADN complémentaire du gène hsp90 chez S. frugiperda, et d'en déterminer les principales caractéristiques. Par ailleurs, le séquençage à grande échelle de la banque a conduit à la constitution d'une banque d'ESTs, permettant l'identification de la quasi-totalité des gènes de protéines ribosomales. L'analyse de leur séquence a révélé l'existence de particularités qui semblent restreintes aux insectes et aux lépidoptères, un résultat inattendu compte tenu de la grande conservation de ces gènes entre espèces.