The overall purpose of this work is to develop systematic methodology for the simultaneous design and selection of processes and molecules (materials). A propertybased approach is used to develop an interface between process and molecular design/selection. In particular, we focus on the problem of designing/selecting materials that are used in the context of a recycle/reuse system of process streams and for energy applications. Fresh and recycled resources (e.g., process streams, biomass, solvents, etc.) are integrated with the process to satisfy property-based constraints for the process units and to optimize the usage of the resources and the design of the process. For molecular design, property operators for mixing streams and group contribution methods (GCM) are used to consistently represent process sources, sinks, and different functional groups on the same property-base. For material selection, property based criteria (e.g., heat rate, high heating value, etc.) are used to bridge the process with material. This consistent representation enables the definition of the optimization problem formulation for product design while taking into consideration the recycle/reuse of process streams. In particular, this dissertation addresses four integrated topics. First, a new graphical approach for material targeting and substitution is presented. This graphical approach offers initial solutions and valuable insights that can be effectively used for conceptual design and for initializing mathematical programming techniques. Second, a mathematical optimization approach is developed along with a decomposition-based global solution procedure for material targeting and substitution using property integration. Third, an implementation approach is developed to synthesize the details of a recycle/reuse process network design based on the targets identified through the graphical and/or the mathematical approaches. Finally, property integration techniques are extended to a broader scope which deals with the lifecycle analysis of biomass utilization for energy generation. A generic model is developed to optimize the types and quantities of the feedstocks used to optimize power generation with biomass-fossil fuel co-fed system. Important issues of biomass growth, harvesting, transportation, processing, and disposal are included. Property-based tracking and constraints are included in the analysis. Also, the issues associated with greenhouse gas (GHG) emissions are incorporated in the analysis. Case studies are solved throughout the dissertation to demonstrate the applicability of the developed procedures.

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Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 185-189).

Single-molecule biosensors offer distinct advantages over their ensemble-averaged counterparts by being able to extract information related to rare targets and specific molecular configurations within a sample. In particular, solid-state nanopores embody a promising single-molecule technique that is based on detecting target molecules by the amount of ionic current they block as they pass through a nanoscale aperture across a thin membrane. In this thesis, I present extensions of the basic nanopore system aimed at addressing some of its main limitations at present, namely: 1) the low rates at which nanopores capture molecules from a bulk volume, which restricts their ability to work with dilute (≲ nM) samples, and 2) the difficulty in using nanopores to distinguish small or closely related molecules by their direct current blockage signatures alone. I begin by describing the design and construction of a nanopore-based instrument that integrates an optical detection channel in parallel with ionic current sensing. A particular emphasis was placed on minimizing the electrical noise contributions of the added optical equipment on the original ionic current channel. Measuring the optical signals of translocating molecules together with their current blockages can improve the discrimination of two fluorescently labelled targets (or two configurations of a single target) that normally produce similar ionic current signatures. I next investigate the combination of nanopore sensing with target pre-concentration, specifically, by embedding a nanopore membrane within a fluidic cell that features an insulator-based dielectrophoretic (iDEP) trap. Applying large (≳ 100 V) AC voltages across the iDEP channels of the cells resulted in the accumulation of polarizable targets (dsDNA, polystyrene beads) at the locations of the membranes, thus pointing toward a convenient method for the detection of ultra-dilute target samples in future nanopore devices. Finally, I introduce improved protocols for the synthesis and nanopore signal analysis of dsDNA-based molecular carriers. In a molecular carrier scheme, in order to enhance the target specificity of the system, target molecules are not sensed directly by a nanopore but instead interact specifically with secondary molecules (“carriers”) to recognizably alter the carrier translocation signals. Here, I present proof-of-principle analyses of DNA carrier experiments that highlight the multiplexing capabilities of our carrier design, which are based on separating targets by their interactions with carriers of different lengths. Developments of the nanopore sensing platform such as those presented in this work, which leverage the intrinsic versatility of solid-state nanopores to be integrated within complex devices and to detect a wide range of target molecules, will play an important role in continuing to increase the precision of single-molecule measurements into the future and to expand their breadth of potential applications.

