Fetal alcohol syndrome (FAS) is a condition caused by prenatal alcohol exposure. The diagnosis of FAS is based on the presence of central nervous system impairments, evidence of growth abnormalities and abnormal facial features. Direct anthropometry has traditionally been used to obtain facial data to assess the FAS facial features. Research efforts have focused on indirect anthropometry such as 3D surface imaging systems to collect facial data for facial analysis. However, 3D surface imaging systems are costly. As an alternative, approaches for 3D reconstruction from a single 2D image of the face using a 3D morphable model (3DMM) were explored in this research study. The research project was accomplished in several steps. 3D facial data were obtained from the publicly available BU-3DFE database, developed by the State University of New York. The 3D face scans in the training set were landmarked by different observers. The reliability and precision in selecting 3D landmarks were evaluated. The intraclass correlation coefficients for intra- and inter-observer reliability were greater than 0.95. The average intra-observer error was 0.26 mm and the average inter-observer error was 0.89 mm. A rigid registration was performed on the 3D face scans in the training set. Following rigid registration, a dense point-to-point correspondence across a set of aligned face scans was computed using the Gaussian process model fitting approach. A 3DMM of the face was constructed from the fully registered 3D face scans. The constructed 3DMM of the face was evaluated based on generalization, specificity, and compactness. The quantitative evaluations show that the constructed 3DMM achieves reliable results. 3D face reconstructions from single 2D images were estimated based on the 3DMM. The MetropolisHastings algorithm was used to fit the 3DMM features to 2D image features to generate the 3D face reconstruction. Finally, the geometric accuracy of the reconstructed 3D faces was evaluated based on ground-truth 3D face scans. The average root mean square error for the surface-to-surface comparisons between the reconstructed faces and the ground-truth face scans was 2.99 mm. In conclusion, a framework to estimate 3D face reconstructions from single 2D facial images was developed and the reconstruction errors were evaluated. The geometric accuracy of the 3D face reconstructions was comparable to that found in the literature. However, future work should consider minimizing reconstruction errors to acceptable clinical standards in order for the framework to be useful for 3D-from-2D reconstruction in general, and also for developing FAS applications. Finally, future work should consider estimating a 3D face using multi-view 2D images to increase the information available for 3D-from-2D reconstruction.

Sensor technologies have the potential to provide a wealth of opportunities for detection and monitoring across the landscape in clinical, food, environmental, and biothreat/biowarfare analyses. Immobilization of biosensors on or in a functional material is critical for subsequent device development and translation to wearable technology. Upon receptor discovery, integration of receptor and transducer determines the performance of the electrochemical biosensor. First, immobilization of biosensors on surfaces is a key step towards development of devices for real world applications. The preparation, characterization, and evaluation of a surface bound transcription factor – nucleic acid complex for analyte detection as an alternative to conventional systems employing aptamers or antibodies are described. The sensor consists of a gold surface modified with thiolated Cy5 fluorophore-labeled DNA and an allosteric transcription factor (TetR) linked to a quantum dot. Upon addition of anhydrotetracycline (aTc) – the target analyte – the TetR-QDs release from the surface-bound DNA, resulting in loss of the Förster resonance energy transfer (FRET) signal. The sensor responds in a dose-dependent manner over the relevant range of 0-200 µᴍ aTc with a limit of detection of 80 nᴍ. The fabrication of the sensor and the subsequent real-time quantitative measurements establish a framework for the design of future surface-bound, affinity-based biosensors using allosteric transcription factors for molecular recognition. Second, the development and assessment of an immobilized quantum dot - transcription factor - nucleic acid complex for progesterone detection as a first step toward such device integration are presented. The sensor is composed of a polyhistidine-tagged transcription factor linked to a quantum dot and a fluorophore-modified cognate DNA and embedded within a hydrogel as an immobilization matrix. The hydrogel is optically transparent, soft, and flexible as well as traps the quantum dot - transcription factor DNA assembly but allows free passage of the analyte, progesterone. Upon progesterone exposure, DNA dissociates from the quantum dot - transcription factor DNA assembly resulting in an attenuated ratiometric fluorescent output via Förster resonance energy transfer. The sensor performs in a dose-dependent manner with a limit of detection of 55 nM. Repeated analyte measurements are also similarly successful. Our approach combines a systematically characterized hydrogel as an immobilization matrix and a transcription factor – DNA binding as a recognition/ transduction element, offering a promising framework for future biosensor devices based upon an allosteric transcription factor. Lastly, the lack of available and functionally validated enzymes is prohibiting the development of redox-based sensors for important analytes. Nicotine is one such analyte of interest, is present in tobacco products, and is one of the most heavily used addictive stimulants. Today, cigarette smoking prevails in 1.1 billion people, causes about 8 million deaths a year worldwide (World Health Organization). It is the leading preventable cause of disease, disability, and death. Cigarette smoking takes a tremendous economic toll, costing the United States nearly 170 billion annually on healthcare. Thus, the detection, quantification, and monitoring of nicotine, the substance responsible for the dependence-forming properties of tobacco product use, is of substantial interest. Herein, the development and assessment of an electrochemical nicotine biosensor, using a known nicotine catabolizing redox enzyme are presented. / 2023-01-19T00:00:00Z

