Three-dimensional (3D) displays project 3D images that give 3D perceptions and mimic real-world objects. Among the rich varieties of 3D displays, multiview displays take advantage of light’s various degrees of freedom and provide some of the 3D perceptions by projecting 2D subsampling of a 3D object. More 2D subsampling is required to project images with smoother parallax and more realistic sensation. As an additional degree of freedom with theoretically unlimited state space, orbital angular momentum (OAM) modes may be an alternative to the conventional multiview approaches and potentially project more images. This research involves exploring the possibility of encoding/decoding off-axis points in 2D images with OAM modes, development of the optical system, and design and development of a multiview colour display architecture. The first part of the research is exploring encoding/decoding off-axis points with OAM modes. Conventionally OAM modes are used to encode/decode the on-axis information only. Analysis of on-axis OAM beams referenced to off-axis points suggests representation of off-axis displacements as a set of expanded OAM components. At current stage off-axis points within an effective coding area are possible to be encoded/decoded with chosen OAM modes for multiplexing. Experimentally a 2D image is encoded/decoded with an OAM modes. When the encoding/decoding OAM modes match, the image is reconstructed. On the other hand, a dark region with zero intensity is shown. The dark region suggests the effective coding area for multiplexing. The final part of the research develops a multiview colour display. Based on understandings of off-axis representation of a set of different OAM components and experimental test of the optical system, three 1 mm monochromatic images are encoded, multiplexed and projected. Having studied wavelength effects on OAM coding, the initial architecture is updated to a scalable colour display consisting of four wavelengths.

Em Leptospira interrogans algumas proteínas com capacidade de ligação aos componentes de matriz extracelular foram identificadas e, em sua maioria, são fatores de virulência. Phage display é considerada uma técnica poderosa na identificação de novos ligantes, inclusive de moléculas adesinas, importantes no primeiro estágio de infecção do hospedeiro. A técnica de shotgun phage display foi utilizada visando à obtenção de ligantes à células de mamíferos. Quatro bibliotecas, por inserção de fragmentos aleatórios obtidos por sonicação do DNA de L. interrogans nos fagomídeos pG8SAET (BBT1 e BBT2) e pG3DSS (BBT5 e BBT6), foram construídas. As bibliotecas BBT1 e BBT5 contém insertos maiores e as BBT2 e BBT6 contém insertos menores, com tamanhos médios de 1500 pb e 350 pb, respectivamente. Após ensaio de panning da BBT5 contra células de mamíferos e soro fetal bovino, as sequências de clones selecionados foram analisadas quanto a orientação correta e se a fusão estava em fase com a proteína pIII. As proteínas codificadas pelos genes LIC11719, LIC10769, LIC13143 e LIC12976 foram selecionadas com estas características. Os genes que codificam a LIC12976, LIC10768, LIC10769 e LIC13418, tiveram sua conservação avaliada em diferentes sorovares da espécie patogênica L. interrogans e no sorovar Patoc da espécie de vida livre L. biflexa. As proteínas LIC12976 (selecionada pela técnica de phage display) e LIC13418 (selecionada por ferramentas de bioinformática) tiveram suas sequências amplificadas por PCR, clonadas em pGEM T easy, subclonadas em vetor de expressão pAE e expressas na fração celular correspondente ao corpúsculo de inclusão em E. coli BL21 (DE3) Star pLysS e E. coli BL21 SI, respectivamente. Após renaturação e purificação destas proteínas por cromatografia de afinidade a metal bivalente, um grupo de cinco animais BALB/c fêmeas foi imunizado. Ambas as proteínas se mostraram imunogênicas com títulos dos soros policlonais 1:256000 e 1:512000, respectivamente. Em ensaio de Western Blot os soros foram específicos no reconhecimento das proteínas recombinantes e as proteínas nativas foram verificadas em extratos de sorovares patogênicos de L. interrogans. Em ensaios de adesão, as proteínas recombinantes aderiram às células A31, LLC-PK1 e Vero e especificamente à laminina. Em ensaios de interferência em células usando laminina houve um aumento da adesão das proteínas recombinantes, o que pode ser explicado pela ligação da laminina às células e uma maior ligação das LICs estudadas. Em ensaio de localização celular usando imunofluorescência e microscopia eletrônica, foi observado que ambas as proteínas se encontram na superfície da L. interrogans. No experimento de desafio animal, a LIC12976 e a LIC13418 não se mostraram protetoras. Este trabalho contribuiu para a identificação das novas adesinas LIC13418 e LIC12976 que podem participar da virulência de leptospiras patogênicas envolvendo a primeira etapa da infecção na interação patógeno-hospedeiro / In Leptospira interrogans, proteins capable to bind to extracellular matrix components have been identified and most of them are important virulence factors. Phage display is a powerful technique to identify new ligands, including adhesin molecules that are important in the first stage of host infection. A shotgun phage display technique was used in order to obtain cell ligands. Four libraries were constructed by inserting random fragments obtained by sonication of L. interrogans DNA into phagemids pG8SAET (BBT1 and BBT2) and pG3DSS (BBT5 and BBT6). The libraries BBT1 and BBT5 contain larger inserts and BBT2 and BBT6 contain smaller inserts, with 1500 bp and 350 bp average sizes, respectively. After panning of BBT5 against mammalian cells and bovine fetal serum, the sequences of selected clones were analyzed for correct orientation and fusion with pIII protein. The proteins encoded by genes LIC11719, LIC10769, LIC13143 and LIC12976 were selected. The genes LIC12976, LIC10768, LIC10769 and LIC13418 were evaluated for their conservation in different pathogenic serovars of L. interrogans and free-living L. biflexa serovar Patoc. Proteins LIC12976 (selected by phage display technique) and also LIC13418 that was selected by bioinformatic tools, were amplified by PCR, cloned into pGEM T easy, subcloned into expression vector pAE and expressed in cellular fraction corresponding to the inclusion body in E. coli BL21 (DE3) Star pLysS and E. coli BL21 SI, respectively. After protein renaturation protocol and purification by affinity chromatography, a group of five BALB/c mice was immunized with the purified proteins. Both proteins were shown to be immunogenic with 1:256000 and 1:512000 polyclonal sera titers, respectively. In Western blot the sera were specific to recognize recombinant proteins and native proteins were detected in pathogenic L. interrogans serovars extracts. In binding assays, recombinant proteins bind to A31, LLC-PK1 and Vero cells and specifically to laminin. In interference cell assay using laminin there was an increase of recombinant protein bindings, which can be explained by the laminin binding to cells and further binding of the recombinant LICs. In cellular localization assay using immunofluorescence and electron microscopy, it was observed that both are surface proteins of L. interrogans. In the animal challenge, the LIC12976 and LIC13418 were not protective. As a whole, this work contributed to the identification of LIC12976 and LIC13418 as new adhesins and they can participate in the virulence of pathogenic Leptospira in the first stage of host pathogen interaction.

