Directed evolution is a powerful method for engineering of specific affinity proteins such as antibodies and alternative scaffold proteins. For selections from combinatorial protein libraries, robust and high-throughput selection platforms are needed. An attractive technology for this purpose is cell surface display, offering many advantages, such as the quantitative isolation of high-affinity library members using flow-cytometric cell sorting. This thesis describes the development, evaluation and use of bacterial display technologies for the engineering of affinity proteins. Affinity proteins used in therapeutic and diagnostic applications commonly aim to specifically bind to disease-related drug targets. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a critical process in various types of cancer and vascular eye disorders. Vascular Growth Factor Receptor 2 (VEGFR2) is one of the main regulators of angiogenesis. The first two studies presented in this thesis describe the engineering of a biparatopic Affibody molecule targeting VEGFR2, intended for therapeutic and in vivo imaging applications. Monomeric VEGFR2-specific Affibody molecules were generated by combining phage and staphylococcal display technologies, and the engineering of two Affibody molecules, targeting distinct epitopes on VEGFR2 into a biparatopic construct, resulted in a dramatic increase in affinity. The biparatopic construct was able to block the ligand VEGF-A from binding to VEGFR2-expressing cells, resulting in an efficient inhibition of VEGFR2 phosphorylation and angiogenesis-like tube formation in vitro. In the third study, the staphylococcal display system was evaluated for the selection from a single-domain antibody library. This was the first demonstration of successful selection from an antibody-based library on Gram-positive bacteria. A direct comparison to the selection from the same library displayed on phage resulted in different sets of binders, and higher affinities among the clones selected by staphylococcal display. These results highlight the importance of choosing a display system that is suitable for the intended application. The last study describes the development and evaluation of an autotransporter-based display system intended for display of Affibody libraries on E. coli. A dual-purpose expression vector was designed, allowing efficient display of Affibody molecules, as well as small-scale protein production and purification of selected candidates without the need for sub-cloning. The use of E. coli would allow the display of large Affibody libraries due to a high transformation frequency. In combination with the facilitated means for protein production, this system has potential to improve the throughput of the engineering process of Affibody molecules. In summary, this thesis describes the development, evaluation and use of bacterial display systems for engineering of affinity proteins. The results demonstrate great potential of these display systems and the generated affinity proteins for future biotechnological and therapeutic use. / <p>QC 20141203</p>

Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references (p.56-59).

Techn. Universiẗat, Diss., 2004--Darmstadt.

