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Orientador: Octavio Henrique de Oliveira Pavan / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-20T21:51:54Z (GMT). No. of bitstreams: 1 Garcia-Canedo_AlejandraMariaGladys_D.pdf: 6144185 bytes, checksum: 1828f8158312f3ff8bd975aac77f9653 (MD5) Previous issue date: 1995 / Doutorado / Genetica / Doutor em Ciências Biológicas

Orientadores: Sonia Nair Bao, Bergamann Morais Ribeiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T02:39:11Z (GMT). No. of bitstreams: 1 Silveira_EniBragada_D.pdf: 8190576 bytes, checksum: 40d63d1dc3a53a42d25cdd9abd266af0 (MD5) Previous issue date: 2005 / Resumo: A família Baculoviridae é constituída por vírus de fita-dupla de DNA que infectam artrópodes, larvas de lepidópteros em sua maioria. No início da década de 90, descobriu-se que baculovírus possuem genes (p35 e iap) capazes de inibir a apoptose em células hospedeiras, uma estratégia para garantir a replicação viral. O mutante vApAg, derivado de Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) e que possui um transposon interposto no gene iap3, induz apoptose em uma linhagem celular derivada de Anticarsia gemmatalis, havendo produção de progênie viral. Já o vírus vP35del, derivado de Autographa californica multiple nucleopolyhedrovirus (AcMNPV) e que possui o gene p35 deletado, também induz apoptose na mesma linhagem celular, porém sem sinais de produção de progênie. Neste trabalho, investigou-se a ocorrência de apoptose in vivo induzida por vApAg e pelo recombinante p35- derivado de AcMNPV ¿ vHSGFP/P35del em larvas de A. gemmatalis. Utilizou-se como modelo de estudo hemócitos infectados via injeção intrahemocélica de vírus extracelulares. Os hemócitos de A. gemmatalis foram caracterizados e o processo infeccioso de AgMNPV, vApAg, vHSGFP e vHSGFP/P35del nestas células foi estudado com base em técnicas morfológicas, tendo sido, ainda, observadas a mortalidade e alterações no desenvolvimento larval induzidas pelos vírus. Foram observados seis tipos de hemócitos: prohemócitos (pr), plasmatócitos (pl), granulócitos tipo 1 (gh1), granulócitos tipo 2 (gh2), oenocitóides (oe) e esferulócitos (sph), os quais apresentam semelhança geral com o conjunto de hemócitos de outros lepidópteros, inclusive com relação ao seu número total (CTH), às proporções dos diferentes tipos ao longo do desenvolvimento (CDH) e à atividade de fenoloxidase. A inativação do gene iap-3 de AgMNPV resulta em apoptose para um número considerável de hemócitos em até 72 horas pós-infecção, com a produção de vírus extracelulares e ocluídos. O tempo médio de morte para larvas infectadas com 100 e 1000 PFU de vApAg é cerca de 2,5 dias maior que para as mesmas doses de AgMNPV. A deleção do gene p35 de AcMNPV resulta em uma apoptose de hemócitos discreta, entre 24 e 72 horas pós-infecção, com alguma produção de vírus extracelulares, havendo redução na proporção de hemócitos infectados ao longo do tempo e redução na infectividade das larvas. Para vHSGFP, uma dose da ordem de 105 vezes maior que de AgMNPV foi necessária para causar uma mortalide próxima a 100%. Desta forma, A. gemmatalis consiste em um hospedeiro semi-permissivo a AcMNPV. Ao contrário de AgMNPV e vApAg, vHSGFP e vHSGFP/P35del, em geral, não causam liquefação dos insetos. Algumas larvas sobreviventes à infecção por vHSGFP e, principalmente, por vHSGFP/P35del, originam pupas anômalas inviáveis. Todos os tipos de hemócitos de A. gemmatalis são susceptíveis à infecção pelos quatro vírus. Pl e gh1 são mais susceptíveis enquanto gh2 raramente são infectados. Sph são especialmente tolerantes aos vírus derivados de AcMNPV, podendo constituir um dos fatores determinantes da baixa infectividade destes vírus em A. gemmatalis. Gh1 e pl fagocitam células, fragmentos celulares, corpos apoptóticos, vírus extracelulares e ocluídos. Hemócitos infectados parecem ser alvos de uma resposta citotóxica causadora de necrose. Os resultados deste trabalho reforçam a hipótese de que a apoptose constitui uma estratégia anti-viral importante em insetos, tendo em vista que os mesmos não possuem imunidade adquirida. A compreensão de aspectos da relação patógeno-hospedeiro, especialmente no que diz respeito aos mecanismos de imunidade de insetos contra vírus, pode contribuir para a otimização dos baculovírus como agentes de controle biológico de pragas e como vetores de expressão de genes heterólogos, bem como colaborar para a descoberta de novos antivirais e para o controle da transmissão de vírus por insetos / Abstract: The Baculoviridae family is composed by double-strand DNA viruses that infect arthropods, generally lepidoptera larvae. In the beginning of the 90's, it was shown that baculoviruses possess genes (p35 e iap) that inhibit apoptosis in host cells - a strategy to guarantee viral replication. The mutant vApAg, derived from Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), which has the iap-3 gene interrupted by a transposon, induces apoptosis in a cell line derived from Anticarsia gemmatalis, with progeny production. The virus vP35del, derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV), which has a deletion in the gene p35, also induces apoptosis in the same cell line, however there is no progeny production. In this work, we have investigated