Molecular mechanisms of bacteriophage phi6 RNA-dependent RNA polymerase and its utilization in biotechnology

Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.

Profitable Customer Management: A Study in Retail Banking

The increasing focus of relationship marketing and customer relationship management (CRM) studies on issues of customer profitability has led to the emergence of an area of research on profitable customer management. Nevertheless, there is a notable lack of empirical research examining the current practices of firms specifically with regard to the profitable management of customer relationships according to the approaches suggested in theory. This thesis fills this research gap by exploring profitable customer management in the retail banking sector. Several topics are covered, including marketing metrics and accountability; challenges in the implementation of profitable customer management approaches in practice; analytic versus heuristic (‘rule of thumb’) decision making; and the modification of costly customer behavior in order to increase customer profitability, customer lifetime value (CLV), and customer equity, i.e. the financial value of the customer base. The thesis critically reviews the concept of customer equity and proposes a Customer Equity Scorecard, providing a starting point for a constructive dialog between marketing and finance concerning the development of appropriate metrics to measure marketing outcomes. Since customer management and measurement issues go hand in hand, profitable customer management is contingent on both marketing management skills and financial measurement skills. A clear gap between marketing theory and practice regarding profitable customer management is also identified. The findings show that key customer management aspects that have been proposed within the literature on profitable customer management for many years, are not being actively applied by the banks included in the research. Instead, several areas of customer management decision making are found to be influenced by heuristics. This dilemma for marketing accountability is addressed by emphasizing that CLV and customer equity, which are aggregate metrics, only provide certain indications regarding the relative value of customers and the approximate value of the customer base (or groups of customers), respectively. The value created by marketing manifests itself in the effect of marketing actions on customer perceptions, behavior, and ultimately the components of CLV, namely revenues, costs, risk, and retention, as well as additional components of customer equity, such as customer acquisition. The thesis also points out that although costs are a crucial component of CLV, they have largely been neglected in prior CRM research. Cost-cutting has often been viewed negatively in customer-focused marketing literature on service quality and customer profitability, but the case studies in this thesis demonstrate that reduced costs do not necessarily have to lead to lower service quality, customer retention, and customer-related revenues. Consequently, this thesis provides an expanded foundation upon which marketers can stake their claim for accountability. By focusing on the range of drivers and all of the components of CLV and customer equity, marketing has the potential to provide specific evidence concerning how various activities have affected the drivers and components of CLV within different groups of customers, and the implications for customer equity on a customer base level.