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This research effort was directed at the development of legibility prediction models for flat-panel electronic displays used under typical office illumination (i.e., fluorescent) and various phases of daylight illumination. The models are intended to permit the manipulation of candidate display technologies and pertinent display characteristics such that legibility is maximized with respect to specified viewing conditions. Further, the legibility prediction models are intended to serve as an aid to the display designer in the intelligent selection of a display device for a known application. Critical legibility parameters were identified and relevant display measurement techniques were developed. A multiple degree of freedom daylight simulation apparatus capable of simulating the desired office and daylight illumination was designed and constructed. Reading speed performance data was collected from 14 subjects representative of both an age 18 to 30 group and an age 50 to 70 group. Each subject performed a numeral identification task using seven candidate flat-panel display technologies under 120 different symbol subtense/illumination/viewing angle arrangements. In addition, complete display characterization through microphotometric and microradiometric measurements was conducted for the seven flat-panel technologies under each of the viewing conditions. The results of the experiment revealed significant differences and complex interactions among the display technologies and viewing conditions which provided an excellent basis for the development of legibility prediction models. Extensive regression analyses were performed in which the human performance data was compared to both the critical legibility parameters and the display characterization measurements. Multiple sets of technology-based legibility prediction equations were successfully derived for each age group along with a generalized flat-panel display legibility prediction model. The models developed permit the evaluation and comparison of candidate display devices based on predicted reading speed as an indicant of legibility for a specified viewing environment. The required display characterization measurements can be collected in the field or under simulated conditions using commercially available hand-held measurement devices. In the absence of specific measures, careful estimation through comparison with similar display measurements contained in this report may be acceptable for certain applications. In their final form, the legibility prediction models serve as an aid in the evaluation and selection of electronic displays for a known application. In addition, the generalized legibility prediction equation developed holds the potential for application to other similar display technologies beyond those flat-panel candidates examined in the present study. Further, use of the models may permit the evaluation of new displays and display concepts without the immediate need for purchase, prototype construction, or complicated simulation. / Ph. D.

Multiple hurdles, such as drug solubility, stability, and physical barriers in the body, hinder bioavailability of many promising therapeutics. Polymeric nanocarriers can encapsulate the therapeutics to protect non-target areas from side effects but also protect the drug from premature degradation for increased circulation and bioavailability. To capitalize on these advantages, the polymer nanoparticle must be properly engineered for increased control in size distribution, therapeutic encapsulation, colloidal stability, and release kinetics. However, each application requires a specific set of characteristics and properties. Being able to tailor these by manipulation of different design parameters is essential to optimize nanoparticles for the application of interest. This study of nanoparticle fabrication and characterization takes us a step closer to building effective delivery systems tailored for specific treatments. Poly(ethylene oxide)-b-poly(D,L-lactic acid) (PEO-b-PDLLA) based nanoparticles were produced to range from 100-200 nm in size. They were fluorescently labeled with a hydrophobic dye 6-13 bis(triisopropylsilylethynyl) pentacene (TIPS pentacene) at an optimal loading of 0.5 wt% with respect to the core. Surfaces were successfully coated with streptavidin to be readily functionalized with various biotinylated compounds such as PD-L1 antibodies or A488 fluorophore. Using the same PEO-b-PDLLA, iron oxide and a conjugated polymer poly(2- methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene) (MEH-PPV) were co-encapsulated to form fluorescently labeled magnetic particles. Using poly(lactic-co-glycolic acid), CRISPR-Cas9 plasmids were encapsulated at 1.6 wt% and most of the payload released within the first 24 hours. The incorporated plasmids were intact enough to have mammalian macrophages successfully express the bacterial protein Cas9. Using similar PLGA based particles, the surface was functionalized with streptavidin and bound to the surface of bacteria as an active carrier for increased penetration of solid tumors averaging ~23 particles per bacterium. PEO-b-PLGA based particles were used in conjunction with a hydrophobic salt former to encapsulate a peptide designed to reduce platelet binding to cancer cells and mitigate extravasation. The peptide encapsulated was increased from < 2 wt% without salt former to 8.5 wt% with the used of hexadecyl phosphonic acid. Although the applications across these projects can be broad, the fundamentals and important design parameters considered contribute to the overarching field of effective carriers for drug delivery. / Ph. D. / There are many reasons why many promising pharmaceutical formulations never make it through regulation and onto market, including low solubility of the drug, low absorbance by the body, and harmful side effects, to name a few. Using polymer drug carriers, these difficulties can be overcome by holding the drug in a more soluble carrier, releasing it on a certain timeline or to a specific location to increase absorbance and decrease side effects. When designing a carrier, the requirements for the product are dependent on the application and the disease of interest. This work looks at the material types and conditions during particle formation to see how it affects the final product to better define and understand how these parameters change the performance. This work shows that the carrier size can be manipulated depending on how much of one material is used versus the other, they can be labeled to fluoresce so they can be tracked during cell and animal studies, and they can be coated with targeting compounds on the surface to increase the specificity of the carrier to localize to a target location of interest. Different particles containing DNA for gene editing, peptides for cancer therapies, and magnetic iron oxides to increase transport across difficult cell barriers have all be fabricated and characterized. The lessons learned through these projects will help guide future work to more effective and efficient delivery of pharmaceuticals to the body.

