Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

All epithelia are characterized by keratins, which make up a type of intermediate filament (IF). In epithelial tumors, which account for the majority of clinical cancers, the loss of cytoskeletal integration is considered one of the first alterations in epithelial metaplasia. This may have something to do with the expression of keratins or rearrangement of keratin filaments. In this study, I employed shotgun proteomic analysis and bioinformatic tools to identify proteins that interact with keratin filaments and thus may contribute to the disintegration of cytoskeleton. Using four airway epithelial cell lines in culture, I confirmed they highly expressed Keratin 14 (K14) and its obligatory partners, Keratin 5 (K5) or Keratin 6A (K6A). This suggests that the predominant IF is made up of K14 paired with K5/K6A. Although samples were enriched in keratin-associated proteins by immunoprecipitation (IP) with an antibody directed against K14 and K17, additional keratins not specifically targeted were also captured. Proteomic analysis revealed a list of non-keratin proteins enriched by IP. Some were associated with actin and microtubules, 23 and 6 proteins, respectively. Most of these were not linearly related to keratin content by abundance, but the motor protein, dynein I heavy chain, showed a Pearson correlation coefficient (CC) of -0.84 with keratin. Similarly, of 54 proteins associated with focal adhesions, intercellular junctions, or membranes, only septin-9 had a CC suggesting its abundance tracked with that of keratins. Finally, I analyzed IP-specific proteins that were cytosolic or had unknown subcellular distribution. A CC of -0.91 was found for one of these proteins, namely 26S proteasome regulatory subunit 8 (Psmc5). Further investigation and validation of the dataset was done by GO Enrichment Analysis. Using a subset of proteins highly concentrated by IP, compared to controls, I found the GO functions predicted were intracellular transport, (open full item for complete abstract)

Committee: Carol Heckman Ph.D (Committee Chair); Michael Geusz Ph.D (Committee Member); Xiaohong Tan Ph.D (Committee Member) Subjects: Bioinformatics; Biology; Biomedical Research; Cellular Biology; Molecular Biology; Oncology

Master of Science, University of Akron, 2012, Polymer Science

A fundamental investigation to study the surface of hair by modeling the wetting behavior of oil on a flat keratin surface was conducted. This study provides a model to study the complex keratin surface in contact with surfactants used in shampoo and conditioner formulations. This study focused on using keratin films as a tool to evaluate the effectiveness of surfactants to wet and dewet oil on hair. Keratin, a natural polymer is the major component of all hair types. Hair is highly crosslinked with disulfide bonds which were extracted from hair fibers by breaking peptide bonds.1,2,3 Extracted keratin has the ability to self-assemble and was casted into films. Two surfaces were investigated, silica glass and keratin films. The keratin was extracted from the hair by using the Shindai Method for protein extraction.1 Surfactants at their critical micelle concentration played an important role in the wetting of oils.4 Surface wetting profiles were studied to model the packing and arrangement of surfactant at the interface of keratin and oils. A model of a monolayer and possibly a bilayer of surfactants on the surface of keratin are suspected to influence wetting properties.5,6 This study demonstrated that keratin films can be used to model the surface of the hair fiber. Changes in contact angle as well as the physical profile of the oil droplets in contact with the keratin surface behave similarly to natural hair fibers. Differences in wetting profiles were distinguishable between anionic and cationic surfactants. The anionic surfactants produced high contact angle reading exhibiting a dewetting response. Wetting and deposition was improved by use cationic surfactants. Differences in wetting was also detectable between two cationic surfactants with different counterions using this modeling study. These results supports the claims of the dual processes of charge-driven adhesion and hydrophobic effects conducted on tassels of hair.6

Committee: Ali Dhinojwala Dr. (Advisor); Gary Hamed Dr. (Other) Subjects:

Master of Environmental Science, Miami University, 2021, Environmental Sciences

Growing industrial activities coupled with inability to effectuate common methods of water treatment has made lead contamination a recurring problem in low- and middle-income countries (LIMCs). Heavy metal contamination of water can significantly impact the health and well-being of populations of people affected by such pollution. The aim of this study was to investigate the use of keratin fibers from human hair as cost-effective, globally-available and sustainable adsorbents to remove lead (Pb2+) ions from water. For this aim, untreated and chemically treated hair were assessed as adsorbents to remove Pb2+ from water. Keratin adsorption capacities for Pb2+ was tested as a function of hair type, extent of chemical treatment, and contact time. Keratin was extracted from hair of persons from three ethnic groups: African (AF), Caucasian (NAM) and Asian (AS) in order to compare adsorption properties of the keratin fibers from three different types of human hair. Batch tests were conducted, and Pb2+ was used as a representative heavy metal for comparing the keratin adsorbents. Pb2+ was measured using Atomic Adsorption spectroscopy (AAS), and the resulting experimental Pb2+ adsorption isotherm data were best fit to the Langmuir isotherm model. While the results obtained in this study suggest that adsorption capacities of keratin from human hair may be affected by the type of hair, the small sample size used in this study did not provide conclusive evidence. For all three hair samples, treated and untreated hair was able to adsorb Pb2+ from water, and the uptake of Pb2+ was significantly affected by the extent of chemical treatment. The highest uptake of Pb2+ by keratin from human hair was with partially treated AS hair. The evaluation of adsorption kinetics indicates that Pb2+ adsorption on keratin follows a pseudo second order kinetics model. From this study, we concluded that human hair keratin can be used as an affordable, sustainable, and globally-available adsorb (open full item for complete abstract)

