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Title: Characterization of the role of His257 of vibrio cholerae ApbE in the flavin transfer reaction
Creator: Yuan, Ming
Date: 2019
Description: ApbE is a novel enzyme that transfers flavin cofactors into subunits NqrB and NqrC of the sodium-dependent NADH dehydrogenase (Na+-NQR). As...
Show moreApbE is a novel enzyme that transfers flavin cofactors into subunits NqrB and NqrC of the sodium-dependent NADH dehydrogenase (Na+-NQR). As the first enzyme of the bacterial respiratory chain, the function of Na+-NQR affects the survival and development of pathogenicity in many disease-causing bacteria, including Vibrio cholerae. Our preliminary studies indicate that His257 plays a key role in the catalytic activity of ApbE, and that it is an essential component in the transfer of FMN to NqrC. In order to further study how His257 is specifically involved in the catalytic reaction of ApbE, we produced and characterized four mutants: H257G, H257E, H257K, and H257T; in the presence of the activator, K+. Our data showed that mutants H257E and H257K present minimal flavin transfer activity. Interestingly, the mutants H257G and H257T showed activity several times higher compared to the other mutants, however, their activities were still smaller when compared to wild-type. The data suggests that His257 has a very important role for ApbE activity, but that it is not essential. Furthermore, steady-state kinetics showed that the mutants have similar substrate KM values with the wild-type. In addition, double reciprocal plots from bi-substrate titrations showed that ApbE follows a sequential kinetic mechanism where a ternary complex is formed during the reaction.
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Title: THE K+ ACTIVATION MECHANISM OF V. CHOLERAE APBE
Creator: Yang, Jun
Date: 2019
Description: ABSTRACTNa+-translocating NADH: quinone oxidoreductase (NQR) is a protein complex that exists in the respiratory chain of Vibrio cholerae....
Show moreABSTRACTNa+-translocating NADH: quinone oxidoreductase (NQR) is a protein complex that exists in the respiratory chain of Vibrio cholerae. This complex can transport sodium ions to the outside of the plasma membrane. NQR has important influences on the survival and pathogenesis of V. cholerae. Two of the subunits of NQR, NqrC and NqrB, has a covalently bound FMN coenzyme. This FMN is necessary for the activity of NQR complex. A protein, alternative pyrimidine biosynthesis protein (ApbE) can transfer the FMN molecule to NqrB and NqrC covalently. And ApbE is also important to some other flavoproteins like the NOS and RNF. The ApbE protein use the FAD as the substrate to transfer the FMN group to the NqrC and NqrB apo-enzyme. Mg2+ is necessary for the activity of ApbE protein. Sodium and potassium ion are not necessary, but potassium ion can increase the activity of the ApbE by about ten times. In order to understand the mechanism of potassium activation of ApbE, several potassium binding sites were identified by molecular docking in this study. Point mutations of the amino acid residues constituting these sites were performed. The FMN transfer activity and affinity to potassium ions of these mutants were measured. The results suggest that when G125 was mutated, the binding of potassium ions was affected. Therefore, the structure composed of P126 and G125 may play a significant role in the activation of ApbE potassium ions.
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Title: Detection Of BAXΔ2 Reading Frame Shift Using A Dual Luciferase Reporter System
Creator: Beatty, Evan Alexander
Date: 2019
Description: While initial studies of the pro-apoptotic Bcl-2-associated X protein isoform Δ2 (BAXΔ2) identified the combination of an alternative splicing...
Show moreWhile initial studies of the pro-apoptotic Bcl-2-associated X protein isoform Δ2 (BAXΔ2) identified the combination of an alternative splicing event and a gene-level mutation as the prerequisites for biosynthesis in microsatellite unstable (MSI+) human colon cancer cells, no similar explanation existed to explain the presence of this protein in normal and normal adjacent tissues. To identify an alternative to the gene-level mutation in the absence of an MSI+ phenotype, we utilized a dual luciferase reporter assay designed to observe epigenetic recoding. Plasmid constructs containing the first two exons encoding BAXΔ2 were either transcribed and translated in vitro or transfected into BAX-negative human colon cancer cells. In both cases, assay of the protein products of the reporter genes demonstrate that a low level (2.82% in vitro, 4.43% in vivo) of all translational events which produce the protein product of an upstream reporter gene also produce the protein product of a downstream reporter gene. This occurs despite the two existing in different reading frames as a result of the BAX exons cloned between them. These results confirm that an epigenetic recoding event is able to salvage the BAX reading frame in cases where exon 2 has been excised, and further narrow down the potential mechanism involved to either transcriptional slippage or programmed ribosomal frameshifting.
