A. James Clark School of Engineering
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The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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Item HISTATIN 5 MODIFICATIONS IMPACT PROTEOLYTIC STABILITY IN THE PRESENCE OF FUNGAL AND SALIVARY PROTEASES(2024) Makambi, Wright Kingi; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Candida albicans, found in the oral cavities of 30-50% of the global population, can lead to oral candidiasis, particularly in immunocompromised individuals like those with HIV or diabetes. The current treatments, small-molecule antifungals, often fall short due to drug resistance and toxicity. To address these challenges, histatin 5 (Hst5), a 24-amino-acid peptide naturally present in human saliva, has been studied as a potential antifungal therapy. Hst5, however, is susceptible to degradation by secreted aspartyl proteases (Saps) produced by C. albicans and salivary enzymes, limiting its potential efficacy as a therapeutic. We have engineered Hst5 variants utilizing rational design in order to understand the interactions with Saps and Saliva. We have also made advancements in developing a novel screening method utilizing the directed evolution technique yeast surface display. Our study employed rational design to modify Hst5, at its lysine residues (K5, K11, K13, and K17), substituting them with leucine or arginine to examine their influence on interactions with Saps (Sap1, Sap2, Sap3, Sap5, Sap6, Sap9, and Sap10). Sap5, Sap6, and Sap10 did not degrade Hst5 at the tested conditions, while Sap1, Sap2, Sap3, and Sap9 did. Some modifications, such as K13L, are particularly susceptible to proteolysis by Sap1, Sap2, Sap3, and Sap9. In contrast, K17L substantially increases the stability and antifungal activity of Hst5 in the presence of Saps. Additionally, although the K11RK17L variant was degraded more than the K17L variant, their antifungal activities were largely similar. The proteolysis products of were also identified by mass spectrometry identifying the [4-24], [1-17], and [14-24] Sap proteolysis products. We also evaluated the proteolytic stability of these variants in saliva. Both K17L and K5R showed improved stability; however, the enhancements were modest, suggesting that further engineering is required to achieve significant improvements. Further experiments evaluated how additional amino acid substitutions at K13 and K17 affect the peptide’s proteolytic stability in the presence of Saps (with and without zinc). Our findings suggest that the positive charge at K13 is important for the proteolytic stability of Hst5, as all other variants tested except K13R reduce overall proteolytic stability. Furthermore, many substitutions at K17, including tryptophan, significantly enhance proteolytic resistance and antifungal activity following incubation with Saps. The K17W variant showed improved stability and antifungal efficacy, maintaining its function even in the presence of zinc and exhibiting stronger antibiofilm activity than the parent Hst5. In addition to the rational design work, we have advanced the development of a directed evolution yeast surface display platform for screening peptides for proteolytic stability. This would allow for the expression of large peptide libraries on the surface of Saccharomyces cerevisiae. Through optimization of expression and display conditions, we determined an induction media at 30°C with a pH of 3.5 and devoid of glucose improved the expression and display of Hst5 peptides on the surface of S. cerevisiae. We also optimized the degradation conditions for Sap2 37°C, a pH not exceeding 7.4, and a Sap2 concentration of 0.78 µg/mL led to the best discrepancy between proteolytically stable variants. Additionally, we found that a 40 amino acid linker between the peptide and the yeast surface provided the best observing proteolytic degradation. Using the optimized system, we showed that yeast surface display can be used to discriminate between peptide variants with different levels of proteolytic stability. This lays the foundation for future work to screen large libraries of peptides for proteolytic stability. From these results, we have gained a deeper understanding of the interactions between Hst5 and Saps, showing that modification at different lysine residues greatly impacts the proteolytic stability of Hst5. Furthermore, we have shown that the yeast surface display platform can be used to screen the proteolytic stability of peptides. Looking forward, this peptide should be engineered for proteolytic stability in saliva. Furthermore, mock screens should be made before screening a library of peptides using the yeast surface display platform.Item EXPERIMENTAL INVESTIGATION OF THE LIPID-BINDING MECHANISM OF OSH4 PROTEIN(2024) Konakbayeva, Dinara; Karlsson, Amy; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recent findings show that intracellular lipid traffic between organelles primarily occurs through a non-vesicular pathway involving lipid transport proteins (LTPs) and is facilitated by areas of close apposition between two organelles so called membrane contact sites (MCS). Oxysterol-binding homologue (Osh) proteins in the yeast Saccharomyces cerevisiae serve as examples of LTPs. Osh proteins are crucial for transporting signaling lipids and are believed to form MCSs. In this study, we examined the binding mechanism of the Osh4 protein, aiming