Les anticorps monoclonaux (mAb) sont depuis les années 2000 devenus des médicaments incontournables et de routine en thérapie et notamment en cancérologie. Le domaine continue à croître très rapidement et devant l’abondance des molécules disponibles, il est de plus en plus important d’apporter des molécules innovantes à haute valeur ajoutée pour la thérapie. Deux grandes approches sont utilisées pour sélectionner ces mAbs : l’hybridation lymphocytaire à partir de souris normales ou humanisées ; Les systèmes de display comme le phage-display. Les intérêts majeurs du phage display sont la rapidité de développement des mAbs, la facilité de manipulation chez E. coli, l’accès aux techniques de "protein engineering". Classiquement, les anticorps sont d’abord sélectionnés sur leur capacité de liaison à l’antigène puis ensuite testés pour leur efficacité fonctionnelle dans des modèles cellulaires. Cependant, seulement une partie de l'activité des anticorps est expliquée par leur liaison à l’antigène et l’activité thérapeutique dépend aussi fortement de leur capacité à recruter le système immunitaire (ADCC) et à activer la cascade du complément (CDC).Ce projet de thèse consiste à développer un nouveau format de banque d'anticorps recombinants combinant la puissance de la sélection par phage display à un criblage fonctionnel au format IgG entières produites en cellules eucaryotes. Ce nouveau système est basé sur des régions initiatrices hybrides contenant à la fois des promoteurs et des séquences signal procaryotes et eucaryotes permettant l’expression dans ces deux systèmes cellulaires, et des évènements de recombinaisons sites-spécifiques transférant le fragment Fab du vecteur de display vers le chromosome d’une lignée cellulaire de mammifère spécialement développée pour aboutir à la sécrétion d’un anticorps humain monoclonal par la cellule. L’approche habituelle de reclonage un par un du vecteur E. Coli au format IgG n’est plus nécessaire puisqu’il se fait directement par transfection. Ce nouveau système rend possible le couplage d’une sélection par phage display à un criblage fonctionnel direct sur une large population de clones monoclonaux humains. / Since 2000, monoclonal antibodies (mAb) have become essential and routine drugs in therapy and particularly in oncology. The field continues to grow very quickly and given the abundance of molecules available, it is increasingly important to bring innovative molecules with a high added value for therapy.Two main approaches are used to select these mAbs: hybridoma technology using normal or humanized mice; display systems such as phage-display. The major interests of phage display are the speed of mAb development, the facilities offered by E. coli and the easy access to protein engineering techniques. Typically, antibodies are first selected on their ability to bind to the antigen, and then tested for their functional efficiency in cellular models. However, only a part of the activity of antibodies is explained by their binding to the antigen, and the therapeutic activity also depends strongly on their ability to recruit the immune system (ADCC) and activate the complement cascade (CDC).This thesis project consists in the development of a new recombinant antibody library format combining the power of phage display selection with functional screening in a whole IgG format produced in eukaryotic cells. This new system is based on hybrid promoter and signal peptide regions allowing expression both in prokaryotic and eukaryotic cells, and a site-specific recombination event that exchanges the Fab between the display vector and the chromosome of an especially developed mammalian cell line resulting in the secretion of a monoclonal human antibody by the cell. The usual approach of recloning one by one from E.Coli vector to an IgG format is no more needed since it is done directly by transfection. This new system makes possible to couple selection by phage display with a direct functional screening of a large population of human monoclonal clones.

Notre laboratoire a développé une stratégie d'isolement de fragments d'anticorps recombinants à partir de primates non humains (Macaca fascicularis) immunisés, en utilisant la technologie des phages. Dans le cadre de cette thèse, une comparaison des séquences d'anticorps de macaques (Macaca Mulatta) et d'anticorps humains a toutefois montré que les anticorps des deux espèces présentent des différences qui rendent souhaitable une étape d'humanisation des anticorps de macaques. Cette stratégie a été utilisée dans le cadre du projet Européen AntiBotABE (www.antibotabe.com) et l'étape de criblage a été adaptée pour isoler des scFv neutralisant de façon croisée les toxines botuliques BoNT/B des sous-types B1 et B2, en utilisant séquentiellement l'holotoxine BoNT/B1 et un fragment recombinant représentant la région C-terminale de la chaîne lourde de BoNT/B2. Le meilleur scFv ciblant les régions C-terminales des chaînes lourdes de BoNT/B1 et BoNT/B2, B2-7, a montré une bonne capacité de neutralisation de BoNT/B1 et BoNT/B2 dans le test ex vivo de paralysie hémidiaphragmatique. Les régions charpentes du scFv B2-7 ont un pourcentage d'identité élevé (80 %) avec leurs homologues humains. Des scFv neutralisant BoNT/A1 en ciblant sa chaîne légère ont aussi été isolés, dont le scFv le plus efficace, 2H8, induit une diminution de 50% de l'activité endopeptidasique à une concentration correspondant à un rapport molaire 2H8/BoNT/A1 de 64000. Les régions charpentes de 2H8 ont également un pourcentage d'identité élevée (88%) avec leurs homologues humains. La versatilité de cette stratégie en fait un outil permettant l'isolement de nombreux autres fragments d'anticorps à visée thérapeutique. / Our laboratory has developed a strategy to isolate recombinant antibody fragments technology from immunized non human primates (Macaca fascicularis) by phage display. In the course of the present thesis, a comparison between macaque (Macaca mulatta) and human antibody sequences has demonstrated that antibodies of the two species are different. This difference makes the humanization of macaque antibodies desirable. The strategy was used in the framework of the European AntiBotABE project, and the screening was adapted to isolate antibody fragments cross neutralizing the B1 and B2 subtypes of botulinum B neurotoxin, by using sequentially the holotoxin BoNT/B1 and a recombinant fragment representing the C-terminal region of the heavy chain of BoNTB2. The best scFv targeting the C-terminal region of BoNT/B1 and BoNTB2 heavy chains, B2-7, demonstrated a high capacity to neutralize BoNT/B1 and BoNT/B2 in the ex vivo hemidiaphragmatic assay. A high identity (80%) between the framework regions of B2-7 and their human homologs was observed. ScFvs neutralizing BoNT/A1 by targeting its light chain were also isolated and among them, the scFv 2H8 induced a decrease of 50% in the endopeptidase activity at a concentration corresponding to a molar ratio of 2H8/BoNT/A1 of 64000. A high identity (88%) between the framework regions of 2H8 and their human homologs was also observed. Our strategy can be used to isolate other therapeutic antibody fragments.