Le transfert d'ADN chez les bactéries est un processus essentiel dans la ségrégation des chromosomes lors de la progression du cycle cellulaire et de nombreuses protéines sont impliquées dans ce processus. Parmi elles, on trouve la famille des protéines SpoIIIE / FtsK, et différentes fonctions leur ont été attribuées. SpoIIIE a été identifiée comme étant essentielle a la sporulation chez Bacillus subtilis. Au cours de la sporulation, un septum de division asymétrique se développe à proximité d'un pôle de la cellule et divise la cellule en deux compartiments : la préspore et la cellule mère. SpoIIIE est responsable de la translocation directionnelle de l'ADN de la cellule mère à la préspore. Ce transport implique l'interaction de SpoIIIE avec des séquences spécifiques qui sont distribuées de forme asymétrique le long des chromosomes (séquences de reconnaissance SpoIIIE ou SRS). Dans cette thèse, je développe des méthodes de molécule unique pour aborder les différents aspects des mécanismes de translocation de SpoIIIE. Cette thèse est divisée en trois sections : (1) les développements et de l'optimisation méthodologiques, (2) la caractérisation de sytox comme un nouveau colorant intercalant l ‘ADN pour les expériences en molécules uniques, et (3) l'utilisation de ces méthodes de molécules uniques pour tester les modèles de translocation d'ADN par SpoIIIE. Pour commencer, j'ai développé deux méthodes de détection et manipulation par molécules uniques: 1) le premier permet la visualisation simultanée de l'ADN et les protéines fluorescentes par microscopie TIRF et à épifluorescence, et (2) l'utilisation de pinces magnétiques dans une configuration transversale qui, couplée à la détection par fluorescence, permet la détection simultanée de l’ extension de l'ADN et la visualisation de la localisation des protéines. Au cours de la thèse, j'ai construit ces configurations optiques, les ai caractérisé, et optimisé. Deuxièmement, j’ai étudié le mécanisme de liaison et des propriétés de fluorescence de sytox, un nouveau colorant d’ADN. Plus précisément, j'ai déterminé que: (1) sytox se lie à l'ADN rapidement dans un processus en deux étapes séquentielles qui implique des interactions électrostatiques; (2) la dynamique rapide de liaison et de dissociation de sytox conduit à un taux extrêmement faible de photoblanchiment , (3) la dégradation de l'ADN par sytox est quatre fois inférieure à celle observée pour d'autres bis-intercalants, tels que YOYO-1, et 4) sytox est un intercalant d'ADN qui augmente la longueur de l'ADN de 43%, et n'affecte pas ses propriétés mécaniques (mesurée par la longueur de persistance). Enfin, pour observer l'interaction entre SpoIIIE et SRS, la protéine SpoIIIE a été chimiquement étiquetée et caractérisée. Substrats d'ADN contenant la séquence SRS ont été préparés et adaptés aux méthodes de molécule unique développées pendant ma thèse. L’observation directe des interactions SpoIIIE-SRS par ces méthodes ont permis de réfuter un des modèles existant. / DNA contains the genetic information of cells. Several cellular processes, including chromosome segregation during cell division and sporulation, and plasmid conjugation require the transport of double-stranded DNA (dsDNA) within and between bacterial cells. SpoIIIE/FtsK/Tra are a family of ring-shaped, membrane-anchored, ATP-fueled, directional motors required to segregate DNA across membranes during sporulation, cell division and conjugation. In particular, SpoIIIE is responsible for packaging the chromosome inside the prespore during the process of sporulation in Bacillus subtilis. This transport is directional and requires that SpoIIIE recognizes highly-skewed octameric sequences (SpoIIIE Recognition Sequences, or SRS) sparsely distributed along the whole chromosome. In this thesis, I developed different single-molecule methods to investigate the molecular mechanism by which SpoIIIE-SRS interactions lead to directional DNA transport. This thesis is divided in three sections: methodological developments and optimization, characterization of sytox as a new intercalating dye for single molecule experiments, and the use of single molecule methods to test the models for directional DNA translocation by SpoIIIE. First, I developed two single molecule methods that involved 1) the simultaneous visualization of DNA and protein by using intercalating dyes and direct protein labels to detect the localization of SpoIIIE on DNA using TIRF and epi-fluorescence microscopy; and (2) the use of a transverse magnetic tweezers setup coupled to fluorescence detection to simultaneously detect DNA extension and visualize protein localization. I built these optical setups, characterized them, and optimized several parameters. Secondly, we investigated the binding mechanism and fluorescence properties of sytox, a new bright, low photo-damage, multi-color DNA labeling agent. Specifically, I determined that: (1) sytox binds DNA rapidly in a two-step sequential process that involves electrostatic interactions; (2) the fast dynamics of binding and unbinding of sytox leads to an extremely low photobleaching rate; (3) DNA degradation by sytox is four-fold lower than that observed for other bis-intercalators, such as YOYO-1; and 4) sytox is a DNA intercalator that increases the DNA length upon binding by 43 %, while not affecting its mechanical properties (measured by the persistence length). Finally, to observe SpoIIIE-SRS interactions, SpoIIIE was chemically labeled and characterized. DNA substrates containing SRS sequence were prepared suitable for the different single molecule approaches undertaken and also characterized. Observation of SpoIIIE-SRS interactions allowed us to conclude that: (1) in the absence of SRS, SpoIIIE can bind DNA non-specifically (2) this first binding event does not require threading through the DNA end or assembly of monomers but rather the binding of a hexamer from an open to a closed conformation, (3) in the presence of ATP, SpoIIIE translocates on DNA and is predominantly located in DNA ends, and (4) can often condense DNA by looping, reconstituting the activity observed in magnetic tweezers assays, (5) when SRS sequences are present, SpoIIIE is redistributed from non-specific sites by a diffusional or 3D looping mechanism and locates SRS sequences where it remains bound with a higher affinity than to non-specific sequences.

