http://hdl.handle.net/11343/159 2019-08-20T20:23:50Z http://hdl.handle.net/11343/227164 Determining the role of doublecortin X (DCX) in cytoskeleton organisation Moslehi, Maryam X-linked Doublecortin (DCX) is a 40 kDa neuron-specific microtubule (MT)-associated protein. The initial identification of inheritable dcx gene mutations and their disruption of cortical layering and brain development in lissencephaly and subcortical band heterotopia have prompted subsequent evaluation of the functions of the DCX protein. Previous biochemical studies have revealed that DCX binds between four tubulin monomers within the polymerised MT lattice to stabilise the lateral and longitudinal contacts along the MT protofilaments. Although DCX has been proposed to regulate MT-related cellular events, many aspects of its mechanism of actions and its regulation of functions are yet to be determined. The DCX protein consists of two MT-interacting doublecortin (DC) domains, DC1 (the N-terminal DC domain) and DC2 (the C-terminal DC domain), linked in tandem via a flexible unstructured region (linker) and additionally flanked by a likely unstructured N-terminal and S/P-rich C-terminal sequences. Since many DCX pathogenic mutations have been predominantly mapped to within the DC domains to thus disrupt the interaction of DCX with MTs, studies of the regulation of the DCX-MT interaction have been undertaken to improve understanding of the functions of DCX. In addition, the DCX termini that flank the DC domains may act as regulators of DCX function, but whereas the S/P-rich DCX C-terminus is known for its phosphoregulatory role in DCX’s functions, a regulatory role of the DCX N-terminus region is largely unknown. The studies presented in this thesis address the critical roles of the DCX N- and C-termini in DCX function. Immunoprecipitation and live-imaging approaches using monkey fibroblastoma COS-1 and human neuroblastoma SH-SY5Y cells have been employed to identify the effects of the DCX N- and C-termini on the association of DCX with the cytoskeleton. The results were combined with live-imaging fluorescence recovery after photobleaching (FRAP) protocol findings to define the regulatory impact of DCX termini on dynamics of DCX in association with the cytoskeletal components, MTs and F-ACT. These studies have been the first to show the dynamic association of DCX with MTs in living cells. The role of the DCX C-terminus has been evaluated by examining the features of DCX lacking its C-terminus. Whereas full length, wildtype DCX shows rapid and complete exchange within the MT network, the exchange of the truncated DCX protein is slowed significantly. Moreover, dynamics of exchange of the C-terminal truncated DCX was unaltered in the presence of a MT-stabilising agent, taxol, or a hyperosmotic stress stimulus, sorbitol, both of which were shown to slow wildtype DCX exchanges rate within the MT network. Thus, DCX dynamically associates with MTs in living cells and its C-terminal region plays important roles in the association of DCX with MTs. To explore the regulatory role of DCX N-terminus, the influence of the only identified phosphorylation site within the DCX N-terminus, DCX S28, on association of DCX with MTs and F-ACT was assessed. Thus, both DCX S28A (phospho-resistant) and DCX S28E (phospho-mimetic) mutants were examined alongside wildtype DCX. For DCX S28E, decreased interaction with MTs but a shift to favour association with both F-ACT and the ACT-binding protein, spinophilin (Spn) was observed. FRAP studies showed that DCX S28E increased the dynamics of association with MTs. Compared to DCX S28E, the associations with MTs and F-ACT were reversed in the presence of DCX S28A. Therefore, these results highlight a new role for DCX S28 as a regulatory switch for cytoskeletal organisation and thus highlight a contribution by DCX-N in the phosphoregulation of DCX function. The impact of a pathogenic mutant within the DCX N-terminus, DCX E2K, on the cytoskeletal association was also studied. Unlike most DC domain pathogenic mutants of DCX, the DCX E2K mutant protein retained its ability to interact with MTs. However, MTs in association with DCX E2K showed a reduced sensitivity to nocodazole-induced depolymerisation as well as slower α-tubulin exchanges rate. Furthermore, the DCX E2K mutant showed increased association with the F-ACT. These results highlight the importance of the N-terminus of DCX in regulating the association with, and the coordination of, the MT and F-ACT networks. Taken together, the studies presented in this thesis have revealed several new features of the regulatory roles of the DCX termini in the association of the DCX protein with MTs and F-ACT. The findings should aid a better understanding of the DCX function in MTs and F-ACT organisation during MT-related cellular events including neuronal cell migration. 2019-01-01T00:00:00Z http://hdl.handle.net/11343/227162 Analysis of Complex Predicates in Mandarin Chinese Hsu, Chi-Pin This project examines complex predicates in Mandarin, which is a language abundant in complex predicates. The approach of syntactic and semantic analyses in this study is based on the Simpler Syntax model developed by Culicover and Jackendoff (2005), which incorporates the LCS (Lexical Conceptual Structure) approach of Jackendoff (1990), with some minor adjustments to account for my analysis of eventuality structure. This study is also supplemented by approaches taken from other previous studies as necessary. This study proposes a two-dimensional classification for Mandarin complex predicates. Complex predicates in this study are classified as monoclausal or polyclausal in the syntactic dimension and as resultatives, depictives, directionals, etc. in the semantic dimension. Monoclausal resultatives and polyclausal serial verb constructions with bleached verbs are given particular focus in the thesis and discussed in detail. Naturally occurring sentences show that some monoclausal resultatives can take imperfective aspect categories. I suggest two sources for the compatibility between these resultatives and the imperfective aspect categories: (a) iterative readings and (b) focus on the process portions of resultatives. A questionnaire undertaken with native speakers shows that compatibility between resultatives and the Progressive Aspect may also be influenced by polarity. The verb na, which has a full verb meaning of ‘take’ with the hand as the default body part, can be used as a bleached verb and employed as an instrument marker in polyclausal serial verb constructions. A corpus of mouth and foot painting artists-related articles are collected for the analysis of na, where the executive body part is not the hand. The analysis of sentences in the corpus shows that the distinction between full and bleached verbs is not categorical and that verb bleaching can be influenced by pragmatic factors such as context. 