Ph.D.

Prior work has indicated that preventing Cdc42 interacting with its downstream effector proteins can reverse Ras-driven oncogenesis. The experiments demonstrating this used the G protein binding region (GBD) of the Cdc42-specific effector ACK, which is 42 amino acids long and binds with 40 nM affinity. The aim of this work was to find a peptide which exhibits similar effects in reversing oncogenic characteristics in cells but with more promising therapeutic properties: for example, a smaller size, improved binding and improved protease resistance. Firstly, the Cdc42-ACK binding interface was characterised thermodynamically, producing a comprehensive dataset of the contribution of individual ACK residues to complex affinity. This information revealed regions of the ACK GBD with higher concentrations of vital interactions but also residues that were unimportant for maintaining affinity. An \emph{in vitro} display technology (CIS-display) was employed to select for shorter peptides with improved binding to Cdc42. By selecting the best binders from libraries based on the ACK GBD, peptides have been generated that are half the length of the full binding domain but bind with affinities approaching the wild-type ($\approx$100 nM). More interestingly, a serendipitous discovery in a na\"{\i}ve cyclic library revealed a 16-residue sequence that binds with 350 nM affinity, subsequently called C1. The biophysical properties of selected peptides were characterised and demonstrate that C1 is reliant on an intramolecular disulphide bond for tight binding. An N-terminal nona-arginine sequence was added to C1 to facilitate entry into cells: internalisation was confirmed by confocal microscopy. Subsequently it was found that C1 reduced signalling through the ACK and PAK effector pathways, negatively impinging on MAPK signalling. Levels of signalling returned to baseline within 24 hours, however, and the transiency of the effect was thought to be linked to peptide degradation: an effect likely in a construct dependent on a disulphide bond for cyclisation and binding affinity. Subsequent maturation of C1 using a focussed CIS-display library has selected second generation peptides. These have been characterized and display binding affinities to Cdc42 of $\approx$20 nM: a 17-fold increase in binding over C1. This has enhanced the affinity to a level even higher than wild-type ACK and should improve the cellular potency of the peptides identified in this selection. %any more?! Having successfully selected peptides both shorter in length and with higher affinity than the wild-type sequence, efforts were channelled into developing a method to cyclize the peptide by a chemical linkage that would be stable in a cellular environment. The most successful approach involved desulphurisation of the disulphide bond to a thioether, making the cyclic linkage non-labile in the reducing environment of the cytosol, while retaining original binding affinity. The peptides developed in this work bind to Cdc42 with nanomolar affinity \emph{in vitro} and can enter cells and impact upon signalling processes connected with oncogenesis. As such, they provide a potential therapeutic lead for future development.

This thesis reports on the design, fabrication, and testing of state-of-the-art, high-performance inverted top-emitting organic light-emitting diodes (OLEDs). The vast majority of research reports focuses on a device architecture referred to as a conventional OLED which has its anode on the bottom of the device and its cathode on the top. Moreover, most conventional OLEDs are bottom-emitting such that light exits the structure through both a semitransparent bottom electrode of indium-tin oxide and a glass substrate. The particular device architecture developed in this thesis is one in which the devices are inverted (i.e. their cathode is on the bottom as opposed to on top) and top-emitting. Despite the advantages that inverted top-emitting OLEDs possess over conventional bottom-emitting OLEDs, their development has been relatively slow. This is because inverted OLEDs have traditionally been hampered by the difficulty of injecting electrons effectively into the device. In this work, a novel method of injecting electrons from bottom cathodes into inverted OLEDs is discovered. In several previous reports, bottom Al/LiF cathodes had been used with the electron-transport material Alq3 to produce inverted OLEDs, but the resulting inverted OLEDs exhibited inferior performance to conventional OLEDs with top cathodes of Al/LiF. A new route for the development of highly efficient inverted OLEDs is shown through the use of electron-transport materials with high electron mobility values and large electron affinities. After systematic device optimization, inverted top-emitting OLEDs are demonstrated that currently define the state-of-the-art in terms of device efficiency. Optimized green and blue inverted top-emitting OLEDs are demonstrated that have a current efficacies of 92.5 cd/A and 32.0 cd/A, respectively, at luminance values exceeding 1,000 cd/m2. Finally, this discovery has enabled the development of the first stacked inverted top-emitting OLEDs ever made, combining all of the advantages offered by an inverted architecture, a top-emissive design, and a stacked structure. These OLEDs have a current efficacy of 200 cd/A at a luminance of 1011 cd/m2, attaining a maximum current efficacy of 205 cd/A at luminance of 103 cd/m2.