L'épigénétique est un composant essentiel du fonctionnement des génomes eucaryotes. Les divers phénomènes épigénétiques modifient l’état chromatinien et participent à la plasticité du génome, mais aussi au maintien de son identité fonctionnelle à travers les générations cellulaires. Parmi ces processus, la méthylation de l’ADN joue un rôle fondamental dans la régulation de l’expression des gènes.Chez les mammifères, la méthylation de l'ADN est associée à la répression transcriptionnelle, et elle remplit au moins trois fonctions essentielles. Premièrement, elle permet de réprimer les séquences répétées afin de préserver l’intégrité du génome. Deuxièmement, la méthylation contrôle l’expression des gènes soumis à l’empreinte parentale, qui sont des régulateurs cruciaux du développement et de la vie adulte. Enfin, la méthylation permet de réprimer certains gènes tissu-spécifiques dans les organes où ils doivent être silencieux. En plus de ces rôles physiologiques, la méthylation est liée au cancer. En effet, des patrons de méthylation anormaux sont fréquemment observés dans les cellules tumorales, et ces anomalies participent à la transformation cellulaire par plusieurs mécanismes.La méthylation exerce ces effets par l'intermédiaire de protéines dédiées, qui reconnaissent spécifiquement l'ADN méthylé et contrôlent la transcription en modulant la chromatine. Trois familles de protéines liant l'ADN méthylé sont connues chez les mammifères, et elles totalisent entre elles neuf membres. De nombreux arguments suggèrent que cette liste est encore incomplète, et que des protéines humaines liant l'ADN méthylé restent à découvrir. Dans cette optique, nous avons opté pour deux types d’approches distinctes, une approche basée sur la littérature et une approche génétique. L’étude des protéines candidates ne nous a pas permis d’identifier de nouvelles protéines liant l’ADN méthylé et l’approche génétique par phage display a révélé deux protéines intéressantes, CHD3 et HMGB1 qui doivent désormais être validées par des approches in vivo et in vitro.Par ailleurs, nous avons entrepris l’étude de la régulation des éléments répétés par la protéine Zbtb4 chez la souris. Les expériences préliminaires indiquent une possible régulation des satellites mineurs par Zbtb4. Le rôle de cette régulation sera, par la suite, approfondi. / Epigenetic phenomena are key contributors to the function of eukaryotic genomes. These processes act on chromatin, and they are used to render the genome dynamic, but also stable throughout successive rounds of cell division. Among epigenetic processes, DNA methylation is especially well known for its role in the regulation of gene expression.In mammals, DNA methylation is strongly correlated with transcriptional repression, and fulfills at least three essential roles. First, it maintains repeated sequences transcriptionally silenced, thus ensuring the stability of the genome. Second, it is responsible for the proper regulation of parentally imprinted genes, which are crucial regulators of embryonic development and adult life. Finally, DNA methylation ensures that some tissue-specific genes are kept inactive in the organs in which they should be repressed. Besides these roles in the physiology of normal cells, DNA methylation has strong links to cancer. Indeed the pattern of DNA methylation on the genome is frequently altered in cancer cells, and these anomalies contribute to transformation by several mechanisms.DNA methylation does not control transcription directly, but instead acts via a set of dedicated proteins that specifically recognize methylated DNA and repress transcription by acting at the chromatin level. At present, three families of such proteins, totalling 9 members altogether, are known in humans. However, several lines of evidence suggest that the list is not exhaustive, and that other human proteins that bind methylated DNA remain to be found. This was the goal of the current project.To this end, we opted for two distinct types approaches, an approach based on literature and a genetic approach. The study of candidate proteins does not allow us to identify new methylated DNA binding proteins and the genetic approach by phage display revealed two proteins of interest, HMGB1 and CHD3 that must now be validated by in vivo and in vitro approaches.Furthermore, we studied the regulation of DNA repeats by Zbtb4 in mice. Preliminary results show a regulation of minor satellites by Zbtb4. The role of this regulation will be analyse further in the future.

