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Impact of autocrine factors on physiology and productivity in Trichoplusia ni serum-free culturesEriksson, Ulrika January 2005 (has links)
The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells. Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation. Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics. In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity. Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well. / <p>QC 20101129</p>
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Regulation of productivity in Trichoplusia ni and Spodoptera frugiperda Sf9 serum-free culturesCalles, Karin January 2005 (has links)
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
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Intraspecific Variation in Natal Plant Secondary Chemistry Leads to Plasticity in Lepidopteran Oviposition BehaviorRyan, Sean F. 19 July 2011 (has links)
No description available.
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