Yes / We report a simple temperature-responsive bioconjugate system comprising superfolder green fluorescent protein (sfGFP) decorated with poly[(oligo ethylene glycol) methyl ether methacrylate] (PEGMA) polymers. We used amber suppression to site-specifically incorporate the non-canonical azide-functional amino acid p-azidophenylalanine (pAzF) into sfGFP at different positions. The azide moiety on modified sfGFP was then coupled using copper-catalyzed “click” chemistry with the alkyne terminus of a PEGMA synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization. The protein in the resulting bioconjugate was found to remain functionally active (i.e., fluorescent) after conjugation. Turbidity measurements revealed that the point of attachment of the polymer onto the protein scaffold has an impact on the thermoresponsive behavior of the resultant bioconjugate. Furthermore, small-angle X-ray scattering analysis showed the wrapping of the polymer around the protein in a temperature-dependent fashion. Our work demonstrates that standard genetic manipulation combined with an expanded genetic code provides an easy way to construct functional hybrid biomaterials where the location of the conjugation site on the protein plays an important role in determining material properties. We anticipate that our approach could be generalized for the synthesis of complex functional materials with precisely defined domain orientation, connectivity, and composition. / Engineering and Physical Sciences Research Council (EPSRC), University of Warwick, National Science Foundation (U.S.) (NSF), United States. Defense Advanced Research Projects Agency (DARPA), Seventh Framework Programme (European Commission) (FP7), European Research Council (ERC)

<div> <p>Fluorescent proteins and the biosensors created with them have been used extensively to monitor chemical species inside and outside of the cell. They have been used to increase our knowledge of cellular function in normal and diseased states. Fluorescent biosensors are advantageous because they can be genetically encoded, do not require exogenous reagents, and can be quantitative. Fluorescent biosensors are also able to measure analytes with high spatial and temporal resolutions, enabling measurements at the scale of physiological events. In this thesis efforts have made to increase the available fluorescent biosensor tools for imaging cellular events. This work includes creation of new sensors for two molecules not yet detectable via fluorescent protein biosensor, acetylcholine and adenosine diphosphate. Efforts were also made to improve the current available biosensors for adenosine triphosphate and cellular redox, to make them more compatible with multiplex and deep tissue imaging. Here I present my work to design, characterize and utilize these fluorescent biosensors.</p> </div> <br>