Mycoplasma hyopneumoniae is the etiologic agent causing chronic pneumonia of swine. The lung lesions of swine produce the slower growth rate and lower feed conversion ratio and finally cause economic loss. Although four genome projects of mycoplasma species had been completed, the genome-sequencing project of M. hyopneumoniae also closed to the finished stage. However, only a few genes and proteins of M. hyopneumoniae have been studied, the molecular pathogenic mechanism remains elusive. The research of molecular vaccine is still preliminary. In order to obtain more information about epitope structures as the basis to develop molecular vaccine against this pathogen, two phage-displayed random heptapeptides libraries were used to identify epitopes recognized by purified IgG of rabbit anti-M. hyopneumoniae hyperimmune serum in this study. Individual phage clones were isolated and verified the binding specificity to the purified IgG by Western blot analysis and competitive ELISA after three rounds of biopanning. The selected clones were further characterized by DNA sequencing analysis and deduced to amino acid sequences. There are six consensus sequences contained tri- to hepta-peptide existing among the selected phage clones by aligning the sequences of foreign amino acids displaying on pIII protein. The consensus sequences may be serving as crucial epitopes of M. hyopneumoniae. By searching the protein database of M. hyopneumoniae deposited in NCBI, some surface proteins were matched by the selected mimotopes. Like P97, the essential protein for attaching to cilia of swine, the deduced epitopes mainly located at a.a. from 365 to 382, 395 to 403 and 436 to 452, the R1 and R2 repeated sequences also matched by the mimotopes. To evaluate the potential of these mimotopes as effective vaccine, several phage clones were chosen to immunize mice by intraperitoneal and intranasal administration. There are specific antibody responses to these mimotopes existing in serum IgG, fecal extracts and bronchoalveolar lavage fluids IgA. The serum IgG subclass profiles analysis reveals that these are mainly existed in IgG1 subclass. Base on the results of IgG subclass profiles analysis in sera, the results suggest that the phage-derived vaccines mainly activate Th2 cellular immunity pathway with the strategy used in this study. The similar results were found in the inactivated vaccine. The Th2 activation will promote the elimination of extracellular microorganism. Western blotting analysis showed that each serum raised by the phage clones could recognize 2 to 5 mycoplasma proteins. With the results of growth inhibition assay, we found that the selected phage clones CS4 and 58 are better vaccine candidates and some proteins (97 kDa¡B56 kDa and 30 kDa) may play crucial roles in block the bacteria growth. The advantage was taken of the natural property of M13 phage to infect E. coli, which is initiated by the N terminal of pIII coat protein binding with the F pili of E. coli. Plaque reduction tests were proposed to demonstrate the humoral immunity responses induced by phage-derived vaccine containing the antibodies specifically against the foreign peptide displayed on pIII coat protein. The present results provide more epitope information of M. hyopneumoniae. The mice immunization results reveal that the phage-displayed mimotopes can be used as potential vaccine candidates. The strategy presented in this study can shorten the time course for vaccine development and provide an alternative pathway for searching vaccine candidates against M. hyopneumoniae.

