Le but de cette thèse fut d’étudier l’influence de l’environnement sur l’évolution des génomes de baculovirus. Nous avons d’abord caractérisé génétiquement la population naturelle d’AcMNPV par séquençage haut-débit et établi par des bioessais la sensibilité de 4 espèces hôtes au virus. Ensuite, une évolution expérimentale de 10 cycles fut mise en place sur les 4 espèces hôtes, à partir d’une population naturelle d’AcMNPV. Elle nous a permis de caractériser phénotypiquement et génotypiquement les lignées de 10ème génération. Cette expérience nous a montré des trade-off de virulence pour chaque lignée : pour augmenter leur virulence pour l’hôte sur lequel elles ont évolué, les lignées ont perdu en potentiel adaptatif généraliste. De plus, la diversité intra-populationnelle a diminué pour toutes les lignées en fonction de la sensibilité des hôtes. Enfin, en corrélant tous ces résultats nous avons mis en évidence des positions spécifiques du génome, impliquées dans l’adaptation à l’hôte. / The purpose of this thesis was to study the influence of the environment on the evolution of baculovirus genomes. We first genetically characterised the AcMNPV natural population by high-throughput sequencing and established the susceptibility of 4 hosts to the virus by bioassays. Then, the AcMNPV natural population was subjected to experimental evolution on the 4 host species for 10 cycles. The 10th generation of the evolved viral lines were then phenotypically and genotypically characterised. This experiment showed a virulence trade-off for each line: to increase their virulence to the host on which they evolved, the lines have lost generalist adaptive potential. Furthermore, intra-population diversity decreased for all the lines regardless of host susceptibility. Lastly, by correlating all these results we found specific genome positions involved in host adaptation.

Diversos estudos têm sido feitos para verificar quais as relações entre a lagarta do cartucho-do-milho, Spodoptera frugiperda, bem como o curuquerê-do-algodoeiro, Alabama argillacea, e potenciais agentes de controle biológico, visto que são importantes pragas de diversas culturas. Pesquisas com parasitoides e predadores de diferentes ordens têm sido desenvolvidas, com esse propósito. A tesourinha Euborellia annulipes apresenta grande potencial, assim como o pentatomídeo Podisus nigrispinus, por serem inimigos naturais desses insetos-praga. O parasitoide Campoletis flavicincta é, também, considerado um potencial agente controlador de S. frugiperda, no campo. Quatro experimentos foram instalados e conduzidos para estudar as relações ecológicas entre as espécies citadas: i) competição entre os predadores por larvas de S. frugiperda; ii) competição entre os predadores por larvas de A. argillacea; iii) competição intraespecífica entre as ninfas dos predadores; iv) teste de preferência por larvas de S. frugiperda previamente parasitadas, ou não, por C. flavicincta. Modelos estatísticos de diferentes tipos foram ajustados aos dados obtidos. Foram utilizados modelos lineares generalizados para dados discretos univariados (contagens e proporções), modelos de análise de sobrevivência para dados de tempo até ocorrência de um determinado evento, modelos aditivos generalizados e modelos multinomiais ordinais para dados de taxas de mortalidade observadas ao longo do tempo. Para verificar a qualidade do ajuste de modelos lineares generalizados, funções que produzem gráficos meio-normais com envelope de simulação foram implementadas no software estatístico gratuito R. Observou-se que as fêmeas de E. annulipes são mais agressivas e a espécie tem vantagem competitiva sobre P. nigrispinus, podendo atuar como predadora intraguilda. Além disso, os predadores têm a capacidade de discriminar entre larvas de S. frugiperda parasitadas ou não por C. flavicincta, sendo que há evidências de que os machos evitam consumir larvas parasitadas. Já as fêmeas não têm preferência e podem consumir o parasitoide, indiretamente, ao predar uma larva parasitada, sendo que isso pode ser um fator estabilizador do sistema. Adicionalmente, a coexistência entre a praga, os predadores e o parasitoide é mais provável quando as taxas de ataque dos predadores são mais altas e a do parasitoide é mais baixa. / Several studies have verified the relations among the fall armyworm, Spodoptera frugiperda, as well as the cotton leafworm, Alabama argillacea, and potential biological control agents, as they are important pests of several crops. Research with parasitoids and predators of different orders has been developed with this purpose. The ring-legged earwig (Euborellia annulipes) shows great potential, as well as the Neotrpical stink bug (Podisus nigrispinus), as they are natural enemies of these pests. The parasitoid wasp Campoletis flavicincta is also considered a biological control agent of S. frugiperda. Four experiments were set up and conducted to characterize the ecological relations among the described species: i) competition between the predators for S. frugiperda larvae; ii) competition between the predators for A. argillacea larvae; iii) intraspecific competition among the predators\' nymphs; iv) choice tests between S. frugiperda larvae that were previously parasitised by C. flavicincta and larvae that were not. Statistical models of different types were fit to the data. Generalized linear models were used to analyse univariate discrete data (counts and proportions), survival analysis models were used to analyse time-until-event data, generalized additive models and ordered multinomial logistic models were used to analyse mortality rates through time. To assess goodness-of-fit, functions that produce half-normal plots with a simulation envelope were implemented for the open-source software R. It was observed that females of E. annulipes are less aggressive and this species has a competitive advantage over P. nigrispinus, and may act as an intraguild predator. Besides that, the predators are able to distinguish parasitised from non-parasitised S. frugiperda larvae, and there are evidence that show that males tend to avoid feeding on larvae parasitised by C. flavicincta. Females, however, have no prefference and may indirectly feed on the parasitoid when predating a parasitised larva, and this might be a stabilizing factor in this system. In addition, coexistence among the pest, predators and parasitoid is most likely when predation rates are high and parasitism rates are low.

