MCDS is an autosomal dominant skeletal dysplasia disorder caused by mutations in collagen X. In most cases, mutations in collagen X result in a misfolded protein which is retained within the ER of hypertrophic chondrocytes, causing increased ER stress. It has previously been demonstrated that increased ER stress causes hypertrophic chondrocytes to de-differentiate in an attempt to avoid the stress. The altered differentiation results in reduced cell hypertrophy and impaired vascular invasion accounting for reduced bone growth. The presence of increased ER stress in hypertrophic chondrocytes is sufficient to cause the MCDS pathology; therefore reducing ER stress may be beneficial in terms of improving the associated pathology. The autophagy enhancing drug carbamazepine (CBZ) has been shown to be capable of reducing ER stress in cells expressing the MCDS-causing p.N617K collagen X mutation. I show in this thesis that CBZ treatment reduced ER stress in HeLa cells transiently expressing a further 3 MCDS-causing collagen X mutations. I have also demonstrated that CBZ treatment induced the degradation of mutant collagen X proteins either through autophagy or proteasomal degradation depending on the nature of the mutation. The drug was tested in vivo using the p.N617K collagen X mouse model of MCDS. In MCDS mice, CBZ reduced the severity of the disease pathology based on histological analyses, restored hypertrophic chondrocyte differentiation toward normal, increased long bone growth rates and decreased the severity of the hip dysplasia. Gene expression analyses on RNA isolated from microdissected hypertrophic chondrocytes revealed that CBZ shifted the pattern of hypertrophic differentiation markers in MCDS mice toward the wild-type pattern, most likely through its stimulation of gene expression associated with intracellular proteolytic pathways. The results presented in this thesis have contributed to the identification of a potential treatment strategy for MCDS- the stimulation of intracellular proteolysis of mutant collagen X. CBZ is FDA approved for the use of epilepsy and bipolar disorder and has a strong safety record in humans. Therefore CBZ could be a potential treatment strategy for MCDS.

Mutations in the COL10A1 gene cause metaphyseal chondrodysplasia type Schmid (MCDS) by triggering ER stress and unfolded protein response (UPR). MCDS is characterised by a mild short-limb dwarfism accompanied by expansion of the cartilage growth plate hypertrophic zone (HZ) and altered differentiation of hypertrophic chondrocytes (HCs). ATF6 is one of the UPR mediators, which exists in two isoforms, ATF6α and ATF6β. Activation and up-regulation of ATF6α was a prominent biochemical sign of ER stress in a mouse model of MCDS, COL10a1 p.N617K. Although ATF6β is induced and activated in response to ER stress in a similar fashion to ATF6α, the role and significance of ATF6β in the pathology of many ER stress-associated diseases including MCDS is unknown. Here we utilized a combination of in vitro and in vivo approaches to define the precise role of each isoform of ATF6 in MCDS.To investigate the functions of ATF6α and ATF6β in vitro, we developed a MCDS cell model system (expressing either the wild type collagen X or one of the following MCDS-causing mutant forms of the protein: p.N617K, G618V, Y598D, and NC1del10) in which the expression of either ATF6α or ATF6β was efficiently silenced using siRNAs. ATF6α knockdown in HeLa cells expressing different MCDS-causing mutations suppressed the increased expression of UPR-associated genes such as BiP leading to an elevated ER stress, based on increased XBP1 splicing and/or ATF4 protein. In contrast, ATF6β knockdown did not significantly affect the mutant collagen X-induced increased expression of UPR-associated genes. Furthermore, the ER stress levels were significantly reduced in the ATF6β knockdown MCDS mutant cells based on the lower levels of XBP1 splicing and/or ATF4 protein detected. We then crossed the ATF6α/β knockout mice models with COL10a1 p.N617K mouse model of MCDS to investigate the function of ATF6α and ATF6β in vivo. Ablation of ATF6α in MCDS mice further- reduced the endochondral bone growth rate, further expanded the growth plate hypertrophic zone, and disrupted differentiation of HCs. Therefore, ATF6α appeared to play a chondroprotective role in MCDS as its deficiency caused an increase in the severity of the disease. Of particular note, the level of ER stress was further increased in the absence of ATF6α in MCDS, based on enhanced activities of PERK and IRE1 signalling pathways in compensation for the ATF6α loss. Paradoxically, ablation of ATF6β in MCDS mice reduced the intracellular retention of collagen X protein, and alleviated the ER stress as judged by the attenuated activities of PERK and IRE1 signalling pathways. The reduced ER stress resulting from deficiency for ATF6β in MCDS mice restored the expression of collagen X mRNA towards normal and improved the differentiation of HCs, causing a mark decrease in the expansion of HZ. The results presented within this thesis greatly increased our understanding of the function of ATF6α and ATF6β and their interplay in the pathogenesis of MCDS. We demonstrated an indispensable beneficiary role for ATF6α but a detrimental role for its closely related isoform, ATF6β, in pathology of MCDS. We also showed that the role of ATF6β should not be ignored. These findings may be used to develop a potential therapeutic strategy for MCDS through targeting and enhancing ATF6α-dependent and/or attenuating/blocking of ATF6β-dependent signalling pathways.