to gain a better understanding of its explicit membrane-binding mechanism.The Osh4 protein possesses an α-helical binding domain known as the amphipathic lipid-packing sensor (ALPS)-like motif. Our approach involved utilizing experimental methods to examine the biophysical interactions of both the ALPS peptide and the full-length Osh4 protein. To investigate the binding interactions of ALPS with membranes of different lipid compositions, we examined its interactions with three different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; has a zwitterionic head group) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS; has a negatively charged head group)—1:1 POPC-POPS, 4:1 POPC-POPS, and 9:1 POPC-POPS—as well as pure POPC. To understand the structural changes in ALPS and model membranes during peptide-membrane interactions, we performed a series of experimental studies. Circular dichroism (CD) was used to study the changes in the secondary structure of ALPS in different environments. The CD data indicated that the α-helical conformation of the ALPS peptide was more pronounced in the presence of POPC-POPS liposomes, especially with a higher content of POPS lipid, compared to liposomes composed entirely of POPC. This observation underscores the significant influence of anionic lipids in the facilitation of peptide folding at the membrane-water interface. X-ray diffraction was utilized to study the changes in membrane structure upon ALPS binds to it. The X-ray diffraction results showed that the ALPS peptide caused thinning of the multilayer with an increased POPS lipid ratio. This could be due to the electrostatic interaction of the positively charged Lys residue in the ALPS sequence with the anionic POPS lipid. We also studied the binding of the peptide to membranes by observing changes in the Trp fluorescence emission spectrum of ALPS upon the addition of liposomes. We observed a blue shift in the fluorescence emission maximum of Trp with higher POPS content. This suggests that the ALPS peptide was experiencing a more hydrophobic and less polar environment in the presence of the liposomes, indicating possible penetration of the peptide into the hydrocarbon region of the bilayer. The blue shifts of Trp emission in the presence of POPS liposomes were higher than those observed with POPC liposomes and suggest that the ALPS peptide binds better to charged POPS lipids, which is consistent with the X-ray diffraction data. We also conducted Trp fluorescence titration and ITC experiments to gain deeper insights into the binding affinity of the ALPS peptide to a model membrane. Using fluorescence data, we estimated the binding constant for the binding of ALPS to liposomes by performing titration measurements of vesicles with the ALPS peptide. Our analysis demonstrated that ALPS binding to 4:1 POPC-POPS lipid membranes had a Kd of 1.88 ± 0.47 μM, which corresponds to a free energy change (ΔG) of -7.82 ± 0.15 kcal/mol. Additionally, the ITC experiments performed with the same vesicles yielded a ΔG of -4.41± 0.04 kcal/mol. This result is slightly less than the ΔG value of -7.82 ± 0.15 kcal/mol obtained from fluorescence spectroscopy titration. The observed discrepancy of -3.41 kcal/mol may indicate the energy associated with the folding of the ALPS peptide. In order to understand how Osh4 forms MCSs between two membranes, we need to examine how the membranes interact with the full-length protein. The first step to achieve this is to produce the protein through recombinant protein production methods. After evaluating two different fusion tags, glutathione S-transferase (GST) and small ubiquitin-related modifier (SUMO), it was found that the SUMO tag resulted in higher protein yield and greater protein purity. Our work lays the foundation for future experiments with the full-length Osh4 protein to improve our understanding of the mechanisms of lipid transport between membranes. Our results emphasize the ALPS peptide’s selectivity for specific lipid environments, particularly its affinity for anionic lipids. We demonstrated that the presence of anionic lipids is crucial for the motif's ability to induce conformational changes upon binding to a membrane, and these conformational changes likely play a critical role in intracellular lipid trafficking and membrane organization.Item Enhanced Throughput Single-Cell Capillary Electrophoresis Mass Spectrometry(2023) Mendis, John Udara; Nemes, Peter; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mass spectrometry (MS) has allowed for the analysis of small molecules and metabolites with high specificity and sensitivity. Capillary Electrophoresis mass spectrometry (CE-MS) is an ultrasensitive analytical technique to process amount-limited samples. Robust high-throughput ultrasensitive CE-MS methods and technologies are needed to be developed to comprehensively study the metabolome or proteome of a sample with a limited amount of material. In this study, we developed an enhanced-throughput multi field amplified sample stacking (M-FASS) method. The resulting approach has a sample processing throughput of 5–10 times that of conventional CE methods. FASS voltage duration and strength were optimized for peak area and peak resolution. The M-FASS CE-MS method was then applied for single cell analysis (SCA) of metabolic differences and gradients in the developing embryo of Xenopus Laevis. The statistical analysis: PCA and Fuzzy-c means