Made available in DSpace on 2015-07-22T20:49:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-24 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A paracoccidioidomicose (PCM) é uma doença sistêmica de caráter granulomatoso, prevalente na América do Sul, causada pelo fungo termodimórfico Paracoccidioides brasiliensis. O fungo apresenta estrutura complexa de proteínas e glicoproteínas e dentre esses componentes, alguns estão envolvidos na patogenicidade da doença. A recuperação da virulência de isolados por meio de passagem in vivo foi realizada com sucesso em nosso laboratório. Os isolados Pb18 atenuado e virulento foram analisados sob diversos ângulos para confirmação dessa modificação. Os resultados de curva de sobrevida, recuperação de CFU de órgão e histologia, mostraram claramente as diferenças no padrão de patogenicidade desses dois isolados. Outras características também foram avaliadas como morfometria, padrão de crescimento e ultraestrutura celular. Análises de expressão gênica diferencial apontaram um perfil de regulação positiva para genes relacionados ao metabolismo de proteínas, de lipídeos e aminoácidos. Algumas moléculas anteriormente descritas como putativos fatores de virulência foram moduladas positivamante, entre as quais podemos citar a calmodulina, proteína kex-like e hsp70. Entretanto, ainda pouco se sabe sobre estes fatores virulência para maioria dos fungos dimórficos, entre eles o P.brasiliensis. Várias técnicas têm sido utilizadas, sem sucesso, para caracterização destas moléculas. Utilizando a metodologia de Phage display, foram selecionados três fagos que se ligam preferencialmente ao isolado Pb18 virulento. Por meio de ensaio de ligação, o fago p04 foi capaz de distinguir graus de virulência de outros quatro isolados. As imagens obtidas por microscopia confocal mostraram que o pep04, acoplado a 6-FAM, foi internalizado somente por leveduras do isolado virulento. Imagens seriadas indicam marcação do meio intracelular, frequentemente associada ou co-localizada à marcação por DAPI. Estes resultados demonstraram que ambos, pep04 e p04, podem ser considerados biomarcadores de virulência na PCM. Para avaliar as consequências das interações destes biomarcadores com células fúngicas, foram realizados ensaios in vitro e in vivo. O fago p04 foi suficiente para impedir a implantação e disseminação do fungo. Além disso, foi capaz de reduzir o número de CFUs recuperadas de animais tratados com este fago, quando comparado ao controle (fago sem inserto). Experimentos in vitro utilizando o pep04 demostraram a atividade fungicida deste peptídeo contra, apenas, o isolado virulento. Desta foram, estes biomarcadores poderão ser utilizados tanto no diagnóstico, quanto na pratica clínica como adjuvante terapêutico. / Paracoccidioidomycosis (PCM) is a human systemic granulomatous disease, prevalent in South America, caused by a thermodimorphic fungus, Paracoccidioides brasiliensis. This fungus presents complex antigenic structure and some of these components have been related with its pathogenicity, of which little is known. The virulence recovery of isolates by passage in vivo was performed in our laboratory. Attenuated and virulent Pb18 isolates were analyzed from various angles to confirm this change. The results of the survival curve, the number of CFUs and histology, showed clear differences in pathogenicity pattern of these isolates. Other features were also evaluated as morphology, growth curve and cell ultrastructure. Analysis of differential gene expression profile showed positive regulation genes related to metabolisms of proteins, lipids and amino acids. Some molecules, previously described as putative virulence factors, were positive regulated, among which calmodulin, kex-like protein and Hsp70. However, the number of defined virulence factors for dimorphic fungal pathogens, up to now, is relatively small. Several techniques have unsuccessfully been employed to characterize these elusive antigenic structures. Using phage display methodology, three peptide-displaying phages that bound preferentially to virulent isolates of P. brasiliensis were selected. By binding assay, p04 phage distinguished predefined degrees of virulence of isolates. Using confocal microscopy, the homologue synthetic peptide (pep04), labeled with 6-FAM, was internalized by only virulent isolate yeast cells. Sequential optical section imaging indicated that the labeling was within the intracellular milieu and frequently close or overlapping DAPI staining. These results showed that both, phage p04 and pep04, can be considered as biomarkers of virulence in PCM since both bound to virulent P. brasiliensis. To evaluate the consequences of interactions between the biomarkers and fungal cells, in vitro and in vivo experiments were performed. The latter demonstrated that p04 phage was sufficient to prevent the implantation of the fungus in the lungs and its migration to spleen and liver. In addition, this phage reduced colony-forming units in the lungs of mice infected with P. brasiliensis as compared to controls. In vitro experiments showed that pep04 exhibited fungicidal activity only against virulent P. brasiliensis, leaving unaltered the growth of the attenuated isolate. Therefore, these biomarkers may be useful tools for prognosis in PCM and may be possibly used in the routine clinical practice as therapeutic drug adjuvants. / TEDE / BV UNIFESP: Teses e dissertações

