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The University of Maryland School of Pharmacy, founded in 1841, is a thriving center for life sciences research and community service. Through its education, research, and service programs, the School of Pharmacy strives to improve the health and well-being of society by aiding in the discovery, development, and use of medicines.
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Regulation of retinoid homeostasis by cellular retinol-binding protein, type 1Retinoic acid (RA) is the main active metabolite of Vitamin A, an essential diet-derived nutrient. RA signaling regulates cell differentiation, proliferation and apoptosis. RA levels are tightly regulated throughout the body via the expression and activity of catabolic and biosynthetic enzymes, and chaperone proteins, including cellular retinol binding protein, type 1 (CRBP1). CRBP1 binds to retinol and retinal, protecting them from non-specific oxidation, and facilitating their delivery to the appropriate enzymes for RA biosynthesis. CRBP1 has been shown to be decreased in disease states that display dysfunctional proliferation and differentiation, including cancers. Reduction of CRBP1 levels directly correlates with reduction in RA and restoration of CRBP1 expression has been shown to increase RA levels and positively impact RA-dependent outcomes. Research on the role of CRBP1 in disease has been limited because of its low abundance and poor immunogenicity. We have developed a targeted, bottom-up proteomics approach for absolute CRBP1 quantitation in complex biological matrices and have utilized this assay to answer important biological questions regarding the role of CRBP1 in regulating RA and RA-mediated signaling. While proper RA homeostasis is essential for biological processes throughout the body, the research in this thesis has focused on its role in the small intestine, heart, and lung. In the small intestine, RA plays an essential role in regulating the gut immune response. In instances of cellular stress in the intestine, RA levels are decreased. We have employed our CRBP1 quantitative assay, along with retinoid metabolite quantitation and quantitative gene expression, to systemically probe the mechanism of disrupted retinoid signaling in intestinal disease via an in vitro model of the small intestine. Proper RA levels are also necessary for growth and development, including heart and lung morphogenesis, and have also been shown to be disrupted in many diseases, such as heart failure and lung cancer. Using a global CRBP1 knock-out mouse model, we have also explored the in vivo effect of loss of CRBP1 on retinoid signaling via multi-omics analysis. Together these studies will help further our understanding of the mechanisms and impact of CRBP1 loss in diseases of the intestine, heart, and lungs.
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Method Optimization of a New Automated Platform for Proteome-Wide Structural BiologyProteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein HOS and dynamics requires integrated approaches, including mass spectrometry (MS), which has evolved into an indispensable tool for proteomics research. One approach readily integrated with MS is protein footprinting. In-cell fast photochemical oxidation of proteins (IC-FPOP) is a protein footprinting method that utilizes hydroxyl radicals to oxidatively modify the side chains of solvent accessible amino acids. Liquid chromatography coupled to mass spectrometry is used to both identify modified amino acids and quantify the levels of labeling. Owing to solvent accessibility changing upon binding or changes in conformations, IC-FPOP can be used to identify protein-ligand and protein-protein interaction sites and regions of conformational change. The method can modify thousands of proteins in a single experiment leading to structural information across the proteome. IC-FPOP modifies proteins on the microsecond timescale making the method suitable to study fast biological processes. However, the single cell flow system developed for initial IC-FPOP experiments had temporal limitations motivating the design for a higher throughput platform. My research describes the development of a new platform for IC-FPOP entitled Platform Incubator with XY Movement (PIXY). PIXY permits IC-FPOP to occur in a sterile system using a temperature-controlled stage top incubator, peristaltic pumps for chemical transport, mirrors for laser beam guidance, and a mobile stage for XY movement. Automated communication amongst the entire PIXY system was made possible using LabVIEW software which allows the analysis of one sample in only 20 seconds. Well over 2000 proteins in HEK cells can be oxidatively modified by IC-FPOP in PIXY. This allows for a greater amount of structural information to be obtained. The capabilities of this high throughput platform permit other cell based experimental applications including fluorescent imaging and time-dependent solution transfer. PIXY’s ability to accommodate automated time points and subsequent changes over time make it a powerful tool for probing protein biochemistry in the native cellular environment.
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Poison Prevention Press 2023University of Maryland, Baltimore. School of Pharmacy. Maryland Poison Center, 2023
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Capillary zone electrophoresis (CZE) for Protein-Oligonucleotide Conjugate Analysis and Characterization.Protein-Oligo molecule is new class of biomolecules containing Protein-Oligo conjugation. The molecule contains two different modalities: protein and Oligonucleotide. At neutral pH, protein may be positively or negatively charged whereas Oligonucleotide is negatively charged. The net charge of molecule is affected by pI of surrounding environment. Microchip based CZE coupled to a MS can be used to identify the major impurities as well as the variants of the final product CZE can resolve. Traditional capillary based CZE can be used to separate and characterize all these species with the more user friendly and robust UV based detection. The unique challenges of the new conjugate require the development of new methods to perform product characterization.
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Effect of Excipients on the Performance of Spray-dried Amorphous Solid Dispersion (ASD) in TabletsAmorphous solid dispersions (ASD) are a proven method of improving the solubility and bioavailability of poorly soluble drugs. Immediate-release tablets are frequently used as the final dosage form for ASDs. The selection of polymers and excipients is critical for the manufacturability and bioavailability of ASD tablets. ASDs were prepared by spray drying; ASD tablets were then generated using a compaction simulator. We first studied the impact of polymer types and drug-polymer ratios on bulk powder properties, morphologies, and compaction behaviors of ASDs. Itraconazole (ITZ) and indomethacin (IND) were used as model drugs, and two polymers were used: hydroxypropyl methylcellulose acetate succinate (HPMCAS) and polyvinylpyrrolidone (PVP). The results indicated that the tabletability increased with decreasing drug loadings, except for ITZ-PVP ASDs. Multivariate analysis revealed that particle surface area was the most significant factor influencing the tensile strength of ASD tablets. Secondly, the contact angle and surface free energy of ITZ ASD tablets containing different HPMCAS grades and drug loadings were evaluated using a Drop Shape Analyzer. A larger contact angle was correlated with a higher dissolution rate, suggesting that contact angle could be a high throughput tool for screening ASDs formulations. Lastly, we investigated the influence of fillers such as microcrystalline cellulose, lactose, mannitol, and starch on drug release and stability of ITZ-HPMCAS ASDs. We discovered that the dissolution performance and physical stability of tablets were influenced by the choice of filler. The results and inferences drawn from this research will provide valuable insights into ASD formulation development downstream tablet production.
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The Regulatory Role of the Cytoplasmic Heme Binding Protein PhuS in Pseudomonas aeruginosaPseudomonas aeruginosa is an opportunistic pathogen that requires iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either system is eventually sequestered by the cytoplasmic protein PhuS, which specifically shuttles heme to the iron-regulated heme oxygenase HemO. Furthermore, a conformational rearrangement upon heme binding is necessary for the protein-protein interaction with HemO and a ligand switch between the heme coordinating ligands (His209 and His212) was proposed ot be required for translocation of heme to HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipation-PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron- responsive sRNAs prrF1, F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, can also modulate PrrF and PrrH levels in response to heme. The dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host. Additional biophysical, genetic and metabolic approaches have been conducted to determine the role of the PhuS heme coordinating residues regulate the mutual exclusivity of heme and DNA binding and the resulting effects on PrrF and PrrH expression.