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Title: MITOCHONDRIA RELOCALIZATION IN CHLAMYDIA TRACHOMATIS INFECTED HFF-1 CELLS
Creator: Shuppara, Alexander Mitchell
Date: 2021
Description: Chlamydia trachomatis is an infectious, gram-negative, obligate intracellular human bacterial pathogen. With over eight hundred million cases...
Show moreChlamydia trachomatis is an infectious, gram-negative, obligate intracellular human bacterial pathogen. With over eight hundred million cases worldwide, C. trachomatis is the most prevalent sexually transmitted infection. It manifests as either trachoma, lymphogranuloma venereum, or other urogenital tract sequelae. As an intracellular pathogen, Chlamydia must scavenge for essential metabolites from establishing networks with its host’s organelles including Golgi apparatus, endoplasmic reticulum, endocytic vesicles, mitochondria, and the cytoskeleton. C. trachomatis was considered an “energy parasite” that is entirely dependent on their host’s ATP production. Yet, recent mitochondrial inhibitor-based evidence suggests that C. trachomatis possess a sodium-based energy gradient for ATP production. Despite this finding, literature on specific interactions between host cell mitochondria and C. trachomatis requires further definition. This project evaluates mitochondrial dynamics changes from C. trachomatis infection in the human foreskin fibroblast cell line, HFF-1. We first defined C. trachomatis growth characteristics in HFF-1 over 36 hours-post infection. Next, we determined changes in mitochondrial dynamics and content throughout infection using immunofluorescent and immunoblotting techniques. observations on infected cells show mitochondrial morphology changes from an elongated appearance at the early stages of infection to fragmented in the late infection stages. Unlike in HeLa cells, HFF-1 remains in a normal distribution throughout the cell and we do not observe mitochondria relocalizing toward the inclusion. By studying mitochondrial relocalization dynamics, new insights into the dynamic and parasitic relationship of Chlamydia and its host can be discovered.
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Title: ENERGY METABOLISM OF CHLAMYDIA PNEUMONIAE
Creator: McMillan, B. Julia
Date: 2021
Description: Chlamydia pneumoniae is a gram-negative bacterium that infects the humanrespiratory tract. It causes acute pneumonia and has been linked to...
Show moreChlamydia pneumoniae is a gram-negative bacterium that infects the humanrespiratory tract. It causes acute pneumonia and has been linked to several chronic diseases including cardiovascular disease, asthma, and some neurological diseases. C. pneumoniae primarily exists in two forms, the elementary body (EB) and the reticulate body (RB). The EB infects host cells and the RB replicates inside them. In order to survive in and out of the host, it was thought that C. pneumoniae RBs obtain host ATP to use for energy, making it an “energy parasite.” However, genomic analysis indicated that it was also possible for C. pneumoniae to create ATP from its own respiratory chain using the Na + pump NADH Ubiquinone Oxidoreductase (Na + -NQR). Neither the details of the energy parasite theory nor the possibility of C. pneumoniae creating its own energy had been experimentally explored. This project used a pharmacological approach to explore C. pneumoniae host energy consumption at various developmental stages, examine a mechanism that the bacterium could use to produce its own energy, and assess the importance of a balanced Na + /H + gradient for energy production and maintaining homeostasis. Based on the genomic analysis, it was thought that C. pneumoniae would rely heavily on host ATP in the EB form but not the RB form, that inhibiting Na + -NQR would slow bacterial growth, particularly in RBs, and that disrupting the Na + /H + gradient would significantly reduce RB infection. The results indicate that in the EB form, C. pneumoniae relies on host ATP and requires a balanced Na + /H + gradient, but disrupting Na + -NQR does not hinder its growth. In the RB form, C. pneumoniae is not dependent on host ATP, nor on its own respiratory chain ATP, and is not impacted by an unbalanced Na + /H + gradient. Therefore, the energy parasite hypothesis appears to apply to C. pneumoniae EBs but not RBs. Furthermore, established C. pneumoniae infections are excellent at compensating for various environmental conditions and sources of energy, which proves challenging for drug design against C. pneumoniae.
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Title: Evaluation of Bax∆2 Positive-Staining in Skin Samples Using Two Immunohistochemical Methods
Creator: Basheer, Sana
Date: 2021
Description: BaxΔ2 is a pro-death and tumor suppressor protein that sensitizes cells to certain chemotherapies. Previous diaminobenzidine (DAB)-based...