clustering analysis revealed cell-to-cell differences among D11, V11, D12, and V12 cells and uncovered 6 distinct metabolite gradients between the four cells in X. Laevis 16-cell embryos. The findings showcase inherent metabolic gradients in the developing embryo.Item ENABLING RAPID PHENOTYPIC DETECTION OF CEPHALOSPORIN RESISTANCE BEYOND THE CENTRAL LABORATORY(2019) Nguyen, Hieu Thuong; White, Ian; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The so-called bacterial “superbugs” are largely resistant to some of the most commonly prescribed antibiotics, including a drug class known as cephalosporins used to treat many hospital and community-acquired infections. This major public health threat has been acknowledged for decades by the Centers for Disease Control (CDC) as a major concern; yet, the detection of superbugs has not been made routine since standard testing practices have been limited to specialized “central” laboratories with sophisticated yet bulky and expensive equipment and highly trained personnel. As a result, the lack of simpler testing methods that can be used in everyday clinics and doctor’s offices can be viewed as a source of error contributing to incorrect antibiotic treatment and poorer patient outcomes, factors that drive even more advanced resistance, depleting our drugs or last resort. In this dissertation, we explore new strategies for simplified methods to test for cephalosporin resistance in order to give higher accessibility in the timely detection of superbugs to support the improvement of patient care. To do this, we take an organic chemistry and biochemical approach to develop new detection molecules that report resistance activity in bacteria expressing extended-spectrum β-lactamase (ESBL) enzymes, one of the most prolific resistance strategies used by superbugs. Next, we describe methods of integrating these detection molecules into practical testing methods, and detail the engineering of simpler assays that allow for rapid readout of ESBL phenotypes using commonplace laboratory plate readers, portable Raman devices, and even handheld personal glucose meters (used for diabetes monitoring) purchased from the drugstore.Item Engineering cell-penetrating peptides for translocation and intracellular cargo delivery in Candida species(2017) Gong, Zifan; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungal infections caused by Candida species, particularly C. albicans and C. glabrata, have become a serious threat to public health. The rising drug resistance has prevented effective treatment and increased the mortal rate. Novel approaches to improve the therapeutic effects of antifungal agents and allow delivery of agents that are not normally cell-permeable are in demand. In order to improve the intracellular delivery of antifungal agents, we have investigated using cell-penetrating peptides as drug carriers for treating fungal infections. CPPs have been widely studied as tools for delivering a variety of molecular cargo into cells, including DNA, RNA, proteins, and nanoparticles. Previous work with CPPs has mainly focused on their uptake in mammalian cells, but CPPs also have potential as drug delivery and research tools in other organisms, including Candida pathogens. We have explored various well-studied CPPs to identify peptides that retain their translocation capability with Candida cells, including pVEC, penetratin, MAP, MPG, SynB, TP-10 and cecropin B. The CPPs pVEC, penetratin, MAP and cecropin B show a higher level in the cytosol adopt direct translocation mechanisms and exhibit toxicity towards C. albicans. Our peptide localization and mechanistic studies allow better understanding of the mode of translocation for different CPPs, which is related to the potential toxicity towards Candida pathogens. To further understand the molecular mechanisms of translocation of CPP, we investigated the biophysical properties of the peptides. CPPs that previously were shown to use direct translocation mechanisms (pVEC, MAP, and cecropin B) exhibit helical conformations upon interaction with cells due to the hydrophobic interaction with the core of bilayers. Membrane associations of peptides that entered cells via endocytosis were controlled by electrostatic forces. Our novel structure characterization methods using circular dichroism with live fungal cells, along with Monte Carlo simulations, allow us to understand how CPPs interact with cell membranes and how the membrane association affects the translocation mechanisms. After beginning to understand the structure-function relationships of CPPs, we engineered two CPPs, pVEC and SynB, to enable better translocation efficacy and manipulation of translocation mechanisms. We tuned the properties of the peptides, including the net charge and the hydrophobicity, to alter intracellular fates and the level of antifungal activity. These results are promising and motivate better peptide engineering for specific purposes. Our work with CPPs and fungal pathogens contributes to the understanding of structure-function relationship of CPPs in Candida species. We have provided the foundation for further peptide engineering and explorations into applications of CPPs in treating fungal infections.Item Dual Quorum Quenching Capsules: Disrupting two bacterial communication pathways that lead to virulence(2016) Rhoads, Melissa Katherine; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Healthcare Associated