During inflammation and thrombosis, leukocytes tether to and roll on vascular surfaces and platelets through selectin molecules under shear flow. This selectin family of cell adhesion molecules includes P-, E-, and L-selectin. The association and dissociation of two or more selectin-mediated bonds under mechanical load produce the rolling motion of the leukocytes. Although much has been uncovered about the properties of selectins, the complete story of the selectin-mediated adhesion process is yet to be told. The goal of this research is to gain a more quantitative understanding of this receptor-ligand binding through the study of the dissociation kinetics of E-selectin-mediated adhesion using flow chamber techniques. From transient tethering experiments, the dissociation rate of E-selectin-mediated adhesion was found to have a triphasic shear dependence at low shear stresses, where the bond transitioned from a slip to a catch then again to a slip bond. This trend was further supported by observations of the average rolling velocity of cells adhering to E-selectin at various shear stresses. A triphasic force dependence of the rolling velocity was revealed that showed that regions of increasing rolling velocity corresponded to the slip bond regime where tether lifetime decreased with increasing shear stress. Decreasing rolling velocity coincided with the catch bond regime, a regime of prolonged tether lifetime with increasing shear stress. An invertible flow chamber was used in hopes of directly quantifying the dissociation rate of rollingly adherent cells on E-selectin to compare it to the dissociation rate data obtained through transient tethering experiments. However, tether formation, which relates to the association rate, and its role in the stability of rolling seemed to be a key factor in the dissociation rate of rollingly adherent cells over the low shear stress range. Overall, these results provide supporting evidence of a shear threshold for E-selectin as well as data to suggest that tether formation, in coordination with off-rate, determine the rolling velocity behavior of cells on E-selectin substrates.

Plant cell culture is an attractive platform technology for production and supply of important plant derived medicinals. A unique characteristic of plant cells is the ability to grow as multicellular aggregates in suspension. The presence of these non-uniform aggregates results in creation of distinct microenvironments, which can induce variations in cellular metabolism (e.g., growth, oxygen consumption and secondary metabolite synthesis). This heterogeneity can lead to unpredictable and suboptimal performance in large scale bioprocesses. One example is the Taxus cell culture system, which produces a widely used chemotherapeutic drug - paclitaxel (Taxol ®). Despite extensive process engineering efforts which have led to increased yields of paclitaxel, Taxus cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces the accumulation of paclitaxel, but to varying extents in culture. A significant negative correlation was observed between paclitaxel level and mean aggregate size of the culture, demonstrating the relevance of measuring, and potentially controlling aggregate size during long term subculture. Understanding the regulation of gene expression can provide rational engineering strategies to control variability and optimize performance of Taxus cell cultures. Biosynthetic pathway gene analyses revealed upregulation of genes upon elicitation with MeJA; results also suggested additional molecular regulatory points outside of the biosynthetic pathway. In order to fully understand Taxus molecular regulation and the relationship to paclitaxel production variability, a transcriptome-wide analysis using next generation sequencing (454 and Illumina) methods was performed. Several pathways outside of paclitaxel biosynthesis were found active upon MeJA elicitation. Global comparison of gene expression amongst cultures accumulating different levels of paclitaxel is being performed to completely understand the interactions amongst the paclitaxel biosynthetic pathway and other complimentary and competing pathways to suggest effective targets for metabolic engineering. This work collectively represents the first molecular studies to understand metabolic regulation in Taxus cell cultures. Apart from inducing paclitaxel biosynthesis, MeJA decreases cell growth in Taxus cell cultures. The MeJA-mediated repression of cell growth was shown to correlate with inhibition of cell cycle progression as evident both at the culture level through flow cytometric analyses and at the transcriptional level by repression of key cell cycle-associated genes. Results from this study provide valuable insight into the mechanisms governing MeJA perception and subsequent events leading to repression of Taxus cell growth.