2019-01-01T00:00:00Z http://hdl.handle.net/11343/227158 Modelling the impacts of Demand Responsive Transport Systems on Mobility Navidi Kashani, Zahra The recent progress in Information and Communication Technology and the ubiquity of smart devices have revolutionised transportation systems in a potentially disruptive manner. It is culminating in Mobility as a Service platforms and facilitated the advent of demand responsive transport systems. Examples of such systems include ride-sourcing, ride-sharing, and car-sharing, which are emerging flexible transportation options with promising features to be the key to a number of transportation problems, filling the gap between public and active transport. However, their sustainable and successful deployment is notoriously difficult to achieve due to the high dynamicity of such systems, which mean high volatility of demand and supply. This work sheds light on the feasibility of these systems, with a focus on spatial aspects, using agent-based modelling and the concept of multi-sided markets. The major outcomes of this work are three folds: first, for the very first time the concept of spatial critical mass is developed and analysed in this work, which can have profound impact on the understanding and design of the future schemes of DRT systems for the operators, and identifying and imposing the apt policies for governments and authorities. Spatial critical mass highlights the distinction of spatial systems from their aspatial counterparts in multi-sided markets and emphasizes the significance of spatial components of such systems. Second, this work presents the first analytical evidence of the advantages of collaboration among the demand responsive transport operators. With the rapidly growing interest in MaaS concept, it is imperative for the involved parties, including transport network operators and governments, to understand the consequences of such schemes to make informed astute decisions on appropriate planning and policies. Third, this work demonstrates the feasibility of using simulation to synthesise the required data for developing a mode choice model. This implies the applicability of the approach in travel behaviour studies involving new travel modes, such as demand responsive transport and autonomous ride-sharing. Finally, in addition to the above-mentioned outcomes, this piece of work could be an initial evidence base to nudge governments towards fully integrated transport systems, in which DRT systems play a key role. 2019-01-01T00:00:00Z http://hdl.handle.net/11343/227148 Empirical studies on firm-level productivity and innovation Lim, William In Chapter 1, I define a novel concept of low productivity spells. Instead of treating productivity as a continuous variable, I define a low-productivity state as poor productivity growth over 2 years consecutively. This provides an alternative insight into understanding firms which pose a potential drag on aggregate productivity growth. Using a competing risk framework, I find that over the course of 1991--2014, the exposure of import competition from China had a significant and negative effect on the exit from these low-productivity spells into high-productivity performance for listed firms in the U.S. On the other hand, this import exposure had no effect on the same firms shutting down. Together, these imply that the growth in import penetration from China had the effect of trapping U.S. manufacturing firms in low-productivity spells for longer. In Chapter 2, I investigate the effect of the America Invents Act (AIA) in 2011 on the patents granted by small firm compared with large firms. In first constructing my dataset, I further bootstrap the algorithm proposed by Autor, Dorn, Hanson, Pisano and Shu (2017) in merging patent data from the United States Patents and Trademarks Office USPTO with firm-level data from COMPUSTAT to better correct for false positives and true negatives, as well as enabling it to be more replicable. With a difference-in-difference framework using de-trended patenting data, I find a negative effect by the AIA on patents granted to small firms compared with large firms. However, this effect appears to be driven primarily by the amount of cash holdings firms have. A triple-difference framework which includes cash holdings finds the AIA had no significant effect on the patents granted to small firms which are cash-poor compared with large firms which are also cash-poor. In Chapter 3, I examine the effect of Chinese import competition on innovation by U.S. manufacturing firms originally investigated by Autor, Dorn, Hanson, Pisano and Shu (2017) but within a framework that differentiates this effect between small and large firms. In addition, I also measure the innovation response by firms using patents granted to them instead of their patent applications per se. With a model without heterogeneous effects, I find that the import penetration growth from China had increased innovation on average by all U.S. manufacturing firms when innovation is measured by the patents granted each firm. In a model with heterogeneous effects, I find this effect to be insignificantly different for large firms compared with small firms, and for cash-rich firms compared with cash-poor firms. © 2019 William Lim 2019-01-01T00:00:00Z