Mucopolysaccharidosis IIIB (MPS IIIB) is a rare, metabolic disorder that results from a deficiency in the lysosomal hydrolase, α-N-acetylglucosaminidase (Naglu). Naglu is a housekeeping enzyme involved in the degradation pathway of heparan sulfate. A deficiency in active Naglu leads to an accumulation of heparan sulfate within the lysosome, initiating a pathological cascade within the cell. Patients with MPS IIIB experience progressive central nervous system degeneration and die within the first few decades of life. Presently, enzyme replacement therapy, which is a standard of care for other lysosomal storage disorders, is an ineffective treatment for MPS IIIB. This is due to impermeability of the blood-brain barrier (BBB) to exogenous recombinant enzymes. A promising approach to this therapeutic obstacle is protein transduction domains. Protein transduction domains have been shown to facilitate the delivery of active enzyme across the BBB in mice. Previously, our laboratory used Spodoptera frugiperda (Sf9) insect cell system to express human recombinant Naglu fused to a synthetic protein transduction domain (PTD4). The purpose was to use PTD4 to the facilitate the delivery of Naglu across biological membranes, including the blood-brain barrier. However, a missing stop codon following PTD4 limited its transducibility. The stop codon was re-introduced and the improved fusion enzyme, Naglu-PTD4X, was stably expressed in Sf9 cells. The overarching goal of this project is to create a large-scale production of human recombinant Naglu that has the potential to be used to treat the neuropathology of patients with MPS IIIB. This project used a three-step purification system to purify Naglu-PTD4X. Uptake of Naglu-PTD4X was assessed in MPS IIIB fibroblasts using a fluorogenic activity assay, immunoblotting, and immunocytochemistry. Our purification system was successful at purifying Naglu-PTD4X to homogeneity with a 26% yield and specific activity of 84,000 units/mg. An increase in Naglu activity was detected in MPS IIIB fibroblasts following incubation with Naglu-PTD4X. Future directions will focus on optimizing immunodetection and conducting BBB penetration studies in murine models. / Graduate / 2020-06-21

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. 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. / QC 20101125

No description available.

Arthropod hemocyanin and phenoloxidase are members of a group of proteins called the Type-3 copper oxygen-binding proteins, both possessing a highly conserved oxygen-binding site containing two copper atoms each coordinated by three histidine residues (Decker and Tuczek, 2000). Despite similarities in their active site, these proteins have very different physiological functions. Phenoloxidase possesses both tyrosinase and o-diphenoloxidase activity, and is predominantly involved in reactions which protect insects from infection (Kopàcek et al., 1995). Hemocyanin is a large multi-subunit protein with a primary function as a respiratory protein, reversibly binding and transporting molecular O2 (Decker and Rimke, 1998; Decker and Tuczek, 2000). Recently, it has been demonstrated in vitro that arthropod hemocyanin possesses an inducible phenoloxidase activity when incubated with denaturants, detergents, phospholipids or proteolytic enzymes. This activity appears to be restricted to only a few subunit types, and it has been hypothesised that it may be accompanied by conformational change which opens the active site increasing access for larger phenolic substrates (Decker and Jaenicke, 2004; Decker et al., 2001; Decker and Tuczek, 2000). This possibly suggests a dual role of hemocyanin in arthropods. The presented thesis deals with two distinct aims. The first was to isolate and sequence a phenoloxidase gene from the insect Spodoptera littoralis (Egyptian Cottonleaf Worm). Despite efforts, progress was hindered by a number of experimental problems which are outlined within the relevant chapters. The second aim was to characterise the mode of SDS induced phenoloxidase activity in arthropod hemocyanin from the ancient chelicerates Limulus polyphemus (horseshoe crab) and Eurypelma californicum (tarantula) and the more modern chelicerate Pandinus imperator (scorpion), using a number of biophysical techniques. The results indicated that the SDS induced phenoloxidase activity is associated with localised tertiary and secondary conformational changes in hemocyanin, most likely in the vicinity of the dicopper centre, thus enhancing access for larger phenolic substrates. Experiments indicate that copper remains associated with the protein during these structural changes; however the nature of the association is unclear. SDS concentrations approximating the CMC appeared critical in causing the necessary structural changes required for a significant increase in the detectable phenoloxidase activity to be exhibited.

