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This thesis investigates the features of dark/difficult heritage sites and their impacts on visitors' engagements with the past. Two former prison sites, Chia-Yi Old Prison and Jing-Mei Memorial Park, in Taiwan are chosen as case studies, and both of them represent two difficult historical periods when Taiwan was governed by regimes from 'outside.' This research examines how Taiwanese citizens visit and interact with these two prison sites, testing the assumption that the use of dark/difficult heritage can be observed not only from the ways that relevant heritage sites are established and managed, but also from the interaction between the heritage and local population. The observed phenomena demonstrate that the public show different attitudes towards the two prison sites and the impacts they bring to them, and the history displayed in the sites provides opportunities and materials for the public to rethink their previous understanding and current society. The two represented histories need to be put in a context to consider the reasons, which the issues of historical distance, comparison between two backgrounds and other potential can together result in the consequence. This thesis is a snapshot of a nation undergoing development and change and heritage performs as epitome of its complex and difficult history. The interactive process illustrates not only the relation of mutual influence between people and dark sites, but also that people become an important medium and potentially influence the society and how the dark sites are understood by the society. It is expectant that in addition to offering a unique example of heritage in East Asia after WWII, this research and the raised case of Taiwan are able to show the crucial role heritage practices play in presenting hidden, uncomfortable pasts and the extent such pasts impact on people's understanding and their society.

Graphene has shown superior mechanical properties, electronic and thermal conductivity, and room temperature quantum hall effect that enables its wide application in nanoelectronics. Due to its gapless feature, other two-dimensional (2D) materials like transition metal dichalcogenides (TMDs) have drawn attention and have been extensively studied for their application in optoelectronics and photonics. However, the atomic defect structures brought in during synthesis will affect these properties and thus a thorough understanding is needed. 4D-STEM is a characterization method that captures the convergent beam electron diffraction (CBED) pattern to allow post-acquisition reconstruction including orientation and strain mapping, position averaged CBED, and ptychography. This doctoral project focused on using 4D-STEM to characterize graphene, MoS2, and WS2 with atomic defective structures by producing a series of maps of the local electronic environment with atomic resolution. Monolayer MoS2 and WS2 were first investigated to form the basis of this project. The projected electric field map was first extracted since it is linearly proportional to the centre of mass shift of the CBED pattern caused by the atomic electric field, which can then be used to calculate the charge density map and electrostatic potential. Simple defective structures were subsequently investigated, leading to a detailed study of the enhanced electric field on TMD zigzag edges and MoS nanowire. Next, more complex structures, bilayer and trilayer twisted graphene were investigated. The electric field map showed that the bilayer with a large twist angle of 28.4° has decoupled based on comparison between the independent atom model (IAM) simulation and experimental results. A study on a backfolded trilayer further showed that, even though the top and bottom layer have a small twist angle of 3°, they show no strong interlayer coupling in the electric field due to a layer with large twist angle in between, though the observation may be obscured by the insufficient sensitivity of the current technique. Post-acquisition reconstruction modes of 4D-STEM were also investigated and compared. Results showed that the annular bright field (ABF), ptychographic phase, and divergence of differential phase contrast (DPC) can identify low and high atomic number atoms simultaneously with high resolution, surpassing the annular dark field (ADF)-STEM mode under the same imaging conditions. Series of reconstructed low angle ADF (LAADF)-STEM images from confined angular range showed potential in locating and identifying surface adsorbed atoms due to their different intensity profile as a function of reconstruction angular range, and combined with simulation, it will be possible to achieve atomic resolution elemental mapping after data acquisition.

Anti-TNF-α therapy has revolutionised the management of inflammatory arthritis, leading to improvement of symptoms and signs of rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis, as well as inhibiting radiographic progression in rheumatoid arthritis, and psoriatic arthritis. However, despite its success in the majority of patients with inflammatory arthritis, about 20-40% of patients are resistant to anti-TNF-α treatment and few patients (10% or less) achieve long-term remission. Several studies suggest that TNF-α receptor 2 (TNFR2) plays an immunoregulatory role in arthritis and that the interruption of TNF/TNFR2 interaction contributes to resistance to anti-TNF-α therapy and to some of the adverse effects associated with anti-TNF-α therapy. The objective of the work presented in this thesis was to gain a deeper understanding of TNFR signalling in rheumatoid arthritis with particular emphasis on a sub-population of lymphocytes known as regulatory T cells (Tregs). Firstly, an association was shown between TNFR2 signaling and TGF-β in regulating the expression of the Treg associated transcription factor, FoxP3. I further assessed the effect of TNFR signaling on CpG methylation at the FoxP3 locus by bisulfite sequencing. The result showed that TNFR2 deficiency leads to increased level of DNA methylation in the proximal promoter of FoxP3. Secondly, I investigated the role of TNFR signaling in arthritis. As expected, TNFR1-/- mice were found to be relatively resistant to arthritis. In contrast to TNFR1-/- mice, higher incidence and increased disease activity were observed in TNFR2-/- mice compared to wild-type mice. In addition, the results showed that TNFR2 deficiency leads to decreased numbers of Tregs, reduced FoxP3 expression of Tregs, reduced expression of immune inhibitory markers and increased production of pro-inflammatory cytokines in arthritis. Thirdly, I investigated the therapeutic potential of DNA demethylating agents in the treatment of RA. It showed that the DNA demethylating drug, decitabine, ameliorated collagen-induced arthritis (an animal model of rheumatoid arthritis) and promoted Treg suppressive function whilst inhibiting pathogenic Th1/Th17 cells. These findings highlight the therapeutic potential of strategies to boost Tregs by TNFR2 agonists or DNA demethylating agents and suggest that it may be possible to achieve long term inhibition of pathogenic inflammation using this approach.