Includes abstract. / Includes bibliographical references. / The cerebral cortex is composed of a thin layer of Grey Matter (GM), functionally subdivided into discrete regions which are connected in a large scale network via White Matter (WM) tracts. With fMRI (Functional Magnetic Resonance Imaging) it is possible to identify cortical regions involved in specific tasks, and with DTI (Diffusion Tensor Imaging) the structural connections between these areas can be mapped. The aim of this thesis is to to identify and track only those WM tracts entering and leaving a GM Region Of Interest (ROI) defined by fMRI.

Motor recovery of hand function in stroke patients requires months of regular rehabilitation therapy, and is often not measured in a quantitative manner. The first goal of this project was to design a system that can quantitatively track hand movements and, in practice, related changes in hand movements over time. The second goal of this project was to acquire hand and finger movement data during functional imaging (in our case we used magnetoencephalography (MEG)) to be used for characterizing cortical plasticity associated with training. To achieve these goals, for each hand, finger flexion and extension were measured with a data glove and wrist rotation was calculated using an accelerometer. To accomplish the first goal of the project, we designed and implemented Matlab algorithms for the acquisition of behavioral data on different handgrips, specifically power and precision grips. We compiled a set of 52 objects (26 man-made and 26 natural), displayed one at the time on a computer screen, and the subject was asked to form the appropriate handgrip for picking up the object image presented. To accomplish the second goal, we used the setup described above during an MEG scanning session. The timescales for the signals from the glove, accelerometer, and MEG were synchronized and the data analyzed using Brainstorm. We validated proper functionality of the system by demonstrating that the glove and accelerometer data during handgrip formation correspond to the appropriate neural responses.

No description available.

Antibody drug conjugates (ADCs) are a rapidly expanding class of therapeutics that combine the targeting capabilities of monoclonal antibodies with the cytotoxicity of highly potent chemotherapeutics. Six of the ten current FDA approved ADCs received approval in the past two years alone. However, all current formulations of ADCs rely on drug conjugation methods that result in heterogeneous ADC populations, both in terms of the number of drugs conjugated to the antibody (drug to antibody ratio, DAR) and the site of conjugation. Variability in DAR and heterogeneity in the sites of conjugation both affect the pharmacokinetics and efficacies of these molecules. There is a significant need for a site specific and stoichiometric method of conjugation that is stable in plasma and does not require the use of harsh reaction conditions which could lead to decreased antibody affinity or downstream toxicity. Supramolecular assembly through the use of coiled-coil domains has been demonstrated through the formation of fibers, hydrogels, peptide “origami,” multimeric single chain variable fragments (scFvs), and split, universal, and programmable chimeric antigen receptor (SUPRA CAR) T cells. Here, we describe the use of a heterotetrameric coiled-coil system in which one set of coiled-coil peptides is expressed on the C termini of an antibody and the other set is chemically conjugated to the highly cytotoxic microtubule inhibitor monomethyl auristatin E (MMAE). The peptides are either positively charged or negatively charged to discourage homotetramerization, but when mixed under physiological conditions, will form a heterotetrameric coiled-coil. In this way, we control the loading of two drug molecules onto the antibody in a site-specific manner. We explore the use of this coiled-coil system for the treatment of non-small cell lung cancer (NSCLC), which only has a 25% five-year survival rate. GPR87 is a receptor overexpressed in NSCLC in both adenocarcinoma and squamous cell carcinoma, as we demonstrate by the staining of primary NSCLC patient samples. We have prepared an αGPR87-MMAE ADC using coiled-coil based supramolecular assembly. Our ADC is non-cytotoxic to a control GPR87 negative cell line and exhibits in vitro IC50s of 6.3 nM and 9.0 nM in the NSCLC cell lines NCI-H358 and NCI-H520, respectively. Preliminary in vivo experiments using the corresponding antibody fluorophore conjugate (AFC) show higher accumulation of the AFC in a stage 4 NSCLC patient derived xenograft tumor vs. other major organs, such as the liver, kidney, heart, and lungs, 72 hours following injection. / 2023-09-24T00:00:00Z