Rotavirus infection causes gastroenteritis, specifically severe gastroenteritis, affecting children younger than five globally, regardless of hygiene and water quality. Current licensed, live, attenuated vaccines do not contain the G9 genotype, which is a prevalent rotavirus strain circulating in sub-Saharan Africa, a region that carries a high rotavirus disease burden. Rotavirus-like particles (RV-VLPs) is an attractive non-live vaccine candidate, which has shown promising results in animal studies. Previously, dsRNA was extracted from a stool sample containing a South African human G9P[6] neonatal strain, and amplified cDNA using a sequence-independent procedure. The consensus sequence was obtained for the genome segments using 454® pyrosequencing. The insect-cell-codon-optimized genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBACquad vector (pFBq). Several combinations of the genome segments were cloned to produce double-layered particles (DLP; pFBqVP2VP6) or triple-layered particles (TLP; pFBqVP2VP6VP7). In the current study, a ΔTLP (pFBqdVP2-VP8*VP6VP7) construct was generated. The first 92 amino acids of VP2 are not necessary for the conformation of recombinant RV-VLPs. The ORF of VP8*, which contains immune important epitopes, was fused to the 5’ end of the dVP2 coding region resulting in a dVP2-VP8* fused protein which was expressed in the presence of VP6 and VP7 to produce ΔTLPs. The Bac-to-Bac® Baculovirus Expression System and Spodoptera frugiperda (Sf) 9 insect cells were used for expression. All the proteins were successfully expressed. VP2, VP6, VP4 and the dVP2-VP8* fused protein were visible on Coomassie stained SDS-PAGE. Expression of VP7 could only be confirmed with western blot analysis. Particle formation, as assessed by transmission electron microscopy (TEM), was observed for DLPs. No TLPs of dVP2-8*/6/7 or VP2/6/7 were visualized due to the lower expression level of VP7 and the lack of calcium supplements during the assembly process. In conclusion, it was possible to produce RV-DLPs derived from the consensus sequence determined for a G9P[6] rotavirus directly from stool without prior propagation in cell culture or virus isolation. This strain contains both the G9 and P[6] genotypes that are currently prevalent in sub-Saharan Africa. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

Rotavirus infection causes gastroenteritis, specifically severe gastroenteritis, affecting children younger than five globally, regardless of hygiene and water quality. Current licensed, live, attenuated vaccines do not contain the G9 genotype, which is a prevalent rotavirus strain circulating in sub-Saharan Africa, a region that carries a high rotavirus disease burden. Rotavirus-like particles (RV-VLPs) is an attractive non-live vaccine candidate, which has shown promising results in animal studies. Previously, dsRNA was extracted from a stool sample containing a South African human G9P[6] neonatal strain, and amplified cDNA using a sequence-independent procedure. The consensus sequence was obtained for the genome segments using 454® pyrosequencing. The insect-cell-codon-optimized genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBACquad vector (pFBq). Several combinations of the genome segments were cloned to produce double-layered particles (DLP; pFBqVP2VP6) or triple-layered particles (TLP; pFBqVP2VP6VP7). In the current study, a ΔTLP (pFBqdVP2-VP8*VP6VP7) construct was generated. The first 92 amino acids of VP2 are not necessary for the conformation of recombinant RV-VLPs. The ORF of VP8*, which contains immune important epitopes, was fused to the 5’ end of the dVP2 coding region resulting in a dVP2-VP8* fused protein which was expressed in the presence of VP6 and VP7 to produce ΔTLPs. The Bac-to-Bac® Baculovirus Expression System and Spodoptera frugiperda (Sf) 9 insect cells were used for expression. All the proteins were successfully expressed. VP2, VP6, VP4 and the dVP2-VP8* fused protein were visible on Coomassie stained SDS-PAGE. Expression of VP7 could only be confirmed with western blot analysis. Particle formation, as assessed by transmission electron microscopy (TEM), was observed for DLPs. No TLPs of dVP2-8*/6/7 or VP2/6/7 were visualized due to the lower expression level of VP7 and the lack of calcium supplements during the assembly process. In conclusion, it was possible to produce RV-DLPs derived from the consensus sequence determined for a G9P[6] rotavirus directly from stool without prior propagation in cell culture or virus isolation. This strain contains both the G9 and P[6] genotypes that are currently prevalent in sub-Saharan Africa. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

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.