Les matériaux inorganiques ont des propriétés uniques à l'échelle nanométrique. Ces propriétés ont généré beaucoup d'intérêt pour fabriquer des nouveaux matériaux utilisant des nano-objets comme unité de construction. Nous avons suivi une approche biomimétique pour la fabrication de dispositifs à base de nanoparticules afin d'améliorer les méthodes actuelles de fabrication top-down et bottom-up. Certaines protéines naturelles se lient en effet spécifiquement à des matériaux inorganiques, et déclenchent notamment la croissance de cristaux inorganiques. Une première étape dans cette approche biomimétique est de comprendre comment des protéines se lient spécifiquement à des nanomatériaux inorganiques. Nous avons exploré ce mécanisme de reconnaissance en sélectionnant des anticorps (les protéines de notre système immunitaire spécialisées dans les interactions avec de nombreuses cibles) contre des matériaux inorganiques par la méthode combinatoire biotechnologique appelée "phage display". Cette technique permet d'obtenir la séquence génétique codante des anticorps sélectionnés se liant à leur cible à partir d'une banque aléatoire d'anticorps. L'analyse statistique des séquences des anticorps sélectionnés fournit de nouvelles informations sur les interactions protéines/matériaux inorganiques. Notre principale conclusion est l'identification de l'acide aminé arginine en tant que contributeur majeur dans les interactions protéine/or. L'ingénierie génétique des anticorps permet de fonctionnaliser ces nouvelles sondes de matériaux inorganiques en vue de leur utilisation pour des applications dans le domaine des nanomatériaux. Les anticorps recombinants sélectionnés et leurs dérivés fonctionnalisés peuvent être exprimés par sécrétion à l'aide d'un hôte eucaryote (Dictyostelium discoideum) mis au point au cours de cette thèse. / Inorganic materials have unique properties at the nanometer scale. These properties have generated a lot of interest among researchers to fabricate novel materials using nano objects as building units. In this PhD thesis, we have attempted to mimick nature in the fabrication of nanoparticle based devices in order to improve upon current top-down and bottom-up nanomaterial fabrication methods. Proteins can specifically bind inorganic materials and trigger crystal growth and thus are considered as the main building units for a biomimetic approach of fabrication. The first step towards mimicking nature is to explore how proteins bind specifically to nanomaterials. We have explored this recognition mechanism by selecting antibodies (the protein binders of our immune system) against inorganic nanomaterials using the combinatorial biotechnology method of phage display. This technique provides us with the genetic sequence of selected antibodies from a random antibody library exposed against a target. Statistical analysis of selected antibody sequences provides new information on proteins/inorganics interactions. Our main finding in this regard is the identification of the amino acid arginine as a major contributor to protein/gold interactions. Additional functionality to these new binders of inorganic materials is obtained by antibody engineering, allowing for their value added use in nanomaterial science applications. Selected recombinant antibodies and their engineered derivatives along with other recombinant protein can be expressed and secreted using a eukaryotic expression platform (Dictyostelium discoideum) developed during this thesis.