Infections (HAIs) in the United States, are estimated to cost nearly $10 billion annually. And, while device-related infections have decreased, the 60% attributed to pneumonia, gastrointestinal pathogens and surgical site infections (SSIs) remain prevalent. Furthermore, these are often complicated by antibacterial resistance that ultimately cause 2 million illnesses and 23,000 deaths in the US annually. Antibacterial resistance is an issue increasing in severity as existing antibiotics are losing effectiveness, and fewer new antibiotics are being developed. As a result, new methods of combating bacterial virulence are required. Modulating communications of bacteria can alter phenotype, such as biofilm formation and toxin production. Disrupting these communications provides a means of controlling virulence without directly interacting with the bacteria of interest, a strategy contrary to traditional antibiotics. Inter- and intra-species bacterial communication is commonly called quorum sensing because the communication molecules have been linked to phenotypic changes based on bacterial population dynamics. By disrupting the communication, a method called ‘quorum quenching’, bacterial phenotype can be altered. Virulence of bacteria is both population and species dependent; each species will secrete different toxic molecules, and total population will affect bacterial phenotype9. Here, the kinase LsrK and lactonase SsoPox were combined to simultaneously disrupt two different communication pathways with direct ties to virulence leading to SSIs, gastrointestinal infection and pneumonia. To deliver these enzymes for site-specific virulence prevention, two naturally occurring polymers were used, chitosan and alginate. Chitosan, from crustacean shells, and alginate, from seaweed, are frequently studied due to their biocompatibility, availability, self-assembly and biodegrading properties and have already been verified in vivo for wound-dressing. In this work, a novel functionalized capsule of quorum quenching enzymes and biocompatible polymers was constructed and demonstrated to have dual-quenching capability. This combination of immobilized enzymes has the potential for preventing biofilm formation and reducing bacterial toxicity in a wide variety of medical and non-medical applications.Item Interference Studies Using Multidimensional Mapping of Cross-Reactive Sensors: Applications in Blood Monitoring of Clozapine(2014) Chocron, Sheryl E.; Ghodssi, Reza; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Point-of-care sensors are used in clinical applications for diagnosing and monitoring health conditions. For example, a point-of-care sensor for therapeutic drug monitoring of the clozapine antipsychotic can reduce burdens from guidelines suggesting routine monitoring of this medication. However, when measuring chemical markers in complex fluids, there are challenges related to decreased sensor performance due to chemical interference. This work presents a methodology for identifying individual interfering species. A set of cross-reactive electrochemical sensors were developed, whose diversified responses provide a fingerprint-type pattern capable of differentiating various species. By mapping the multidimensional responses, patterns from complex solutions were discerned and matched to those of individual species. Applying this methodology to clozapine sensing in blood, a major source of chemical interference was identified. The understanding matrix components that cause interference can guide the design of reliable sensing systems and can be integrated with pattern recognition tools that can account for it.Item Evaluation of Optical Sensor Platforms for Multiplexed Detection of Proteins(2014) Spindel, Samantha; Sapsford-Medintz, Kim E; White, Ian; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This work investigated optical sensor platforms for protein multiplexing, the ability to analyze multiple analytes simultaneously. Multiplexing is becoming increasingly important for clinical needs because disease and therapeutic response often involve the interplay between a variety of complex biological networks involving multiple, rather than single, proteins. Moreover, one biomarker may be indicative of more than one disease, similar diseases can manifest with similar physical symptoms, and monitoring a disease requires the ability to detect subtle differences over time. Multiplexing is generally achieved through one of two routes, either through spatial separation on a surface (different wells or spots) or with the use of unique identifiers/labels (such as spectral separation - different colored dyes, or unique beads - size or color). We looked into combining both spatial separation and unique labels to further expand the multiplexing capabilities of surfaces. Our original research resulted in one of the few demonstrations of reactive semiconductor nanocrystal immunoassays for multiplexed analysis within a single well on a microtiter plate. Innovative planar surface fluorescent immunoassays were developed for both spatial and spectral multiplexing using Quantum Dots and prospective incorporation into a Point-of-Care (POC) device involving an evanescent wave scanner. These assays used standard microscope slides combined with flow cells and were designed to markedly reduce the amount of sample and reagents needed as compared to standard 96-well plate assays. The platform was