apoptosis in vivo induced by vApAg and by a p35- recombinant derived from AcMNPV - vHSGFP/P35del in A. gemmatalis larvae. Hemocytes were the model chosen for study and the infection occurred by intrahaemocoelic injection of budded viruses. The hemocytes were characterized and the infective processes of AgMNPV, vApAg, vHSGFP and vHSGFP/P35del in these cells were studied based on morphological approaches. It was also observed larval mortality and the effects on larval development caused by these viruses. Six types of hemocytes were observed: prohemocytes (pr), plasmatocytes (pl), granular hemocytes type 1 (gh1), granular hemocytes type 2 (gh2), oenocytoids (oe) and spherulocytes (sph). They presented great similarity to the hemocytes pool from other lepidoptera, including the total number of these cells in circulation (THC), the proportions of each type during larval development (DHC) and phenoloxidase activity. xix The inactivation of the iap-3 gene in AgMNPV resulted in apoptosis induction for a great number of hemocytes until 72 hours post infection, with the production of budded and occluded viruses. The mean time to death was delayed in 2.5 days in average for insects inoculated with 100 and 1000 PFU of vApAg, when compared to the same doses of AgMNPV. The deletion of p35 in AcMNPV resulted in a milder apoptosis, between 24 and 72 hours post infection, with some production of budded viruses. It caused a reduction in the proportion of infected hemocytes along the time of infection and reduced larval infectivity. For vHSGFP, a viral dose in the order of 105 times higher than for AgMNPV is needed to cause a mortality ratio around 100%. Therefore, we consider A. gemmatalis as a semi-permissive host to AcMNPV. The recombinants vHSGFP and vHSGFP/P35del generally do not cause melting of insects, what normally occurs for insects infected by AgMNPV and vApAg. Some surviving larvae infected by vHSGFP and, mainly by vHSGFP/P35del, originate anomalous pupae. All hemocytes types from A. gemmatalis were susceptible to infection by the viruses studied. Pl and gh1 are the most susceptible cells, while gh2 are rarely infected. Sph are especially resistant to AcMNPV-derived viruses. This can constitute one of the factors that determine the low infectivity of AcMNPV in A. gemmatalis larvae. Gh1 and pl phagocytose cells, cell fragments, apoptotic bodies, budded viruses and occluded viruses. Infected hemocytes appear to be targets for a cytotoxic response that causes necrosis. These results reinforce the hypothesis of apoptosis is an important anti-viral strategy in insects, which do not present acquired immunity. A better comprehension of host-pathogen relationships, specially the ones related to insect immunity against viruses, may contribute to an optimization of baculoviruses as biological control agents and heterologous expression vectors, as well as to the iscovery of new antiviral drugs and to the control of virus transmission by insect / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Abstract Type IX collagen is a quantitatively minor component of cartilage collagen fibrils. Although a few mutations have been associated with multiple epiphyseal dysplasia, recent evidence suggests involvement of type IX collagen in a wider spectrum of phenotypes. The functional role of this molecule remains undetermined, in part due to difficulties in obtaining high amounts of intact protein. To facilitate more efficient mutation screening and comparison of the genomic organization of the human genes encoding the α1(IX), α2(IX) and α3(IX) polypeptides, their genomic structures were characterized. Complete nucleotide sequences were determined for the COL9A2 and COL9A3 genes along with sequences for all the exon boundaries in the COL9A1 gene. Putative transcription control elements were identified and the alternative promoter region was characterized in the human and mouse COL9A1 genes. Mutation screening was performed for the COL9A3 gene and two apparently neutral 9-bp deletions within the COL1 domain were identified. These are the first deletions within a triple-helical domain of any collagen that are not associated with a disease phenotype. An insect cell expression system with an exogenous source of prolyl 4-hydroxylase was used to produce heterotrimeric human type IX collagen. The recombinant protein consisted of the three a chains in a 1:1:1 ratio and showed correct folding and high thermal stability. Up to 10 mg of secreted protein could be purified from a litre of culture medium. The expression system was used to analyze the chain association of type IX collagen in cellulo. Although the chains are capable of homotrimerization, a preference for heterotrimer formation was noted.The neutral deletion was characterized further using the insect cell system. Mutant α3(IX) chains carrying a deletion of one Gly-X-Y triplet were shown to form correctly folded heterotrimers with the wild-type α1(IX) and α2(IX) chains. The results suggest a function for the NC2 domain in neutralizing the effect of the deletion. This work provides a novel means for the analysis of type IX collagen mutations and their protein-level effects, and should enable future studies to be made of the structure-function relationship in type IX collagen.