Holoscopic 3D imaging “Integral imaging” was first proposed by Lippmann in 1908. It has become an attractive technique for creating full colour 3D scene that exists in space. It promotes a single camera aperture for recording spatial information of a real scene and it uses a regularly spaced microlens arrays to simulate the principle of Fly’s eye technique, which creates physical duplicates of light field “true 3D-imaging technique”. While stereoscopic and multiview 3D imaging systems which simulate human eye technique are widely available in the commercial market, holoscopic 3D imaging technology is still in the research phase. The aim of this research is to investigate spatial resolution of holoscopic 3D imaging and display technology, which includes holoscopic 3D camera, processing and display. Smart microlens array architecture is proposed that doubles spatial resolution of holoscopic 3D camera horizontally by trading horizontal and vertical resolutions. In particular, it overcomes unbalanced pixel aspect ratio of unidirectional holoscopic 3D images. In addition, omnidirectional holoscopic 3D computer graphics rendering techniques are proposed that simplify the rendering complexity and facilitate holoscopic 3D content generation. Holoscopic 3D image stitching algorithm is proposed that widens overall viewing angle of holoscopic 3D camera aperture and pre-processing of holoscopic 3D image filters are proposed for spatial data alignment and 3D image data processing. In addition, Dynamic hyperlinker tool is developed that offers interactive holoscopic 3D video content search-ability and browse-ability. Novel pixel mapping techniques are proposed that improves spatial resolution and visual definition in space. For instance, 4D-DSPM enhances 3D pixels per inch from 44 3D-PPIs to 176 3D-PPIs horizontally and achieves spatial resolution of 1365 × 384 3D-Pixels whereas the traditional spatial resolution is 341 × 1536 3D-Pixels. In addition distributed pixel mapping is proposed that improves quality of holoscopic 3D scene in space by creating RGB-colour channel elemental images.

Polyclonal antibodies against the first two enzymes of the Arabidopsis thaliana flavonoid biosynthetic pathway were developed using conventional and phage antibody technology. cDNAs from Arabidopsis coding regions of chalcone synthase (CHS) and chalcone isomerase (CHI) were sub-cloned in frame into a bacterial expression vector as fusions with glutathione Stransferase (GST) using standard directional cloning techniques. Analysis of crude extracts of Escherichia coli containing GST .. CHS or GST .. CHI fusion protein indicated that the cells expressed equivalent amounts per volume of culture. CHS and CHI were purified to near homogeneity, yielding approximately 100 micrograms of GST .. CHS and 1 milligram of GST-CHI per liter of culture. The purified fusion proteins were injected into chickens and polyclonal lgY·s were purified from egg yolk Accumulation of CHS and CHI, as well as products of the pathway, were compared during the first eight days of Arabidopsis development. CHS and CHI are sequentially induced and reach maximal accumulation levels by day 5. Anthocyanidin levels are offset by one reaching maximal levels at day 6. The fusion proteins were also used to screen a phage-display library for Fabl fragments that recognize CHS and CHI epitopes. Preliminary data indicated that enrichment of phage displaying antibodies against CHS and CHI was successful. Phage-derived antibodies against CHS and CHI provide valuable tools for future experiments addressing Western blot analysis, immunolocalization experiments, and disruption of the flavonoid biosynthetic pathway by introduction of the corresponding genes into transgenic Arabidopsis plants. / Master of Science

No description available.

