Thesis (Ph.D.)--University of Tennessee Health Science Center, 2001. / Title from title page screen (April 10, 2008). Research advisor: Harry W. Jarrett, Ph.D. Document formatted into pages (xv, 162 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 152-162).

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (site viewed July 9, 2007). PDF text: 146 p. : ill. UMI publication number: AAT 3245358. Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed July 9, 2007). PDF text: 318 p. : ill. UMI publication number: AAT 3249672. Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (M.S.)--Montana State University--Bozeman, 2004. / Typescript. Chairperson, Graduate Committee: Mary J. Cloninger. Includes bibliographical references (leaves 133-138).

The diagnostic and pharmaceutical industries rely on tools for characterizing, discovering, and developing bio-molecular interactions. Diagnostic assays require high affinity capture probes and binding specificity for accurate detection of biomarkers. Selection of drug candidates depends on the drug residency time and duration of drug action. Further, biologic drugs can induce anti-drug antibodies, which require characterization to determine the impact on the drug safety and efficacy. Label-free biosensors are an attractive solution for analyzing these and other bio-molecular interactions because they provide information based on the characteristics of the molecules themselves, without disturbing the native biological systems by labeling. While label-free biosensors can analyze a broad range of analytes, small molecular weight analytes (molecular weight < 1kDa) are the most challenging. Affinity measurements for small molecular weight targets require high sensitivity and long-term signal stability. Additional difficulties occur with different liquid refractive indices that result from to temperature, composition, or matrix effects of sensor surfaces. Some solutions utitlize strong solvents to increase the solubility of small molecules, which also alter the refractive index. Moreover, diagnostics require affinity measurements in relevant solutions, of various refractive indices. When a refractive index difference exists between the analyte solution and the wash buffer, a background signal is generated, referred to as the bulk effect, obscuring the small signal due to surface binding in the presence of large fluctuations due to variations of the optical refractive index of the solutions. The signal generated by low molecular weight analytes is small, and conventional wisdom tends toward signal amplification or resonance for detection of these small signals. With this approach, Surface Plasmon Resonance (SPR) has become the gold standard in affinity measurement technologies. SPR is an expensive and complex technology that is highly susceptible to the bulk effect. SPR uses a reference channel to correct for the bulk effect in post-processing, which requires high precision and sophisticated temperature control, further increasing the cost and complexity. Additionally, multiplexing is desirable as it allows for simultaneous measurements of multiple ligands; however, multiplexing is only possible in the imaging modality of SPR, which has lower sensitivity and difficulty with referencing. The Interferometric Reflectance Imaging Sensor (IRIS) is a low-cost, optical label-free bio-molecular interaction analysis technology capable of providing precise binding affinity measurements; however, limitations in sensitivity and usability have previously prevented its widespread adaptation. Overcoming these limitations requires the implementation of automation, compact and easy-to-use instrumentation, and increased sensitivity. Here, we explore methods for improved sensitivity and usability. We achieve noise reduction and elimination of solution artifacts (bulk effect) through engineered illumination uniformity and temporal and spatial image processing. To validate these methods, we experimentally analyze small molecule molecular interactions to demonstrate highly sensitive kinetic binding measurements, independent of solution refractive index. / 2023-09-24T00:00:00Z

No description available.

Affinity maturation increases the affinity of B-cell derived antibodies to their cognate antigens. In this study, we characterized the kinetic, structural, dynamic and thermodynamic evolution of antibodies during affinity maturation. Through single B-cell cell sorting, paired heavy and light chain sequencing, phylogenetic analysis, antibody expression, and physicochemical characterization, we were able to longitudinally analyze the stages of affinity maturation of anti-PA (B.anthracis protective antigen) antibodies. Following repeated immunizations, we observed up to an 10,000-fold increase in antibody affinity, mainly through a decrease in the off-rates. For detailed maturation analysis, we chose three antibodies lying along a single clonal branch--the clone’s unmutated common ancestor (UCA), a medium affinity antibody (MAAb) appearing after second immunization, and a high-affinity antibody (HAAb) appearing after third immunization. Most of the mutations that occur between the UCA and HAAb resulted in key changes to structural conformation. In particular, mutations change residues in the CDR-H3 region inducing the folding of the CDR-loops into a conformation that is more complementary to PA. This advantageous new antibody conformation is preserved in the unbound state, indicating that though the UCA and MAAb appear to use an induced fit and/or conformational selection mechanism, the HAAb is more rigidly lock-and-key. Thermodynamic results support this interpretation. In the first maturation step from UCA to MAAb, enthalpic improvement indicates optimization of noncovalent interactions. The second step from MAAb to HAAb predominantly involves entropic improvement by which the advantageous conformation made accessible in the first step is made more dominant via the narrowing of effectively accessible conformations, which allows better contact with PA. This is also reflected by a less significant improvement in the enthalpic component of PA-binding. Studies examining the evolving protein-dynamic characteristics further support this interpretation. In summary, we observed that a single energetic component is not responsible for increased affinity in the maturation pathways we studied. From UCA to MAAb, affinity increases through optimization of noncovalent interactions. From MAAb to HAAb, affinity increase is achieved through changes that stabilize the favorable conformation in the unbound state. A better understanding of affinity maturation can have implications for antibody engineering and vaccine development.