Post-surgical abdominal adhesions, initially a fibrinous matrix linking two organs or tissues which matures to scar tissue, form after nearly 90% of abdominal or pelvic surgeries. Adhesions cause a number of serious post-operative complications, including small bowel obstructions, female infertility, chronic pain, and reoperation complications. Adhesions typically are not diagnosed until long after formation when the patient presents complication-related symptoms. Both the diagnosis and treatment require another invasive surgery and these procedures lead to further adhesion formation. This inability to non-invasively assess adhesion formation means that most promising anti-adhesion agents do not advance to human trials. To address this problem, we have developed a polymerized-shell microbubble (PSM) as an ultrasound (US) contrast agent to target fibrin, an early marker of adhesion formation that is not present in the healthy peritoneum. We have characterized the in vitro stability and fibrin binding properties of these PSMs. For adhesion applications, microbubbles require long term stability on the order of days. We can modify the degree of shell polymerization of the PSMs to achieve stability up to 1 week. Achieving long-term stability through shell modification impacts the acoustic properties of the microbubbles, therefore, there is a trade-off between bubble stability and echogenicity. Furthermore, the fibrin-binding dynamics of our targeted peptide are not well understood, and we have performed systematic studies on this property. Our results show these fibrin-targeted PSMs can be successfully fabricated with two methods of production. We have shown the long-term stability of these PSMs and that they bind selectively to fibrin. All together, these studies indicate that fibrin-targeted PSMs can both aid in the early diagnosis of surgical adhesions and help overcome this major roadblock preventing clinical trials. / 2023-09-24T00:00:00Z

The Covid-19 pandemic has brought to the world’s attention how a pandemic can burden hospitals all over the world, especially in regions where the healthcare system is already overburdened and low on resources. Low-resource settings would benefit considerably from point of care tests that can generate quick results, which would allow hospitals and/or clinics to properly allocate resources for treatment. In this thesis, we designed a blood separation system that: separates plasma from whole blood efficiently and is cheaper and more efficient than the gold standard of centrifugation. The blood separation system allows for the pooling and collection of plasma which can then be used to run a diagnostic test. A centrifuge separates blood by creating a gravity field generated by high-speed rotation. In this study, we used fidget-spinners, toys that can be manually spun with the flick of the finger, to generate a gravitational field strong enough to separate plasma from whole blood. We also designed an automated spinner, made of affordable parts, to make the fidget-spinner method of separating blood more efficient and easier to use. The spinner includes a 12V DC motor that can spin at up to 12000 rpm with no load and 3D printed parts that attach to the motor and hold the blood samples. Blood volumes ranging from 25 – 100 μL were used to test the separation methods. Using the spinner manually resulted in a plasma yield of around 30-40% with plasma purity of 65-75%. With a motorized spinner, plasma yield and purity were similar to the yield and purity of centrifuged samples. These findings provide support for the feasibility of low resource- based methods to separate whole blood in regions where a centrifuge is not available.

No description available.