Committee: Catherine Almquist (Advisor); Jonathan Levy (Committee Member); Justin Saul (Committee Member) Subjects: Environmental Science

Master of Science (MS), Ohio University, 2018, Biological Sciences (Arts and Sciences)

Roads are ubiquitous in the United States, and their ecological effects are conspicuous. Turtles are among the vertebrate taxa most affected by roads because of their low vagility and use of road and road-side habitats. In 2013, Wayne National Forest in southeastern Ohio was bisected by a new highway, affecting a road-naive population of eastern box turtles (Terrapene carolina carolina), a species of concern in Ohio and vulnerable throughout its range. The goal of this study was to evaluate ecological, physiological, and behavioral effects of proximity to this new road in this road-naive population of turtles. We used a control-impact study to evaluate potential ecological and physiological effects of proximity to roads, employing radio-telemetry to assess space use, movement behavior, and habitat selection. We used novel bioassay techniques to analyze indicators of chronic stress (across the prior several months) using corticosterone stored in nail keratin. Overall, we found no significant differences in home range sizes, habitat preferences, or corticosterone concentrations between road-side and control sites. While our work suggests that proximity to roads has limited indirect influence on the ecology and chronic stress responses of eastern box turtles, and that road-naive turtles demonstrated avoidance of a high-traffic highway, the road network likely continues to contribute to population declines through direct mortality, and further inquiry is needed to assess road effects, particularly in the areas of stress endocrinology and impacts on demography.

Committee: Viorel Popescu (Advisor) Subjects: Animal Sciences; Animals; Biology; Conservation; Ecology; Endocrinology; Wildlife Conservation; Wildlife Management

Master of Science, Miami University, 2018, Chemical, Paper and Biomedical Engineering

Five to ten percent of bone fractures are classified as critical size defects (CSD), which are fractures that will not fuse without medical intervention. Currently the preferred course of treatment is autografts or allografts however both have high complication rates and require aggressive surgeries. BMP2 is a drug known to promote bone growth when implanted in vivo and is currently used for some orthopedic treatments. However, the amount administered and release rate are critical to its therapeutic success. Keratin hydrogels have been shown to be excellent implant materials, promoting cell proliferation and integration. Direct ink write (DIW) 3D printing allows for hydrogel materials to be 3D printed in customizable shapes using various materials, but printing requires knowledge of the fluid properties of the hydrogel. In this study we show that the printing pressure and speed for printing a hydrogel can be predicted. And we show that DIW printing can be used to tune a drug's release profile if the diffusivity is low enough.

Committee: Jessica Sparks (Advisor); Justin Saul (Committee Member); Jason Berberich (Committee Member); Jens Mueller (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy, University of Akron, 2018, Polymer Science

Biological adhesives are sticky secretions or structures produced by several organisms in nature to serve roles such as locomotion, prey capture and defense. These adhesives stick in a variety of environmental conditions and can maintain their adhesion exceptionally well. The present work focuses on understanding one such environmental factor, `humidity' and presents its correlation with the material composition in influencing the adhesion mechanism in two diverse biological attachment systems: Capture silk and Gecko setae. Understanding adhesion in these natural systems is essential with respect to humidity since many synthetic materials including glues fail in presence of water. The first and second studies focus on the glue laden capture silk produced by web building spiders. In the first study, we explored the capture silk of cobweb weaver `black widow spider' known as `gumfoot glue'. We first investigated the chemical composition of the glue and for the first time reported that it is majorly a combination of hygroscopic organic salts (low molecular mass compounds, LMMCs) and novel glycoproteins, apart from previously known peptides. Next, we correlated the glue composition with humidity based macro and molecular level studies and showed the synergistic role of LMMCs and glycoproteins in adhesion across the range of humidity conditions. Based on the first study which showed the presence and importance of diverse LMMCs in capture silk adhesion, we designed our second study in understanding the role of LMMCs in the capture silk. Based on hypothesis that LMMC's compositions control the maximum adhesion and viscosity trends across species, we designed the study in which by using Solution-State NMR, we first analyzed the water-soluble extract of glues for four different spider species from diverse habitats and found extract belonging to each species is a distinct combination of organic LMMCs present in varied proportions. Next, we studied the water uptake of (open full item for complete abstract)