L’ADN et l’ARN double brin (ADNdb, ARNdb) subissent des transitions de surétirement avec des forces d’environ 60 pN. Nous effectuons des mesures de force à l'aide d'un piège optique à double faisceau contenant deux billes reliées par une seule molécule. Un brin du duplex est attaché aux deux extrémités aux deux billes, tandis que l’autre brin n’est attaché qu’à une seule extrémité. Quatre cas différents sont comparés: ADNdb, ARNdb, hybride ARN- ADN avec ADN sous tension et hybride ADN- ARN avec ARN sous tension.Un surétirement se produit pour les quatre duplex. La différence la plus remarquable est que les ARNdb présentent un plateau lisse, alors que les autres duplex présentent des motifs en dents de scie. Nous constatons que les ARNdb s'étirent par un mécanisme différent et expliquons pourquoi cette propriété pourrait aider les structures d'ARN à s'assembler et à jouer leurs rôles biologiques.Un surétirement de l'hybride ARN-ADN libère progressivement un brin d'ARN. Les structures formées au sein de cet ARN naissant sont visibles dans le signal de force lors du re- recuit. Pour la première fois à notre connaissance, nous imitons donc et étudions le repliement de l'ARN co-transcriptionnel dans un test in vitro. En se concentrant sur le stade précoce de l’assemblage des grandes sous- unités ribosomales de E. coli (domaines I-II de l’ARNr 23S et des protéines r L4, L13, L20, L22, L24), on observe plus souvent un recuit partiel avec les protéines r. Nos résultats indiquent que les cinq protéines r de liaison précoce agissent comme des auxiliaires de repliement bien avant que l’ARN 23S complet ne soit transcrit. / Double-stranded DNA and RNA (dsDNA, dsRNA) undergo overstretching transitions at forces around 60 pN. We perform force measurements using a dual-beam optical trap that holds two beads linked by a single molecule. One strand of the duplex is attached at both extremities to the beads, while the other strand is attached only at one extremity. Four different cases are compared: dsDNA, dsRNA, RNA-DNA hybrid with DNA under tension, and DNA-RNA hybrid with RNA under tension. Overstretching occurs for all four duplexes. The most remarkable difference is that dsRNA exhibits a smooth plateau, while the other duplexes show saw-tooth patterns. We find that dsRNA overstretches by a different mechanism and explain why this property could help RNA structures to assemble and play their biological roles.Overstretching the RNA-DNA hybrid progressively liberates an RNA strand. Structures formed within this nascent RNA are seen in the force signal upon re-annealing. For the first time to our knowledge, we thus mimic and study co-transcriptional RNA folding in an in-vitro assay. Focusing on the early stage of E.coli large ribosomal subunit assembly (domains I-II of 23S rRNA and r-proteins L4, L13, L20, L22, L24), partial re-annealing is observed more frequently with r-proteins than without. Our results indicate that the five early- binding r-proteins act as folding helpers well before the entire 23S RNA is transcribed.