The retroviral structural protein Gag is the primary driving force of assembly of retroviruses. Gag contains three major domains: the N-terminal Matrix domain (MA) for association with the inner leaflet of the plasma membrane (PM), the Capsid domain (CA) for Gag multimerization, and the C-terminal Nucleocapsid domain (NC) for packaging two copies of genomic RNA. The Gag-RNA, Gag-Gag, and Gag-membrane interactions together result in viral assembly. The work presented in this thesis focuses on human immunodeficiency virus type-1 (HIV-1) and Rous sarcoma virus (RSV) Gag interactions with membranes, aiming to understand how Gag binds to the inner leaflet of the PM and how the biophysical properties of the PM contribute to Gag assembly. For membrane association, Gag may exploit electrostatic interactions, hydrophobic interactions, recognition of specific lipid headgroups, protein multimerization, and sensitivity to membrane order. HIV-1 Gag is naturally myristoylated while RSV Gag is not. HIV-1 MA is reported to be sensitive to membrane order, but the mechanism remains elusive. In a comparative study to probe effects of membrane charge and membrane order on Gag-membrane interactions, I developed model membranes with equal phosphatidylserine (PS) concentrations in both disordered (Ld) and ordered (Lo) phases. I found that RSV MA membrane association is primarily based on electrostatic interactions, being only sensitive to membrane charge but not to membrane order. In contrast, myristoylated HIV-1 MA, and proteins containing hydrophobic regions, such as MARCKS, all show preference for disordered membranes. Phosphatidylinositol 4,5-bisphosphate (PIP2) comprises approximately 2 mol% of total inner leaflet lipid. HIV-1 is hypothesized to assemble at PIP2-rich microdomains. To probe whether PIP2-rich domains exist under physiological conditions, I exploited inner leaflet model membranes and used biophysical methods such as self-quenching. I discovered that PIP2 forms clusters at remarkably low concentrations in a multivalent cation-dependent manner. Under physiological cation conditions, free PIP2 and cation-bridged PIP2 clusters can co-exist. Interestingly, I found that HIV-1 not only targets PIP2 clusters, but also further enriches PIP2 at assembly sites as Gag multimerizes in model membranes. I propose that PIP2 cation-bridged cluster formation and protein-induced PIP2 clusters could explain the distinct pools of PIP2 in biological membranes.

Alternative splicing is critical to many cellular functions and appears to be primarily regulated through co -transcriptional mechanisms, where spliced-transcript identity is determined during transcriptional elongation. For HIV-1, alternative splicing is essential since the viral genome encodes multiple overlapping reading frames. Using quantitative single-cell imaging, we find that HIV-1 splicing is not co-transcriptionally regulated. Strikingly, HIV-1 mRNA appears to be spliced post-transcriptionally in a serial cascade. We propose a sensitive new assay to detect in vivo post-transcriptional splicing. The assay result shows that HIV-1 mRNA does not require transcription for spliceosome assembly. This serial splicing cascade appears crucial for the HIV-1 Rev negative-feedback circuit and in agreement with mathematical models. We experimentally verify the counter- intuitive prediction that Rev over-expression leads to reduced HIV-1 p24 expression. Serial post-transcriptional splicing may provide a novel target for antivirals and, more generally, an alternate gene-regulatory mechanism