Committee: Ali Dhinojwala Dr. (Advisor); Mesfin Tsige Dr. (Committee Chair); Todd A. Blackledge Dr. (Committee Member); Miyoshi Toshikazu Dr. (Committee Member); Joy Abraham Dr. (Committee Member) Subjects: Biology; Biophysics; Materials Science; Polymers

Master of Science, Miami University, 2017, Chemical, Paper and Biomedical Engineering

Age-related Macular Degeneration (AMD) is a retinal disease responsible for 8.7% of blindness globally, and it is predicted that 196 million people will be affected by the year 2020. Further, there is currently no cure for AMD. Early stage AMD is usually triggered by dysfunction of the retinal pigment epithelium (RPE), and the Bruch's membrane is often invaded by abnormal angiogenesis around the retina as the disease progress. A biomimetic tissue engineered retinal scaffold may be beneficial in understanding disease etiology and also a possible restorative treatment for AMD. As a primary step toward the development of a tissue engineered retinal scaffold, this thesis focuses on the conceptualization, design, fabrication, and characterization of a scaffold system that combines electrospun polycaprolactone (PCL) fibers to mimic the Bruch's membrane and keratin aerogel to provide structural support reminiscent of the retinal choroid. Results of this work demonstrate that PCL/keratin fibrous-aerogel shows morphological similarity to the native retinal Bruch's membrane under SEM, tunable compressive modulus within a range similar to the native retinal extracellular matrix, and minimal toxicity to ARPE-19 cell line. Importantly, the formation of cell monolayers suggests that such system may be suitable as a model system for testing key parameters associated with retinal injury and/or regeneration.

Committee: Justin Saul (Advisor); Michael Robinson (Committee Member); Coffin Douglas (Committee Member) Subjects: Biomedical Engineering

Master of Science, Miami University, 2016, Computational Science and Engineering

Recombinant human bone morphogenetic protein-2 (rhBMP-2) can be used clinically to promote bone healing as an alternative to bone grafting treatment. The rhBMP-2 can stimulate cellular differentiation of osteoprogenitor cells to promote bone healing. However, delivery of rhBMP-2 is a challenge since rhBMP-2 has a short half-life and has therefore been delivered from collagen sponges implanted at the injury site. While this has led to effective bone regeneration, ectopic bone growth associated with the rapid degradation of collagen and subsequent rhBMP-2 release are clinical problems. We are investigating keratins as alternative rhBMP-2 carriers. Keratins are structural intermediate proteins and can be extracted from human hair. Oxidatively extracted keratin (KOS) cannot achieve disulfide crosslinks whereas reductively extracted keratin (KTN) can form disulfide crosslinks. The rate of degradation of keratin can be tuned by mixing keratose and kerateine in varying ratios. The hypothesis guiding this thesis is that keratin can be formulated with varying ratios of KOS and KTN to modulate the rate of scaffold degradation and thereby control the releasing rate of rhBMP-2. The in vivo release kinetics of rhBMP-2 was assessed by a critically-sized rat femur defect model. The biodistribution of rhBMP-2 after implantation in the critically-sized femur model was assessed in the vital organs.

Committee: Justin Saul (Advisor); Lei Kerr (Committee Member); Michael Robinson (Committee Member) Subjects: Academic Guidance Counseling; Biomedical Engineering

Master of Science, Miami University, 2014, Chemical, Paper and Biomedical Engineering

In the United States, 450,000 burn injuries receive medical treatment per year1 with approximately 2% experiencing wound infection2. Current clinical paradigms such as Epicel, Apligraf, and Alloderm may provide wound coverage and promote wound healing, but do not incorporate antibiotics to fight microbial infection. Thus, dressings or materials that can promote healing while preventing infection are an important unmet clinical need. One possible solution is a biomaterial loaded with antibiotics. Off-the-shelf use of hydrogels made from keratin, collagen, and fibronectin proteins are attractive because of their physiochemical capacity to facilitate skin healing by promoting appropriate cellular responses. This thesis focuses on keratin hydrogels due to their ability to resist proteolytic degradation, promote vascularization, and support cellular ingrowth. The principal hypothesis of these experiments is that antibiotic loaded keratin hydrogels promote sustained release in a manner correlated with their degradation. To investigate this hypothesis, this thesis developed high performance liquid chromatography (HPLC) methods for detection of antibiotics in the presence of keratin proteins. Specifically, the results of this study helped to quantify antibiotic release from keratin hydrogels, and to characterize previous results indicating the sustained release of three specific antibiotics, cephazolin, ciprofloxacin, and neomycin. The results of these studies demonstrate the suitability of keratin hydrogels for antibiotic release and support conducting pre-clinical animal testing as well as possible FDA submissions for future human trials.

