Phage display technology has emerged as the dominant approach in antibody engineering. Here I describe my work in developing a high-throughput method of reliably generating intracellular antibodies. In my first data chapter, I present the first known high-throughput pipeline for antibody-phage display libraries of synthetic diversity and I demonstrate how increasing the scale of both target production and library selection still results in the capture of antibodies to over 50% of targets. In my second data chapter, I present the construction and validation of a novel scFv-phage library that will serve as the first step in my proposed intrabody pipeline. Antibodies obtained from this library will be screened for functionality using a novel yeast-two-hybrid approach and have numerous downstream applications. This high-throughput pipeline is amenable to automation and can be scaled up to thousands of domains, resulting in the potential generation of many novel therapeutic reagents.

Peptide recognition domains (PRD) bind to short linear motifs on their biological partners and are found in several cellular pathways including those found to be critical in tumorigenesis. In this study, I aimed to generate peptide probes against PRDs present on proteins involved in ovarian cancer. Using bioinformatics, I identified 66 potential PRDs present on these proteins. I then used peptide phage display to successfully generate peptides against 27 of the 66 domains. To validate my results, I performed an extensive literature review and structural analysis. For several cases, the phage-display derived binding preferences are similar to previously reported studies. However, for a subset of domains, I identified non-canonical binding preferences that have not been reported previously in literature. The binding preferences obtained in this study can be used to design intracellular probes for studying the role of these PRDs in biological pathways important in ovarian cancer.

The availability of cost-effective high throughput screening assays combined with an enhanced understanding of oncogenesis has driven the development of more potent, specific, and less toxic anti-cancer agents. At the forefront of these advances are immunoglobulin molecules and their fragments. However, difficulties in producing antibodies in sufficient quantity and quality for commercial application have driven the development of alternative systems that can produce antibodies efficiently and cost-effectively. This thesis focuses on the engineering of an antibody fragment referred to as a single chain variable fragment (scFv), which consists of antibody light and heavy chain variable domains fused together by a peptide linker. Although the use of scFvs circumvents many of the issue of full-length antibody production, they still possess their own unique set of difficulties, including stability. In this thesis, we explored the following strategies to increase scFv stability. First, we increased the number of linkers used to join the variable light and heavy domains. We constructed two linear and two cyclic permutated scFvs that contained additional peptide linkers. Two linear permutated scFvs, named Model 1 and Model 3, showed increased stability with calculated melting temperatures (Tms) exceeding that of the unpermutated scFv. The two cyclic scFvs were less stable with Tms less than that of the unpermutated scFv. Second, we mutated light and heavy variable domains by introducing prolines or mutating glycine to alanine in the variable domain framework regions. Sites for proline mutations and glycine to alanine mutations were identified and scFvs containing the mutations were purified and their thermal stability tested. Unfortunately, there were no discernible differences between purified scFv mutants and the control scFv. Third, we designed a new selection/screening strategy using phage display and yeast two-hybrid assays to identify complementarity determining regions on scFvs that increased intracellular stability. We used this strategy to isolate anti-Abl-SH3 scFvs. Transient expression of scFvs in K562 cells indicated that two anti-Abl-SH3 scFv decreased viability.

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In this paper, a three-dimensional (3D) integral imaging display for augmented reality is presented. By implementing the pseudoscopic-to-orthoscopic conversion method, elemental image arrays with different capturing parameters can be transferred into the identical format for 3D display. With the proposed merging algorithm, a new set of elemental images for augmented reality display is generated. The newly generated elemental images contain both the virtual objects and real world scene with desired depth information and transparency parameters. The experimental results indicate the feasibility of the proposed 3D augmented reality with integral imaging.

Virtual Reality är ett media med gamla rötter, men samtidigt en ny teknologi för allmänheten. Ett av problemen med VR är att det kan frammana visst obehag då det brukas, så kallad simulatorsjuka (Kennedy m.fl., 1993), vilket forskare studerar i syftet att förstå hur det undviks. I denna undersökning har en artefakt tagits fram i form av en spelprototyp, av slaget 2,5D-side-scrollingspel till VR, och använts för att studera uppkomsten av simulatorsjuka. Artefakten använder en head-mounted display för att uppnå VR. Tre testpersoner deltog i undersökningen, och för att mäta symptomen användes ett beprövat frågeformulär samt kvalitativa intervjuer. Data visar på att en tidigare hypotes om intryckskonflikt (Kolasinski, 1995, s.7) är inkorrekt, samt att konventionella orienteringsmekaniker har vissa fördelar. För framtida studier bör simulatorsjuka undersökas från fler vinklar och/eller med ett större urval av testpersoner.

Dynamic, refreshable tactile displays offer a method of displaying graphical information to people who are blind or visually impaired. Texture, which is already used as an effective method to present graphical information in physical tactile diagrams, conceivably constitutes the best way to present graphics through a tactile display. This thesis presents the design of a new low-cost haptic matrix display device capable of displaying graphical information through virtual textures. The perception of virtual textures through the display is examined through three main experiments. The first two experiments examine the perception of square wave gratings through the device. The final experiment examines the effect of texture adaptation when using the device, and compares it to exploration with a handheld probe and the bare finger. The results show that haptic matrix displays can be used to display graphical information through texture and offer guidelines in the production of such textures.

A finger-worn haptic device capable of rendering 2-D graphics through vibrotactile feedback is presented. The device development is presented from its initial stages of being a stylus design using a photo-interrupter optical sensor and pager-motor actuator to a small case worn on the finger using a RGB color sensor and a piezoelectric actuator. Testing of the latest prototype design shows that it has a spatial sensitivity (<2mm) comparable to natural touch (~1mm) and can be used to output a variety of vibrotactile textures. The design can be expanded for a multiple finger, independent device, while remaining affordable (<$100) and highly portable (<500g).

The astonishing diversity of floral form in angiosperm plants is driven in large part by preferences of pollinators for various floral traits, including learned preferences. Remarkably, almost all of a vast literature on learning and memory in pollinators relates to nectar as a reward, even though bees and many flies, beetles, and butterflies must collect pollen. In this study, we asked if bees formed preferences for plant species from which pollen had been collected successfully. Using absolute conditioning, we gave pollen foraging bees experience with plant species that offered only pollen rewards. Naive bees generally showed modest preferences, whereas experienced bees adopted strong preferences for those species over alternative species not previously experienced. Learned preferences were retained for at least 24 h, consistent with preferences learned with nectar rewards. These experience-mediated changes in preference raised the possibility that bees formed associations between particular floral features and pollen rewards. We therefore asked if learned preferences required that bees successfully collect pollen. Using differential conditioning, we determined that learned preferences were strongly influenced by receipt of a pollen reward. In a final experiment, we characterized the importance of 2 floral features, the corolla and the anther, in the expression of learned preferences. Although experience altered responses to both floral parts, responses to anthers were influenced more strongly. We discuss recent evidence in the literature for associative learning with pollen rewards and propose that learned preferences in the context of pollen collection have played an important role in floral display evolution.

Store image research has attempted to identify factors contributing to formation of positive or negative perceptions of stores by consumers. These factors include tangible and intangible elements. Of the tangible factors, store atmosphere (including store layout and congestion level) is often identified as contributing to store image. Intangible factors influencing store image include emotional or psychological reactions that consumers have in response to various tangible store factors. One of these emotional responses is retail crowding. Retail crowding is a state of psychological stress occurring in consumers in response to perceived high density levels in stores. Crowding literature suggests that environmental cues, including layout and density level, contribute to this stress level. The overall purpose of this study was to expand on current research by incorporating the concept of retail crowding with store image research.