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A baculovirus (Spodoptera frugiperda) system was designed to express recombinant canine thyroid stimulating hormone (rcTSH). The efficacy of rcTSH was measured against pituitary derived bovine thyroid stimulating hormone (bTSH) through a series of in vitro and in vivo experiments. Initial purification of rcTSH was performed in order to characterize the hormone for further analyses. Ion exchange columns and tangential flow membranes were chosen based upon the traits of the rcTSH molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels visualized by Coomassie blue, silver stain, and western blot demonstrated the effectiveness of the purification process. Primary cell, static tissue, and perifusion tissue cultures were employed to investigate in vitro thyroid cell/tissue response to rcTSH and bTSH. Canine thyroid cells were liberated from tissue samples, cultured, then exposed to TSH treatments in which media was subsequently harvested and measured for cyclic adenosine monophosphate (cAMP), a second messenger in the TSH downstream signaling cascade. The cAMP concentrations measured were sporadic and not consistent with expected concentrations for treatments or controls. For the static tissue culture, slices of bovine thyroid tissue were incubated and exposed to a series of media-only wash steps as well as treatment steps using varying concentrations of rcTSH. Unfortunately, the experiment was compromised resulting in the slow release of thyroxine (T4) for all samples due to tissue death. Perifusion experiments conducted on bovine thyroid tissue compared the release of T4 due to bTSH and rcTSH stimulation in a dynamic system. Unable to perform statistical analysis due to small sample sizes, graphical representation demonstrated stimulatory effects by bTSH and rcTSH when compared to control. Biological assays were used to compare the in vivo efficacy of rcTSH to bTSH which included 3 species (goldfish, rat, and canine). Results from mammalian experiments when subjected to analysis of variance (ANOVA) resulted in the rejection of the null hypothesis of equal means (P<0.05) between control, bTSH, and rcTSH treatments. Further analysis by Tukey's W procedure demonstrated the stimulatory actions of rcTSH and bTSH to be similar (P>0.05) to each other while greater (P<0.05) than control at the 4 and 6 hour post injection time.

Minimising the time from 'scientific breakthrough'to clinical trial of a 'drug candidate' protein is a critical component leading to a successful product release. Crystallographic characterisation has become a standard requirement prior to clinical trial requiring milligram quantities of protein. The optimisation of protein expression systems is therefore of great commercial and social importance and represents a significant technical challenge. Without it, making enough protein for crystallography can quite literally take years. Baculoviral expression of recombinant protein by infection of an insect cell host is a well established technique in modern biotechnology. Although a limit to recombinant protein production in batch culture exists the mechanism has not been demonstrated. In particular, there has been no discussion of how biomass accumulation kinetics relate to the system limits in terms of final recombinant protein yield. The central aim of this thesis was therefore to quantitatively account for the dynamic behaviour in macromolecular compartments after baculovirus infection of insect cells, the rationale being that a rudimentary level of mechanistic structure can greatly enhance our ability to capture transient behaviour. The catalytic mass dictates the rate of total biomass accumulation in the baculovirus expression vector system (BEVS) and is directly proportional to the total RNA content of both baculovirus infected and uninfected Sf9 cells. During infection the total RNA concentration reaches a catalytic limit causing a switch from exponential to zero order mass accumulation kinetics. Importantly, this extends to individual cells as confirmed using a population balance model for the cell volume distribution after the switch to linear growth. By flow cytometry, a positive correlation between RNA content and cell size post infection validated this modelling assumption. The rate of mass accumulation slows down during the first 12 hours post infection (hpi). This is consistent with the decrease in both specific consumption rates of glucose and oxygen when using cell mass rather than cell number as a basis. A decrease in the geometric standard deviation (óg) of the cell volume distribution during the first 12 hpi indicates that cells enter the lower growth rate at times inversely proportional to their volume. Using several approaches no obvious biological mechanism to account for the empirical model was identified. The use of óg kinetics provides a novel tool for characterising the relative behaviour of infected cells in the BEVS. The effect of multiplicity of infection (MOI) on virus timing events between cultures was also tested. Little or no effect of MOI was observed on the timing of virus induced events during synchronous infections. The óg kinetics did