Committee: Justin Saul PhD (Advisor); Jason Berberich PhD (Committee Member); Neil Danielson PhD (Committee Member) Subjects: Engineering

Master of Science, Miami University, 2013, Computational Science and Engineering

Treatment of volumetric muscle loss (VML) is a significant challenge in warfighters due to the severity of the injuries usually caused by improvised explosive devices (IEDs), to which there are currently few viable clinical treatment options. Keratin hydrogels offer a potential solution to this problem as carriers of cells and growth factors. Human epithelial keratins are the intermediate structural proteins in cells and can be extracted from several sources including human hair. Their intrinsic characteristics, such as high levels of cysteine residues and resulting disulfide bonds, make them candidates for biomaterial-based carriers of growth factors and cells. We hypothesized that the formulation hydrogels composed of two forms of keratin (keratose that lacks disulfide bonds and kerateine that contains disulfide bonds) could be used to tailor their degradation profiles. Such control over degradation is important in tissue engineering approaches to allow for growth factor release, provide for cell delivery, and ultimately allow for tissue regeneration. In this work, we assessed both material properties of (e.g. rheology, degradation, and protein release) and biological response (e.g. cell viability) to keratin hydrogel formulations of keratose-kerateine mixtures. The results of this thesis work indicate that modulation of intrinsic mechanical properties of the hydrogel will allow spatiotemporal drug and cell delivery that may ultimately promote skeletal muscle regeneration.

Committee: Justin Saul PhD (Advisor); Lei Kerr PhD (Committee Member); Jessica Sparks PhD (Committee Member) Subjects: Biomedical Engineering; Chemical Engineering; Materials Science

Master of Science, Miami University, 2013, Chemical, Paper and Biomedical Engineering

Bacterial infection is common following traumatic injuries in both military and civilian populations. Limitations with current antibiotic treatments (e.g., antibiotic resistance) necessitate new delivery systems that can achieve local delivery while promoting tissue healing. Keratins are a family of natural fibrous structural proteins that are derived from various sources including human hair. Recently, keratin biomaterials were demonstrated to support release of Ciprofloxacin at a rate correlated to that of the material degradation. The goal of this thesis is to further investigate the applicability of these effects to other common antibiotics. Specifically, we investigated delivery of four antibiotics from keratin biomaterials against three strains of bacteria commonly associated with infected tissues. Broth inhibition assays were used to assess bioactivity and antibiotic release from the keratin materials was measured. In general, antibiotic release correlated with keratin degradation, providing a system that can be used for local antibiotic delivery while promoting tissue healing.

Committee: Justin Saul PhD (Committee Chair); Annette Bollmann PhD (Committee Member); Douglas Coffin PhD (Committee Member) Subjects: Biomedical Engineering; Chemical Engineering

PhD, University of Cincinnati, 2006, Pharmacy : Pharmaceutical Sciences

It has been long known that hydration strongly affects transport and mechanical properties of keratinized membranes. The most studied keratin structures are human stratum corneum, wool and hair. The effect of hydration on transport, however, has not been studied in human nails. This investigation is aimed at filling this gap. In the first phase of the work we have determined the equilibrium water sorption isotherm for human nail. The isotherm obtained in our laboratory was compared with available literature data for nail and other keratinized tissues. Results were described in terms of theoretical sorption models used in natural polymers and food systems including Guggenheim-Anderson-de Boer and D'Arcy-Watt isotherms. Of the models tested, D'Arcy-Watt gave the best description over the entire range of hydration. We further investigated transport properties in nail of water and the antifungal drug, ketoconazole as a function of nail hydration which was controlled by adjusting the relative humidity on one or both sides of the tissue. Diffusivity of water in nail was studied using both vapor phase and liquid phase sorption-desorption techniques. The values obtained ranged from D = 3.160 × 10-7 cm2/s at a water volume fraction phi1 = 0.4107 to D = 7.68 × 10-10 cm2/s at phi1 = 0.0431. They are interpreted in terms of a free volume theory. Transport parameters of ketoconazole were obtained by conducting permeation experiments using a single chamber diffusion cell designed specifically for nail permeation studies. Flux of ketoconazole solvent-deposited onto the nail from ethanol increased consistently with an increase in water content in the nail. Diffusivities estimated from these data can be described by Fujita theory. The findings show that hydration provides enormous increases in water transport through nail, but only a modest (2-3 fold) increase in ketoconazole permeation under conditions attainable in vivo. Thus, nail penetration enhancement by hydration is not large (open full item for complete abstract)

Committee: Gerald Kasting PhD (Committee Chair); Pankaj Desai PhD (Other); Paul Lehman (Other); Giovanni Pauletti PhD (Other); Adel Sakr PhD (Other); Randall Wickett PhD (Other) Subjects: Health Sciences, Pharmacy