This thesis documents the design and implementation of an augmented reality (AR) application that could be extended to support group brainstorming tasks remotely. Additionally, it chronicles our investigation into the helpfulness of traditional collaborative cues in this novel application of augmented reality. We implemented IdeaSpace, an interactive application that emulates an affinity diagramming environment on an AR headset. In our application, users can organize and manipulate virtual sticky notes around a central virtual board. We performed a user study, with each session requiring users to perform an affinity diagramming clustering task with and without common collaborative cues. Our results indicate that the presence or absence of cues has little effect on this task, or that other factors played a larger role than cue condition, such as learning effects. Our results also show that our application's usability could be improved. We conclude this document with a discussion of our results and the design implications that may arise from them. / Master of Science / Our project was aimed at creating an app for modern augmented reality headsets that could help people perform group brainstorming sessions remotely from each other. We were also interested in finding out the benefits or downsides of some of the design decisions that recent research in remote augmented reality recommends, such as lines showing where a user is focusing and visualizations for a user's head and hands. In our app, which we dubbed IdeaSpace, users were faced with a virtual corkboard and a number of virtual sticky notes, similar to what they might expect in a traditional brainstorming session. We ran three-person study sessions comparing design techniques recommended by literature to an absence of such techniques and did not find they helped much in our task. We also found that our application was not as usable as we had hoped and could be improved in future iterations. We conclude our paper discussing what our results might mean and what can be learned for the future.

No description available.

Due to the rising health care costs and with the advent of biogenerics, there is a growing demand to develop new and reliable techniques to characterize proteins and biopharmaceuticals. In addition, characterization aids in understanding the intricate relationship between a protein's structure and its function. To address this challenge, two protein structural parameters, 1) amino acid surface area and 2) amino acid microstructure, were chosen to be investigated. Two chromatographic techniques, 1) ion exchange chromatography (IEC) and 2) immobilized metal affinity chromatography (IMAC), were used to characterize the above-mentioned protein structure parameters. The model protein chosen for our work is T4 lysozyme. The protein consists of 164 amino acids with molecular weight ~ 18 kD. SYBYL 7.1 software was used to generate in silico point mutants. Two categories of protein variants (point mutants) were generated using site-directed protein mutagenesis. The goal for generating point mutants was to obtain mutants that vary in the two structural parameters. The first category point mutants vary in the surface accessibility of a surface accessible histidine residue. The second category point mutants predominantly vary in protein net charge and the amino acid microstructure. In total, seventeen point mutants were generated: 1) category I consists of seven variants that vary predominantly in their histidine surface accessibility, and were obtained by replacing a charged amino acid residue at different locations on the surface of the protein molecule, and 2) category II consists of ten variants that vary in both net charge and charge distribution were obtained by replacing charged and neutral amino acid residues at different locations (different microenvironments) on the protein surface. PCR technique was used to generate the point mutants. Gene and protein sequencing were employed to confirm the veracity of point mutation. CD and Lysozyme activity assays were performed to determine whether or not the 3D structure of all the protein variants was intact. Zonal analysis was used to obtain the binding strength values of all seventeen variants in IMAC with copper as the immobilized metal ions, and gradient elution method was used to obtain the relative retention times (rRT) values of all the variants in IEC. The seven lysozyme variants generated in category I each contains one surface histidine residue. In IMAC, there is a correlation between the surface accessibility of the lone surface histidine and the protein's binding strength with R²⁺= 0.76. In IEC, the correlation between the protein's microstructure, which predominantly consists the surface accessibility of the histidine residue, and the protein's retention times was R²⁺= 0.95. However, there were few outlier variants (e.g. variant K83H) which did not follow the correlations. The variations presented by few outlier variants can be attributed to the presence of intramolecular bonds, which restrict the mobility of the amino acid side chains and subsequently hinder the specific interaction between the amino acid residue and chromatographic media. For category II variants, short and medium range charge perturbations around the sole histidine residue in T4 lysozyme were engineered within 15 Ã distance of histidine. There was a strong correlation (R²⁺ = 0.96) between the theoretical (DeltaDeltaGElec) values, calculated using simple Coulomb's law, and the experimental (DeltaDeltaGB) values, which were obtained by measuring the protein binding strength values using IMAC. Similar correlation (R²⁺= 0.93) was obtained between the change in net charge (-2 to +2 units) and the relative retention times in IEC. Similarly, there were few variants (e.g. S136K, R76D) that did not follow the trends. The deviations of the few outlier variants can be attributed to the presence of unique microstructure effects around the histidine residue. These microstructure effects were quantified in IMAC as (DeltaDeltaGMicro), and in IEC they were quantified by the change in rRT values. In summary, all seventeen variants had different binding strengths and rRT values indicating the variation in the protein structure around the histidine residue. Our work reveals that it is possible to capture the microstructural effects of a protein through the combination of protein molecular modeling and simple chromatographic experiments. / Ph. D.