The development of ubiquitous mobile computing devices has fueled the need for dynamic reconfigurable networks. Mobile ad-hoc network (MANET) routing protocols facilitate the creation of such networks, without centralized infrastructure. One of the challenges in the study of MANET routing protocols is the evaluation and design of an effective routing protocol that works at low data rates and responds to dynamic changes in network topology due to node mobility. Several routing protocols have been standardized by the Internet Engineering Task force (IETF) to address ad-hoc routing requirements. The performance of these protocols are investigated in detail in this thesis. A relatively new approach to ad-hoc routing using the concept of a Minimal Connected Dominating Set (MCDS) has been developed at Virginia Tech. The OSPF-MCDS routing protocol is a modified version of the traditional Open Shortest Path First (OSPF) wired routing protocol which incorporates the MCDS framework. Enhancements to the protocol implementation to support multiple-interface routing are presented in this thesis. The protocol implementation was also ported to ns-2, a popular open source network simulator. Several enhancements to the implementation and simulation model are discussed along with simulation specifics. New scenario visualization tools for mobility pattern generation and analysis are described. A generic framework and tutorial for developing new ad-hoc routing simulation models are also presented. The simulation model developed is used to compare the performance characteristics of OSPF-MCDS to three different standardized MANET routing protocols. Simulation results presented here show that no single protocol can achieve optimal performance for all mobility cases. Different observations from simulation experiments are summarized that support the likely candidate for different mobility scenarios. / Master of Science

In some organizations four generations work together creating a unique culture. Millennials are the second largest generation currently in the workforce. Organizational culture is affected by interpersonal communication. Interviews were conducted with 17 workers between the ages of 19 and 30. Interviews consisted of 22 open-ended questions regarding daily tasks, organized activities, and interpersonal interaction. The perspective of organizational culture was evaluated through personal, task, social, and organizational rituals. Membership categorization was used to determine common vocabulary used to identify with coworkers. Duck’s theory on attraction was used to evaluate interpersonal behavior seeking to determine psychological attractors acting as catalysts for relationship building. Data found suggests that Millennials create a culture similar to the tribal example suggested in previous research. Rituals act as forms of cultural dissemination and strengthening. The use of membership categorization devices reflected the structure of the organization and relationships between coworkers. Using Duck’s attraction theory, an analysis reflected the identification factors that act as catalysts for relationships. Psychological attraction was linked to common interests

Wireless mobile ad-hoc networks consist of a collection of peer mobile nodes that form a network and are capable of communicating with each other without help from stationary infrastructure such as access points. The availability of low-cost, com-modity network interface cards (NICs) has made the IEEE 802.11 medium access control (MAC) protocol the de facto MAC protocol for wireless mobile ad-hoc net-works, even though it is not optimal. The IEEE 802.11 MAC protocol is designed to have stations share a single channel in a given network. However, many of the IEEE 802.11 physical (PHY) layer specifications define multiple channels and allow the simultaneous, non-interfering use of some of these channels. Therefore, multiple communications can occur at the same time, offering the opportunity to increase the effective network capacity. We present an innovative routing protocol that utilizes multiple channels to im-prove the performance of wireless ad-hoc networks. The basic idea of the protocol is to use multiple channels so that multiple useful transmissions can occur simultane-ously, thus increasing network capacity. The proposed scheme requires minor changes to existing proactive ad-hoc routing protocols and no modifications to the current IEEE 802.11 MAC protocol. To reduce inefficiencies due to periodic updates in the proactive routing protocols, the proposed scheme divides the network layer into control and data planes. Nodes send routing updates using the control channel and user packets using the data channel. To demonstrate the multi-channel routing scheme, we extend the Destination-Sequenced Distance-Vector (DSDV), Open Shortest Path First-Minimal Connected Dominating Set (OSPF-MCDS), and Optimized Link State Routing (OLSR) protocol to multiple channel (MC) versions, denoted as DSDV-MC, OSPF-MCDS-MC, and OLSR-MC, respectively. Simulation results for DSDV-MC, OSPF-MCDS-MC, and OLSR-MC are presented and experimental results for OLSR-MC are presented. Simulation results indicate that DSDV-MC and OSPF-MCDS-MC effectively exploit multiple channels to improve network capacity. Goodput, the throughput consider-ing only useful error-free packets, increases with an increased number of available channels as the number of nodes and network load increase in both single-hop and multiple-hop networks. Experimental results with OLSR-MC also support that the proposed scheme increases network capacity without modification to the MAC proto-col in a real implementation. Although simulation and experimental results show that proposed scheme im-proves network capacity by exploiting multiple channels, problems exist with channel distribution. We introduce a new metric, the Channel Distribution Index (CDI) to in-vestigate these issues. The CDI indicates the fairness of the channel distribution. We identify the channel convergence problem, where a particular channel is over-utilized, and propose a channel reallocation scheme to mitigate the impact of the channel convergence problem using the CDI. / Ph. D.