Tumor angiogenesis, the ability of tumor cells to stimulate blood vessel growth, is one the most critical steps of tumor progression. To support the growth of the expanding tumor, the “angiogenic switch” is turned on, which is often triggered by hypoxia (i.e., low oxygen)-mediated events such as expression of vascular endothelial growth factor (VEGF), causing normally quiescent endothelial cells to proliferate and sprout. An emerging picture of angiogenesis suggests that while governed by complex mechanisms, cell adhesion molecules (CAMs) plays a pivotal role in the regulation of angiogenesis. Our laboratory recently identified multiple previously unknown proteins including, transmembrane and immunoglobulin domain containing 1 (TMIGD1) and immunoglobulin-containing and proline-rich receptor 1 (IGPR1). Immunoglobulin-containing and cysteine-rich receptor 1 (IGCR1) represents the third remember of IGPR-1 family proteins. To investigate the expression and function of IGCR1, we have developed a rabbit polyclonal anti-IGCR1 antibody and demonstrated that IGCR1 is expressed in the endothelial cells of human blood vessels. To examine possible function of IGCR1, we have generated porcine aortic endothelial (PAE) cells over-expressing IGCR1. We demonstrate that IGCR1 expression in PAE cells inhibited cell proliferation and capillary tube formation as measured by colorimetric MTT and matrigel tube formation assays, respectively. In contrast, over-expression of IGCR1 in PAE cells inhibited cell migration as measured by wounding assay. Taken together, this study identifies IGCR1 as a novel regulator of angiogenesis. Given, angiogenesis is a highly coordinated cellular processes controlled spatially and temporally by a myriad of cell surface receptors and ligands, IGCR1 by modulating the rate of endothelial cell proliferation and migration, plays a significant role in the formation of blood vessels. / 2018-07-11T00:00:00Z

Charge transport dynamics through molecular scale materials is of common interest to both scientific and engineering disciplines. Putting molecules on nanoscale break junctions is the most straightforward setup to study charge transport dynamics through single molecules. Electromigration process can provide a simple and easy method of forming metallic oxide nanogap junctions. By using silver oxide thin films to form such nanogap junctions, silver nanoclusters (Ag2~Ag8) are also formed in-situ within the junctions. Formed silver nanoclusters strongly and stably electroluminesce under DC, AC, and customized pulse train excitation. By detecting extremely sensitive feedback, i.e. photons, two interesting behaviors of single molecule charge transport dynamics were revealed: 1) asymmetric charge transport and 2) discrete energy level tunneling. The discrete energy level tunneling of field emitted electrons yields negative differential resistance (NDR). Combined with photoconductivity and optical reduction of silver oxide to form silver nanoclusters, junction-asymmetry and NDR can be very useful in both electronic and optoelectronic applications such as on-demand electronics fabrication, single photon sources, and nanoscale photon detectors.

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