indicate virus events occur 5 hours earlier at a MOI of 100 compared to a MOI of 20 plaque forming units per cell. There was however, no significant evidence of earlier death kinetics when measured using Trypan blue dye exclusion to measure cell membrane integrity. Virtually no effect of MOI on virus timing was observed using â-galactosidase production profiles. The viral DNA mass (vDNA) was measured using real time quantitative PCR (RTQ PCR) and has a doubling time of 2 hours. A vDNA template limited replication model fit the data well. Viral replication proceeds from 6 until 24 hpi with the average infected cell accumulating between 12 000 and 84 000 vDNA copies when replication stops. In theory, a dynamic equilibrium could have been present after the commencement of virus budding but this was not the case. At least 62% of the total DNA increase post infection is viral. No more than 16% of the total vDNA produced actually bud from the infected cell. This overproduction of vDNA is probably due to the wild type history of the virus, which normally occludes virions in a crystalline polyhedrin matrix within the cell nucleus as part of its life cycle. The approach taken here provides a framework for characterisation of both viral and total mass accumulation with the use of a few simple intracellular macromolecular pools. This thesis demonstrates that the BEVS limit in batch culture is not simply a result of the exhaustion of an amino acid using a case study of amino acid consumption by uninfected Sf9 cells for a 300 hour culture period. Future attempts to identify the system limits and will require the linkage of a mechanistic model with a more extensive and accurate analysis of important metabolites and specific intracellular species.

The bertha armyworm (Mamestra configurata) is a major pest of canola and other oilseed crops. A promising control agent for this species is the baculovirus Mamestra configurata nucleopolyhedrovirus (MacoNPV). Baculoviruses are insect-specific viruses. Infections initiate in the host midgut following ingestion of virus particles called occlusion bodies. For a productive infection to occur, the occlusion bodies must dissolve to release the infectious occlusion-derived virions. These virions must pass through the peritrophic matrix, a protein-chitin meshwork that lines the midgut of most insects and provides protection against abrasion and pathogen invasion. The mechanism by which the baculovirus virions transit the peritrophic matrix is unknown. Following the initial infection of midgut cells, a second virion phenotype, the budded virus, is released from infected cells and establishes a systemic infection within the insect. The 11K group of genes, which are conserved among baculovirus species and other insect-infecting viruses, encode proteins with a predicted chitin-binding domain. The degree of conservation of these genes among insect-infecting viruses suggests that they may play a role in insect infectivity. It is possible that the gene products could be involved in an interaction between the baculovirus occlusion-derived virions and the peritrophic matrix or the chitin-secreting cells of the midgut epithelium, and therefore may be involved in initial oral infectivity. The two 11K genes from MacoNPV (ORF 118 and ORF 164), and their homologues in a second species of baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV [ORF145 and ORF150]) were expressed in a baculovirus expression system. The ability of the proteins, Maco118, Maco164, Ac145, and Ac150, to bind to chitin was assessed in vitro using chitin-coated beads. Each of the four proteins binds to chitin, and hydrophobic interactions mediate the binding. Other binding mechanisms are likely involved, but were not determined in this project. To determine the function of these proteins, a series of gene knockout and repair constructs was produced for AcMNPV ORF 145 and ORF 150 using an established bacmid system. An analysis of the knockout and repair constructs using quantitative real-time polymerase chain reaction showed that deletion of either ORF 145 or ORF 150 had no effect on the rate of budded virus production or viral DNA replication. Oral and injection bioassays were performed in Trichoplusia ni larvae to determine if there were differences in infectivity between the knockout, repair, and wild type constructs. Injection assays, in which budded virus from each construct was injected directly into the insect haemocoel, therefore bypassing the midgut and peritrophic matrix, indicated that there was no statistical difference in infectivity between the knockout, repair, and wild type constructs at a dose of 15 TCID50 U per larva. Oral bioassays, in which larvae were fed occlusion bodies from each virus construct, indicated that there was no statistical difference in mortality rates between the knockout, repair, and wild type constructs. The results from this study indicate that although the baculovirus 11K genes are highly conserved among baculovirus species, and the 11K gene products from MacoNPV and AcMNPV interact with chitin, they are not required for oral infectivity in T. ni larvae, and likely serve another function in the baculovirus infection cycle.