<p>DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. </p><p>To expand the knowledge of protein–DNA recognition, we have utilized<i> in vitro</i> evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. </p><p>Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring <i>K</i>_D values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure.</p><p>mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. </p><p>The two <i>in vitro</i> selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.</p>

DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. To expand the knowledge of protein–DNA recognition, we have utilized in vitro evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring KD values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure. mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. The two in vitro selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.

Allting började med att jag fick i uppdrag av Euromaint Industry i Skövde att konstruera en alfanumerisk display i syfte att ersätta en utgången display som inte längre nytillverkas. Jag fick i uppdrag att välja ut en modern, lämplig grafisk display och bygga ett interface mellan den nya displayen och den industriella maskin som displayen skall sitta på. Efter att ha letat hos någraelektronikleverantörer kom jag fram till att en TFT-skärm från det japanska företaget Kyocera var den som passade bäst. Skärmen hade ett VGA-liknandeinterface och min uppgift blev att sätta mig in i hur VGA fungerar. Efter att ha konstaterat att det krävdes en snabbare krets än en microcontroller för att använda VGA, var det endast en programmerbar logikkrets, en FPGA, som gällde. Denna FPGA sköter nu ensam om såväl VGA-interfacet som inläsningen av informationen från den industriella NC-maskinen. / Everything started when I got a task from Euromaint Industry in Skövde, Sweden, to develop an alphanumerical display that could replace an old one, which was sold out. I got a task to choose a modern, suitable, graphical display and develop an interface between the new display and the industrial machine, which the old one was connected to. I have searched for a display at some suppliers of electronic components and I have found a TFT-display from the Japanese company Kyocera. The display had an interface similar to VGA so I had to study VGA to see how it works. Then I realized that I needed a faster circuit than a microcontroller. Then I chose a programmable logic circuit, an FPGA, to control the VGA-sweep. Today the FPGA-circuit controls the whole system.

<p>Allting började med att jag fick i uppdrag av Euromaint Industry i Skövde att konstruera en alfanumerisk display i syfte att ersätta en utgången display som inte längre nytillverkas. Jag fick i uppdrag att välja ut en modern, lämplig grafisk display och bygga ett interface mellan den nya displayen och den industriella maskin som displayen skall sitta på. Efter att ha letat hos någraelektronikleverantörer kom jag fram till att en TFT-skärm från det japanska företaget Kyocera var den som passade bäst. Skärmen hade ett VGA-liknandeinterface och min uppgift blev att sätta mig in i hur VGA fungerar. Efter att ha konstaterat att det krävdes en snabbare krets än en microcontroller för att använda VGA, var det endast en programmerbar logikkrets, en FPGA, som gällde. Denna FPGA sköter nu ensam om såväl VGA-interfacet som inläsningen av informationen från den industriella NC-maskinen.</p> / <p>Everything started when I got a task from Euromaint Industry in Skövde, Sweden, to develop an alphanumerical display that could replace an old one, which was sold out. I got a task to choose a modern, suitable, graphical display and develop an interface between the new display and the industrial machine, which the old one was connected to. I have searched for a display at some suppliers of electronic components and I have found a TFT-display from the Japanese company Kyocera. The display had an interface similar to VGA so I had to study VGA to see how it works. Then I realized that I needed a faster circuit than a microcontroller. Then I chose a programmable logic circuit, an FPGA, to control the VGA-sweep. Today the FPGA-circuit controls the whole system.</p>

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Includes bibliographical references (leaves 174-179). Also available via the Internet

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