optimized for detecting Chicken IgG and Staphylococcal Enterotoxin B (SEB); SEB is commonly used in the literature to characterize the performance of biosensor platforms. The planar surface fluorescent immunoassays were applied to a real-world public health need to detect renal injury. Two emerging novel biomarkers, Kidney Injury Marker-1 (KIM-1) and Neutrophil Gelatinase Associated Lipocalin (NGAL), were investigated for their potential to detect injury earlier and with more specificity than current methods using serum creatinine (SCr). Detecting these medically-relevant markers using planar surface fluorescence immunoassays could potentially allow for more rapid diagnosis of acute kidney injury (AKI), among other uses.Item Low-Cost Paper-Based Assays for Multiplexed Genetic Analysis using Surface Enhanced Raman Spectroscopy(2013) Hoppmann, Eric Peter; White, Ian M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In order to improve human health it is critical to develop low-cost sensors for chemical detection and healthcare applications. Low-cost chemical detectors can enable pervasive monitoring to identify health threats. Rapid yet accessible infectious disease diagnostics have the potential to improve patient quality of care, reduce healthcare costs and speed recovery. In both cases, when multiple targets can be detected with a single test (multiplexing), accessibility is improved through lowered costs and simplicity of operation. In this work we have investigated the practical considerations and applications of ink-jet printed paper surface enhanced Raman spectroscopy (SERS) devices. SERS enables specific simultaneous detection of numerous analytes using a single excitation source and detector. Sensitive detection is demonstrated in several real-world applications. We use a low-cost portable spectrometer for detection, further emphasizing the potential for on-site detection. These ink-jet printed devices are then used to develop a novel DNA detection assay, in which the multiplexing capabilities of SERS are combined with DNA amplification through polymerase chain reaction (PCR). In this assay, the chromatographic properties of paper are leveraged to perform discrimination within the substrate itself. As a test case, this assay is then used to perform duplex detection of the Methicillin-resistant Staphylococcus aureus (MRSA) genes mecA and femB, two genes which confer antibiotic resistance on MRSA. Finally, we explore statistical multiplexing methods to enable this assay to be applied to perform highly-multiplexed detection gene targets (5+), and demonstrate the differentiation of these samples using partial least-squares regression (PLS). By averaging the signal over a region of the SERS substrate, substrate variability was mitigated allowing effective identification and differentiation, even for the complex spectra from highly multiplexed samples which were impossible to visually analyze.Item A Microfluidic Programmable Array for Label-free Detection of Biomolecules(2011) Dykstra, Peter Hume; Ghodssi, Reza; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)One of the most promising ways to improve clinical diagnostic tools is to use microfluidic Lab-on-a-chip devices. Such devices can provide a dense array of fluidic components and sensors at the micro-scale which drastically reduce the necessary sample volumes and testing time. This dissertation develops a unique electrochemical sensor array in a microfluidic device for high-throughput, label-free detection of both DNA hybridization and protein adsorption experiments. The device consists of a patterned 3 x 3 grid of electrodes which can be individually addressed and microfluidic channels molded using the elastomer PDMS. The channels are bonded over the patterned electrodes on a silicon or glass substrate. The electrodes are designed to provide a row-column addressing format to reduce the number of contact pads required and to drastically reduce the complexity involved in scaling the device to include larger arrays. The device includes straight channels of 100 micron height which can be manually rotated to provide either horizontal or vertical fluid flow over the patterned sensors. To enhance the design of the arrayed device, a series of microvalves were integrated with the platform. This integrated system requires rounded microfluidic channels of 32 micron height and a second layer of channels which act as pneumatic valves to pinch off selected areas of the microfluidic channel. With the valves, the fluid flow direction can be controlled autonomously without moving the bonded PDMS layer. Changes to the mechanism of detection and diffusion properties of the system were examined after the integration of the microvalve network. Protein adhesion studies of three different proteins to three functionalized surfaces were performed. The electrochemical characterization data could be used to help identify adhesion properties for surface coatings used in biomedical devices or for passivating sensor surfaces. DNA hybridization experiments were performed and confirmed both arrayed and sensitive detection. Hybridization experiments performed in the valved device demonstrated an altered diffusion regime which directly affected the detection mechanism. On average, successful hybridization yielded a signal increase 8x higher than two separate control experiments. The detection limit of the sensor was calculated to be 8 nM.