Ribonuclease P (RNase P), a Mg2+-dependent catalytic ribonucleoprotein (RNP) complex, cleaves the 5'-leader from precursor tRNAs (pre-tRNAs). It is made up of a catalytic RNase P RNA (RPR) and a varying number of RNase P proteins (RPPs) depending on the source: one in bacteria, at least four and nine in archaea and eukarya, respectively. Since the four archaeal RPPs (POP5, RPP30, RPP21 and RPP29) have eukaryotic homologs, archaeal RNase P can be used as an experimental surrogate for the biochemically intractable eukaryotic variant. This dissertation documents a division of labor among archaeal RPPs that aid the cognate RPR's catalysis, and highlights their roles in chemical cleavage, substrate binding, cleavage-site selection and Mg2+ coordination.Previous studies established that archaeal POP5 and RPP30 function as a heterodimer (POP5•RPP30) as do RPP21 and RPP29 (RPP21•RPP29). I assessed the effect of these RPP pairs, either individually or together, on the cleavage step by using single-turnover kinetic measurements. The kinetic parameters obtained from these experiments showed that while POP5•RPP30 is solely responsible for enhancing the RPR’s rate of pre-tRNA cleavage by 60-fold, RPP21•RPP29 increases substrate affinity by 15-fold.We found that the protein-rich archaeal and eukaryal RNase P exhibit better fidelity than the bacterial counterpart while cleaving pre-tRNAGln and its mutant derivatives, which lack consensus structures/sequences. To dissect which archaeal RPPs contribute to this increased fidelity, I examined cis cleavage of pre-tRNAGln by Methanocaldococcus jannaschii (Mja) RPR, a strategy that allowed me to calculate the rates of correct and aberrant cleavage. I showed that RPP21•RPP29 rescued the RPR’s mis-cleavage without affecting the RPR’s rate of cleavage; in addition to promoting the correct cleavage, POP5•RPP30 enhanced cleavage rate by 600-fold for both correct and aberrant cleavage. Interestingly, RPP21•RPP29 specifically increases the rate of correct cleavage by 3-fold in the presence of POP5•RPP30 indicating a synergistic effect between the two RPP pairs. Collectively, these results suggest that protein-rich RNase P display better fidelity while allowing flexibility in substrate-recognition.Finally, I determined that deletion of a universally conserved uridine (-U) in a bulge-helix structure in archaeal RPR weakens Mg2+ coordination and lowers activity, a defect completely rescued by their cognate RPPs under multiple-turnover conditions. Cis cleavage of pre-tRNATyr by Mja -U RPR revealed an 80-fold decrease in the cleavage rate compared to the wildtype; both Mja RPP pairs partially ameliorate this defect and narrow the difference to ~12-fold, indicating their ability to coordinate catalytically relevant Mg2+ ions. In addition to archaeal RPPs rescuing an RPR mutation, we (in collaboration with the laboratory of Dr. Mark Foster, OSU) found that an N-terminal deletion mutant of RPP29, which fails to bind RPP21, is functional upon addition of RPR. Together, these findings illustrate the cooperative subunit interactions critical for generating the functional structure of archaeal RNase P. Overall, my results demonstrate the functional coordination among the RPR and different RPPs, and help rationalize why distinct protein cofactors were recruited by an ancient RNA enzyme during the transition from a primordial RNA to the extant RNP world.

Several studies have documented the existence of sexual harassment in workplaces in society-at-large. This pilot study seeks to determine whether sexual harassment is higher within the hotel industry than in workplaces in society-at-large, whether men and women react differently to sexual behaviors in the workplace, and whether those differences vary by department. In addition, the study seeks to determine hotel employees knowledge of the existence of policies and procedures on sexual harassment within one hotel property, to identify the initiators of sexually harassing behaviors, to suggest implications for management, and to provide a data base for future research. Sixty-nine employees of one hotel property were telephone interviewed by using a 30 question survey designed for hotels that was based on the 1985 Gutek questionnaire. The responses were numerically coded and compiled by the Ci2 program. The data was transferred to Lotus 123, and then into Minitab for statistical analysis. The results were compared with the findings of the 1985 Gutek study of society-at-large. The results of this study indicated sexual harassment is a widespread and deeply felt problem in the surveyed property. The findings confirm (1) that a higher proportion of hotel industry employees than of individuals in society-at-large experience sexually harassing behaviors in their workplace, and (2) that there exists a gender difference in reactions to sexual behaviors from the opposite sex in the workplace. This study found male respondents in food preparation appear to have a higher tolerance for sexual behaviors in the workplace than do their counterparts in other departments. Among women respondents, there is very little variation by department in their reaction to sexual behaviors in the workplace. This study also found 100% of male and female respondents have knowledge of the existence of policies and procedures on sexual harassment in the hotel property. Further, co workers are the initiators of most sexually harassing behaviors. The study recommends the commitment of upper management must be scrupulously adhered to and exemplified by every manager and made known publicly to all employees bom in writing and in practice. It is management's responsibility to observe, to listen, to reassure the employee, and to act. This study also recommends the traditional approach of training only managers is no longer sufficient. The results of this study point to the need for direct training of non-supervisory employees regarding sexual harassment. Furthermore, more research in this sensitive topic needs to be done. A problem of access to and cooperation from the hospitality industry need to be addressed before more research can be conducted.