Durant cette thèse nous avons, dans un premier temps, mis au point un protocole robuste pour quantifier l’échange de ligands à la surface de nano-objets, en prenant les quantum dots comme particules d’étude. Cette méthode se base sur la sensibilité de détection d’un élément radioactif, le tritium. Pour étudier l’échange de ligands, nous avons synthétisé de l’acide oléique tritié, dont l’équivalent non-marqué est un composé très couramment utilisé dans la chimie des QD. Nous avons élaboré un protocole permettant de déterminer la densité de ligands à la surface de quantum dots de type CdSe zinc blende par une méthode innovante. Par ailleurs, nous avons réalisé une étude permettant de déterminer la capacité qu’a un ligand à remplacer l’acide oléique présent à la surface des QD. On a ainsi obtenu une échelle des forces relatives de liaison de diverses fonctions chimiques suivant leur aptitude à déplacer l’acide oléique. Cette étude devrait permettre d’améliorer la compréhension et la mise au point des protocoles utilisés pour la préparation des QD. Dans un second temps, nous avons développé deux stratégies d’encapsulation de QD afin de les solubiliser dans l’eau avec comme objectif des applications en imagerie in vivo. Ces deux approches tentent de répondre aux nombreuses exigences liées à l’utilisation de QD en milieux biologiques. Dans cette optique, nous avons synthétisé des amphiphiles polymérisés d’une part et des amphiphiles perfluorés d’autre part. Des lots de QD, de type CdSe/CdS/ZnS émettant dans le visible, solubilisés selon ces deux stratégies ont été préparés et leur stabilité éprouvée dans plusieurs conditions. Nous avons ensuite appliqué l’encapsulation à des QD de type CuInS2/ZnS émettant dans le proche infrarouge. Des études préliminaire sont pu être réalisées par d’imagerie de fluorescence chez la souris afin d’évaluer in vivo ces nouvelles formulations de QD. / In this thesis we have, at first, developed a robust protocol to quantify the exchange of ligands on the surface of nano-objects, using quantum dots in this study. This method is based on the detection sensitivity of a radioactive element, tritium. To study the exchange of ligands, we synthesized tritiated oleic acid, whose unmarked equivalent is commonly used for the synthesis of QD. Thanks to an innovative method, we have developed a protocol to determine the density of ligands on the surface of zinc blende CdSe quantum dots. In addition, we conducted a study to determine the ability of a ligand to replace oleic acid on the surface of the QD. A scale of relative bond strengths of various chemical functions according to their ability to displace oleic acid has been obtained. This study is expected to improve the understanding and the development of protocols for the preparation of QD.In a second step, we developed two encapsulation strategies of QD in order to obtain colloidal stability in water with in vivo imaging applications as final aim. Both approaches attempt to answer the requirements for the use of QD in biological media. In this context, we have synthesized photopolymerisable amphiphilic compounds on the one hand and perfluorinated amphiphilic compounds on the other. Batches of CdSe/CdS/ZnS QD emitting in the visible, solubilized by these two strategies have been prepared and their stability tested in several conditions. We then applied the encapsulation strategy developed to CuInS2/ZnS QD emitting in the near infrared. Some preliminary studies have been carried out by in vivo fluorescence imaging in mice to assess these new QD formulations.

Two types of fluorescent molecules had been designed and synthesized to improve the analytical infrastructure of capillary electrophoretic separations. First, a hydrophilic version of the permanently cationic acridine-based fluorophore, HEG2Me2-DAA was synthesized. HEG2Me2-DAA has a lambda^ex max of 490 nm which matches the 488 nm line of the commonly used argon ion laser. The emission spectra of HEG2Me2-DAA are pH-independent. HEG2Me2-DAA was used in capillary electrophoresis with an aqueous background electrolyte and was found to be free of the detrimental peak tailing of the acridine orange-based fluorophore that was caused by adsorption on the inner wall of the fused silica capillary. Bovine serum albumin was labeled with excess of the designed amine reactive reagent and the lowest concentration at which the tagged bovine serum albumin was tested was 15 nM. Chicken ovalbumin was also labeled with FL-CA-PFP and analyzed by capillary isoelectric focusing (cIEF) with LIF detection. The pI values of the tagged proteins shifted in the alkaline direction by about 0.02 compared to the pI values of the non-tagged proteins. A tri-functional probe intended to enable selective enrichment and selective detection of a variety of molecules (e.g., natural products, pharmaceuticals, inhibitors, etc.) was also designed and synthetized by combining FL-CA with biotin and an azide group in a "proof-of-principle" level experiment. In cIEF, the profile of the pH gradient can only be determined with the help of pI markers. A large set of pyrene-based fluorescent pI markers was rationally designed to cover the pI range 3 to 10. To prove the feasibility of the proposed synthetic approach, the subgroup of the pI markers having the greatest structural complexity was synthesized and characterized. The classical zone electrophoretic pI determination methods failed due to severe chromatographic retention of the APTS based pI markers on the capillary wall. Exploratory work was done to design a new pI value determination method that combines the advantages of the immobilized pH gradient technology of the OFFGEL instrument and the carrier-ampholyte-based IEF technology. The method aspects of cIEF have also been improved in this work. The new segmented loading method yielded a more linear pH gradient than the previously known cIEF methods. To exploit a unique property of the newly developed fluorescent pI markers, we used them as pyrene-based ampholytic carbohydrate derivatizing reagents. The pI4 carbohydrate derivatization reagent proved advantageous over 8-aminopyrene-1,3,6-trisulfonic acid (APTS): the pI4 conjugates have higher molar absorbance at 488 nm than the APTS conjugates and become detectable in positive ion mode of MS affording better detection sensitivity.