Alzheimer's disease (AD) is part of a unique class of diseases because it is unlike the conventional diseases which are caused by viruses or bacteria. It is a "molecular disease" caused by the aggregation of a small protein (peptide) called β-amyloid (Aβ). Significant genetic, pathological, and biochemical evidence link AD to the aggregation (self-assembly) of the Aβ, especially the 40- or 42-residue isoforms (called Aβ(1-40) and Aβ(1-42) respectively) that are considered to be the most disease relevant. It is known that complex aggregation pathways produce a variety of aggregated Aβ structures, eventually producing amyloid fibrils (filamentous protein nanostructures) that deposit into plaques in the brain. Understanding the molecular structures of Aβ oligomers (aggregates that composed of 2 to roughly 100 molecules) and underlying assembly pathways will advance the fundamental understanding of AD at the molecular level. Furthermore, Aβ aggregation in vivo most likely occurs at peptide concentrations (nanomolar) which are much lower than concentrations required for Aβ aggregation in vitro (micromolar). One possible reason that has been proposed is that aggregation in vivo is catalyzed by interactions between Aβ and surfaces of lipid rafts or cell membranes. In this work, we have utilized solid state nuclear magnetic resonance (NMR) and molecular modeling to study the molecular structure of 150 kDa Aβ(1-42) oligomers formed by the interaction between sodium dodecyl sulfate (SDS) micelles and Aβ (1-42) peptide and compared to Aβ(1-42) fibril structures. Finite pulse radio frequency driven recoupling (fpRFDR) solid state NMR experiments on Aβ(1-42) oligomers reveal chemical shifts of labeled residues that are indicative of β-strand secondary structure. Results from 2D dipolar assisted rotational resonance (DARR) experiments indicate intermolecular proximity between I31 aliphatic and F19 aromatic carbons, which is contrary to models of Aβ oligomers proposed previously by other groups. In contrast, Aβ(1-42) fibrils have shown similar secondary and quaternary structures as oligomers. The most prominent structural differences between Aβ(1-42) oligomers and fibrils were observed through measurements of inter-molecular 13C-13C dipolar couplings observed in PITHIRDS-CT experiments. PITHIRDS-CT data indicate that, unlike fibrils, oligomers are not characterized by in-register parallel β-sheets. A hypothesized model is built based on the structural information we obtained from NMR characterization. We have further reported new solid state NMR constraints which indicate antiparallel intermolecular alignment β-strands within the 150kDa Aβ(1-42) oligomers and this result is consistent with our hypothesized model . Specifically, 150 kDa Aβ(1-42) oligomers with uniform 13C isotopic labels at I32, M35, G37 and V40 exhibit β-strand secondary chemical shifts in 2D fpRFDR NMR spectra, spatial proximities between I32 and V40 as well as M35 and G37 in 2D DARR spectra, and close proximity between M35 Hα and G37 Hα in 2D CHHC spectra. 2D DARR result of oligomer sample prepared with 30% labeled peptide further indicate the I32-V40, M35-G37 contacts are intermolecular. In addition, we employ molecular modeling to compare the newly derived experimental constraints with previously proposed geometries and come up with three candidate molecular models for arrangement of C-terminal region of Aβ(1-42) molecules into oligomers. Furthermore, we have developed a new technique to study how Aβ aggregation could be influenced by environmental factors such as interaction with different surfaces based on a surface patterning technique called microcontact printing (µCP). This approach allows easy measurement of thickness of the adsorbed layer by transferring the adsorbed layer form the polydimethylsiloxane (PDMS) stamp surface to a glass substrate. It also enables characterization of the face of the adsorbed peptide layer in contact with the hydrophobic stamp surface. Characterization of this face is normally not compatible with the conventional surface characterization techniques. Our results have shown that this face exhibits significant higher thioflavin T fluorescence than the face exposed to water, suggesting β-sheet formation induced by the hydrophobic PDMS surface. In addition, we have evaluated the intrinsic stability of the adsorbed layer by printing the layer on a sacrificial layer and have shown that by chemically crosslinking the adsorbed peptide, stable particulate microstructures in water can be obtained. Moreover, co-micropatterning of the different aggregated states of Aβ(1-40) (monomers and fibril fragments) is demonstrated, which may have potential applications for future study of Aβ aggregation or the interaction between different Aβ species. / A Dissertation submitted to the Department of Chemical and Biomedical Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2015. / May 18, 2015. / Aβ oligomer structure, Amyloid-β, microcontact printing, self-assembly, solid state NMR / Includes bibliographical references. / Anant Paravastu, Professor Directing Dissertation; Timothy Logan, University Representative; Jingjiao Guan, Committee Member; Subramanian Ramakrishnan, Committee Member.