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.

The recent increase of affordable virtual reality (VR) head-mounted displays has led to many new video games and applications being developed for virtual reality environments. The improvements to VR technology has introduced many new possibilities, but has also introduced new problems to solve in order to make VR software as comfortable and as effective as possible. In this report, different methods of displaying text and receiving text input in VR environments are investigated and measured. An interactive user study was conducted to evaluate and compare the performance and user opinion of three different text display methods and four separate virtual keyboard solutions. Results revealed that the distance between text and user, with the same relative text size, significantly affected the ease of reading the text, and that designing a good virtual keyboard for VR requires a good balance of multiple factors. An example of such factors is the balance between precise control and the amount of physical exertion required. Additionally, the results suggest that the amount of previous experience with virtual reality equipment, and typing skill with regular physical keyboards, can meaningfully impact which solution is most appropriate. / Den senaste tidens ökning av prisvärda virtual reality (VR) glasögon har lett till en ökning av spel och applikationer som utvecklas för virtual reality miljöer. Förbättringarna av VR tekniken har introducerat många nya möjligheter, men även nya problem att lösa för att skapa VR mjukvara som är så bekväm och effektiv som möjligt. I den här rapporten undersöks och mäts olika metoder för att visa samt ta emot text i VR miljöer. Detta undersöktes genom utförandet av en interaktiv användarstudie som utvärderade och jämförde effektiviteten och användaråsikter kring tre olika metoder för att visa text samt fyra olika virtuella tangentbordslösningar. Resultatet visade att avståndet mellan användaren och texten, med samma relativa textstorlek, avsevärt påverkade lättheten att läsa texten, samt att designen av ett bra virtuellt tangentbord för VR kräver en bra balans mellan flera faktorer. Ett exempel på sådana faktorer är balansen mellan noggrann kontroll och den fysiska ansträngning som krävs. Resultatet tyder även på att mängden av tidigare erfarenhet med virtual reality utrustning samt skicklighet att skriva med vanliga fysiska tangentbord betydligt kan påverka vilka lösningar som är mest passande för situationen.