Nuclear receptors are ligand activated transcription factors, where upon binding with small molecule ligands, these proteins are involved in the regulation of gene expression. To date there are approximately 48 human nuclear receptors known, involved in multiple biological and cellular processes, ranging from differentiation to maintenance of homeostasis. Due to their critical role in transcriptional regulation, these receptors are implicated in several diseases. Currently, 13% of prescribed drugs in the market are NR ligands for diseases such as cancer, diabetes and osteoporosis. In addition to drug discovery, the mechanism of function, mobility and trafficking of these receptors is poorly understood. Gaining insight into the relationship between the function and /or dysfunction of these receptors and their mobility will aid in a better understanding of the role of these receptors. The green fluorescent protein (GFP) has revolutionized molecular biology by providing the ability to monitor protein function and structure via fluorescence. The fluorescence contribution from this biological marker is the chromophore, formed from the polypeptide backbone of three amino acid residues, buried inside 11-stranded â-barrel protein. Synthesis of GFP derivatives of is based on the structure of the arylmethyleneimidazolidinone (AMI), creating a molecule that is only weakly fluorescent. Characterizing these AMI derivatives for other proteins can provide a powerful visualization tool for analysis of protein function and structure. This development could provide a very powerful method for protein analysis in vitro and in vivo. Development of such fluorescent ligands will prove beneficial for the nuclear receptors. In this work, libraries of AMIs derviatives were synthesized by manipulating various R groups around the core structure, and tested for their ability to serve as nuclear receptor ligands with the ability to fluoresce upon binding. The fluorogens are developed for steroidal and non-steroidal receptors, two general classes of nuclear receptors. Specific AMIs were designed and developed for steroid receptor estrogen receptor á (ERá). These ligands are showed to activate the receptor with an EC50 of value 3 ìM and the 10-fold activation with AMI 1 and AMI 2 in comparison to the 21-fold activation observed with natural ERá ligand, 17â-estradiol. These novel ligands were not able to display the fluorescence upon binding the receptor. However, fluorescence localized in nucleus was observed in case of another AMI derivative, AMI 10, which does not activate the receptor. Such ligands open new avenues for developing fluorescent probes for ERá that do not involve fluorescent conjugates attached to a known ERá ligand core. AMIs were also characterized for non-steroidal receptors,specifically the pregnane x receptor (PXR) and retinoic acid receptor á (RARá). To date, fluorogens which turn fluorescence upon binding and activate the receptor have not been developed for these receptors. With respect to PXR, several AMI derivatives were discovered to bind and activate this receptor with a fold-activation better than the known agonist, rifampicin. The best characterized AMI derivative, AMI 4, activates the receptor with an EC50 of value 6.3 ìM and the 154-fold activation in comparison to the 90-fold activation and an EC50 value of 1.3 ìM seen with rifamipicin. This ligand is not only able to activate PXR but also displays fluorescence upon binding to the receptor. The fluroscence pattern was observed around the nucleus. Besides AMI 4, 16 other AMI derivatives are identified that activate PXR with different activation profiles. Thus, a novel class of PXR ligands with fluorescence ability has been developed. The AMI derivatives able to bind and activate RAR, also displayed activation profiles that were comparable to the wild-type ligand, all trans retinoic acid. These ligands activated the receptor with an EC50 value of 220 nM with AMI 109 in comparison to an EC50 value of 0.8 nM with the natural ligand for RARá. When these ligands were tested for fluorescence in yeast, the yeast were able to fluoresce only in the presence of the receptor and the AMI derivative, indicating that these agonists also have the ability to fluoresce.

The use of polymers in electro-optical devices, especially light-emitting diodes (OLEDs), has become very popular in recent years, due to their ease of processability. The major drawback of using polymers in these systems is their time-consuming synthesis when trying to improve upon their physical properties. For example, each time a new color or better conducting properties are desired, a new monomer must be synthesized. To circumvent these problems, the system described in this work is designed to connect the well-known chromophore aluminum tris(8-hydroxyquinoline) (Alq3) to a norbornene monomer unit, followed by polymerization using ring-opening metathesis polymerization (ROMP), thus allowing for the processability of a polymer while maintaining the fluorescent properties of the metalloquinolate. The benefit of this system is that the monomers can be easily altered in order to tune color emission or to enhance the polymer properties. Some of the alterations include changing the metal center from aluminum to zinc in order to improve electron injection, adding substituents to the 8-hydroxyquinoline ligand in order to tune the emission color, and copolymerizing the Alq3-monomer with other norbornene monomers containing either a hole- or an electron-transport material side-chain to improve conductivity. These alterations lead to improved device performance and, more importantly, to a new method of designing polymeric systems for use in electronic devices.