Therapeutische Erfolge von IVIG sind gut dokumentiert, aber die zu Grunde liegenden molekularen Mechanismen sind noch nicht vollständig erforscht. Molekulare Analysen unseres Labors über die Interaktion von IVIG mit Fabs von Patienten, die an einer autoimmunen Thrombozytopenie (ITP) leiden zeigten, dass die am häufigsten selektierten Fab von den V3-23 und V3-30 VH-Keimbahngenen abstammten. Eine weitere Studie mit IgG und IgM Phagen-Display Bibliotheken von einem gesunden Spender zeigten ebenfalls eine bevorzugte Reaktivierung von IVIG mit Fabs vom Ursprung der V3-23 und V3-30 Gene. Es konnte gefolgert werden, dass diese Interaktion von IVIG mit Fabs von diesen zwei VH-Genen weder alleine auf den Gesundheitsstatus des Spenders zurückzuführen war, noch auf eine zuvor erfolgte Behandlung mit IVIG. Diese Dissertation wurde unter Verwendung der Phagen-Display Technologie unternommen, um die molekulare Interaktion von IVIG mit Antikörpern zu erforschen, die von einem Patienten kloniert wurden, der an einem systemischen Lupus erythematodes und rheumatischem Fieber leidet. Die Resultate waren mit den früheren Studien zu vergleichen, insbesondere mit den Daten eines Patienten, der zu der ITP einen Lupus entwickelte. 23 Fabs, welche 7 unabhängige Klone repräsentierten, wurden isoliert. Im Gegensatz zu von Patienten mit ITP abstammenden Klonen reagierte keines von den in dieser Studie selektierten Fabs mit Thrombozyten. Die über IVIG gebundene Fab-Phagen stammten hierbei ausschließlich von den V3-23 und V3-30 VH-Genen ab. Darüber hinaus wurde beobachtet, dass von diesen Fabs verschiedene CDR3 Regionen einschließlich verschiedenen D- und JH-Gensegmenten benutzt wurden. Die Ergebnisse zeigten weiterhing, dass die Bindung von IVIG an die Fabs unabhängig von der Leichten Kette war. Ihrem Keimbahngen-Ursprung entsprechend hatten die Fabs Aminosäuren an Positionen in den FR1, FR3 und im 3'-Ende von CDR2, die dafür bekannt sind, dass sie für die Bindung des B-Zell-Superantigens Staphylococcus Protein A (SpA) essentiell sind. Es wurde gezeigt, dass sich zwar einige von den Fabs stark an SpA banden, aber keine Korrelation in der Intensität zur Bindung mit IVIG vorlag. Einige Fabs zeigten eine schwache Bindung an HIV gp120, einem anderen B-Zell-Superantigen. Zusammenfassend lässt sich aus der vorliegenden Studie und den vorherigen Ergebnissen schließen, dass ein Anteil von IVIG wie ein B-Zellen Superantigen funktionieren könnte, das für die Bildung und Regulation des normalen B-Zellen Repertoires wichtig ist. Der Bindungsmechanismus scheint ähnlich, aber nicht identisch mit dem der anderen getesteten B-Zellen-Superantigene zu sein. / The beneficial therapeutic effects of IVIG are well documented, but the underlying molecular mechanisms are not fully understood. Recent investigations from our laboratory into the molecular analysis of Fabs bound by IVIG from patients suffering from autoimmune thrombocytopenia revealed that the most frequently selected Fabs originated from the V3-23 and V3-30 VH germline genes. A subsequent study with IgG and IgM phage display libraries from a healthy donor also demonstrated a preferential reactivity of IVIG to Fabs of V3-23 and V3-30 origin. That study revealed that the unique reactivity of IVIG to Fabs of these two VH gene loci was not restricted to the autoimmune nature of the donors, neither to previous treatment with IVIG. One of the thrombocytopenia patients developed lupus. This study was undertaken to study the molecular interaction of IVIG with antibodies selected from a patient suffering from systemic lupus erythematosus and rheumatic fever using phage display technology, and to compare the results with the previous studies. Twenty-three Fabs representing seven independent clones were isolated. In contrast to ITP-derived clones, none of the Fabs selected in this study reacted with platelets. The Fab phages bound by IVIG were sequenced in order to determine their VH gene usage and clonal relatedness. V3-23 and V3-30 VH genes were found to be exclusively utilized by the Fab phages bound by IVIG. Moreover, different CDR3 regions including different D and JH gene segments were observed to be used by these Fabs. The results further showed that the binding of IVIG to the Fabs was independent of the light chain since different light chains were observed to be associated with the VH3 immunoglobulins. Detailed sequence analysis of the Fabs revealed the presence of amino acid residues at positions within FR1, FR3, and the 3' end of CDR2 that are known to be contacted by the B cell superantigen Staphylococcus protein A (SpA). Some of the Fabs were shown to bind strongly to SpA, but there was no correlation with the binding-intensity to IVIG. Some bound very weakly to HIV gp120, another B cell superantigen. This study, together with previous results, suggests that a subset of IVIG may function as a B cell superantigen that may significantly shape the B cell repertoire. The binding mechanism appears to be similar but not identical to the other tested B cell superantigens.