Tie1 é um receptor tirosina quinase expresso em células endoteliais importante em angiogênese, formação de vasos sanguíneos a partir de vasos pré-existentes. Este receptor pertence a uma família pequena composta por apenas dois membros (Tie1 e Tie2) para os quais angiopoietinas foram identificadas como ligantes de Tie2. No entanto, Tie1 continua a ser um receptor órfão, sem ligantes identificados até o momento. Sendo assim, é difícil compreender completamente as propriedades biológicas de Tie1 e seus mecanismos moleculares em angiogênese sem um ligante identificado. Entretanto, como sugerido através de estudos de deleção gênica, este receptor é uma molécula essencial na angiogênese, apresentando um papel importante no desenvolvimento da vascularização da retina e desenvolvimento de tumores. O nosso objetivo foi estudar a participação do domínio extracelular de Tie1 na neovascularização e, no processo, identificar possíveis ligantes para este receptor. Através da tecnologia de phage display, identificamos um peptídeo específico e seletivo para Tie1, sugerindo a existência de um sítio de ligação único neste receptor. Mostramos que este peptídeo é capaz de inibir a proliferação de células endoteliais induzida por Ang1, um ligante bem caracterizado de Tie2 que também modula a atividade de Tie1. Além disso, também mostramos que este peptídeo inibe a angiogênese in vivo num modelo animal bastante relevante para estudo de doenças humanas, o modelo da retinopatia induzida por oxigênio. Uma vez que este peptídeo liga-se a um sítio único e seletivo para Tie1, hipotetizamos que o mesmo poderia mimetizar possíveis ligantes naturais deste receptor. Para identificá-los, proteínas com mimetopo cruzado com este peptídeo foram identificadas em extrato proteico de diferentes linhagens celulares. Tais proteínas são possíveis candidatos a interação com Tie1. Em resumo, demonstramos que o domínio extracelular de Tie1 é importante para a angiogênese patológica e identificamos proteínas como possíveis ligantes deste receptor, o que poderá contribuir para um melhor entendimento da participação de Tie1 na formação de vasos. O peptídeo aqui identificado poderá ser ainda uma ferramenta útil para o desenvolvimento de novas terapias anti-angiogênicas com importantes aplicações à saúde humana. / Tie1 is a tyrosine kinase receptor expressed by endothelial cells and important in angiogenesis, the formation of new blood vessels from pre-existing ones. This receptor belongs to a small family of receptors composed of two members only (Tie1 and Tie2) to which angiopoietins have been identified as ligands for Tie2. On the other hand, Tie1 is still an orphan receptor with no ligand identified to date. Thus, it is difficult to assess Tie1 mechanism of action in neovascularization without a known ligand. Nevertheless, gene deletion studies have shown that Tie1 is essential in angiogenesis, and plays an important role in retinal and tumoral vascularization. The aim of our study was to evaluate the participation of Tie1 extracellular domain in angiogenesis, and in the process, to identify putative ligands for this receptor. Utilizing phage display, we have identified and characterized a Tie1 specific and selective ligand peptide, which suggests the existence of a binding site unique to this receptor and not shared by other family members. We show that this peptide prevents endothelial cells proliferation, induced by angiopoetin-1, a ligand for Tie2 but which also modulates Tie1 activity. Using a well-accepted mouse model for human diseases, the oxygen induced retinopathy model, we show that this peptide inhibits angiogenesis in vivo. Since this peptide maps to a unique binding site in Tie1, we hypothesized that it might mimic a natural ligand for this receptor. To identify them, proteins with cross reactive epitopes with an anti-peptide sera were identified by proteomic approaches. These proteins are thus possible ligands for Tie1. In summary, we have shown that Tie1 extracellular domain is important in angiogenesis and we have identified putative ligand for this receptor, which might contribute to a better understanding of the molecular mechanisms associated with Tie1 in blood vessel formation. The peptide here characterized may also be an important tool for the development of novel anti-angiogenesis therapeutic approaches for disesase with an angiogenic component.

This study addresses an individual's performance, relating it to eyesight changes due to the aging of the individual and to color computer screens used for computer-based-instruction not designed specifically for older students. This study determines how existing research in gerontology, human-computer interface, and color use in visual graphics can be applied to the design of computer screen displays containing color text and backgrounds and how various color combinations will affect performance by adult learners forty years of age and older. The results of this research provide software developers and instructional designers guidelines to use when designing computer screen displays for use in instructional computing settings involving older adults.