儀式作為人類社會中最重要的體驗活動，在歷經時代的考驗與價值觀的轉變汰換後，流傳下來的內涵與核心意義雖然不變，但表現形式、媒材不斷隨著時代發展與時俱進，本研究欲以政治大學畢業典禮薪火相傳儀式為例，利用體驗設計為框架，將儀式原有內涵作為敘事基礎，藉由數位內容的製作，以及互動設計結合當代科技的運用，重新策劃並執行互動儀式體驗，進而探討傳統儀式與互動儀式體驗的轉變與差異，因此本研究目的歸納為以下三點： 1. 以體驗設計為框架，儀式內涵為敘事核心，利用互動設計結合展示科技製作數位內容，建立互動儀式媒介、互動儀式情境，進而重新策劃、製作、展演虛實整合的互動儀式體驗。 2. 分別從表演者、主動參與者、被動參與者、製作執行者的角度，探討互動儀式體驗與傳統儀式的的轉變與差異。 3. 歸納出互動體驗設計應用於儀式展演的策劃與執行要點。 研究結果發現，互動儀式體驗透過當代互動科技的應用，讓儀式參與者能即時主動加入儀式展演的內容創作，因此相較於傳統儀式能帶來更多的參與感與歸屬感，雖然多數的儀式參與者仍偏好被動觀賞的形式，但集體共創的展演內容能引起在場儀式參與者的共鳴，進而創造儀式當下的回憶，因此深化體驗的感受，也讓儀式不只是流於形式的過程。 / Ceremony is one of the most important experiences in human society. Although the principles and core elements may remain the same, styles of ceremonies and technologies used in the ceremonies have been developed as time progress and values change. The purpose of this study was to discuss the transformation and difference between traditional ceremony and interactive media-introduced ceremony by a case study of passing the flame in National Chengchi University commencement ceremony. This study covers below three topics: 1. By utilizing experience design as framework and the narrative in ceremony as the core, preparing digital content that combines interaction design and exhibition display technology, establishing interactive media and environment, an interactive ceremony experience was re-curated, prepared, and executed. 2. Discussion of transformation and difference between interactive ceremony experience and traditional ceremony from views of performers, active participants, passive participants, and executors. 3. Key factors of application of interactive experience design in ceremony curation and execution. After field observations, digital content analysis, and interviews with some participants, the results revealed that real-time interactive experiences in ceremony can bring more sense of participation and belonging than used to. Despite most of participants preferred just watching the performance, participants could get more connection from exhibition content that contributed by themselves. Therefore, the memory created at the moment could bring stronger feelings and experience and not letting the ceremony become a mere formality.

(in English): This thesis is conceived as a metareview of research on differences in perception, understanding and retention of text on various display media. It summarizes the results of the most interesting and most relevant research on this topic conducted since the 80s to the present. Even though it is difficult to summarize the results of individual studies, mainly because of differences in research methodology and differently chosen tested samples, we can say that the main finding of this metareview is that display technologies that are available these days have no negative effect on eye fatigue, reading speed, perception, understanding or retention of text. Theoretical part of this thesis is followed by practical part that consists of three short experiments performed on a small sample of participants. First of those experiments studies differences in reading comprehension and retention among high school students, second experiment focuses on differences in reading speed on different media and subjective evaluation of eye fatigue, and the third experiment is an online form about subjective preferences of study materials among learners.