Show moreBaxΔ2 is a pro-death and tumor suppressor protein that sensitizes cells to certain chemotherapies. Previous diaminobenzidine (DAB)-based staining revealed that Bax∆2 is found in all organs, including breast, colon, and skin tissues. In the skin, the Bax∆2 positive cells were mainly found in the basal cell layer of the epidermis with a few Bax∆2 positive cells in the connective tissue of the dermis, although their cellular identity was unknown. Previous literature has shown that melanin, which is found throughout the cells of the epidermis, is a brown color that provides no visual contrast to the DAB staining. While the DAB-based immunostaining showed cells that appeared to be Bax∆2 positive, this result needed to be confirmed. For this, a set of human skin samples from normal and cancerous tissue of various patients was examined. The co-staining of these samples for Bax∆2 and basal cells using immunofluorescence revealed that the apparent Bax∆2-positve DAB staining in epidermal basal cells and squamous cell carcinoma as false-positive, but the Bax∆2 positive cells found in the dermal connective tissue were not false positive—which is consistent with both previous DAB-based and fluorescence-based immunostaining. Using co-immunostaining for Bax∆2 with different cellular markers, the Bax2-positive cells in the connective tissue were identified potentially as macrophages and fibroblasts. Further studies are required to confirm the identity of the Bax∆2 positive cells in the connective tissue.
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Title: THE EFFECTS OF MODIFIED SURFACES ON INSULIN CRYSTALLIZATION
Creator: Hammadi, Okba Tahar
Date: 2021
Description: Engineered nucleation features (ENFs) were designed with the hope to improve the efficiency of protein crystallization and increase...
Show moreEngineered nucleation features (ENFs) were designed with the hope to improve the efficiency of protein crystallization and increase reproducibility both in quality and quantity. These ENFs were tested with human insulin as the protein of choice since it has flexible parameters, only one cofactor, and a large amount of commercially available crystal ready protein. Insulin crystallization on the ENFs will produce more crystals while also having a reduced crystallization on-set time compared to the control glass surface. The ENFs were compared to control surfaces under similar conditions and observed over time to record both onset-times and end times. The ENFs performed markedly better in on-set times, having an overall 87%-time reduction when compared to the control drops. The drops placed on the ENF produced more than 2.5x the number of crystals in the control drops.
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Title: Biophysical and Computational Characterization of CinDel Edits of Dystrophin
Creator: Stojkovic, Vladimir
Date: 2022
Description: Duchenne muscular dystrophy (DMD) is a degenerative genetic disease caused by a genetic defect that results in the absence of dystrophin, a...
Show moreDuchenne muscular dystrophy (DMD) is a degenerative genetic disease caused by a genetic defect that results in the absence of dystrophin, a protein with an important stabilizing role in muscle cells. DMD causes progressive muscle degeneration leading to the loss of ambulation, and typically results in death before the third decade of life. Treatments for DMD aim to restore dystrophin expression and typically do so by producing edited or modified dystrophins. The only FDA approved therapy, exon skipping, produces dystrophin edits at exon boundaries but emerging therapeutic approaches like gene replacement therapy and CRISPR-Cas9-based gene editing techniques like CinDel allow for greater flexibility and are not constrained to exon boundary edits. However, understanding of what makes a “good”, functional edit is limited so it is not clear how to make use of this increased flexibility to produce optimal edits which are believed to be necessary for robust treatment. In an effort to improve understanding of the biophysics of these non-exon edits, we have embarked on a mixed experimental and computational study of a set of CinDel edits in the D19-D21 region of the dystrophin central rod domain. First, we have conducted an Alphafold structure prediction-based screen of a subset of possible edits in this region and selected one edit for follow-up characterization. We then compared this computationally-selected edit to three other heuristically designed edits experimentally and computationally by molecular dynamics simulations. We found that the computationally selected edit is significantly more thermodynamically stable than the other edits in the cohort. This edit also generally exhibited more favorable properties in MD simulations across multiple measures such as helicity, STR-junction unwinding and conformational variability.
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Title: Synthesis and Photophysical Characterization of Novel Aromatic Triplet Dyes for Photodynamic Therapy Applications
Creator: Morgan, Jayla A
Date: 2022
Description: Photodynamic therapy is a biomedical approach to treating specific types of cancerous tumor cells and harmful bacteria. The core principle of...
Show morePhotodynamic therapy is a biomedical approach to treating specific types of cancerous tumor cells and harmful bacteria. The core principle of photodynamic therapy involves the usage of a photosensitizer, which is an agent with the capability of transforming molecular, triplet state oxygen, into a reactive oxygen species upon a reaction with near-infrared (NIR) light. The reactive oxygen species has been demonstrated to cause apoptosis among harmful cells without damaging cancer free cells. The effectiveness of photodynamic is highly dependent upon the identity of the photosensitizer; a powerful and efficient photosensitizer should be non-toxic, exhibit high light absorption capabilities, and should produce large amounts of the reactive oxygen species. A novel chromophore bis-iodo-dipyrrolonaphthyridine-dione was demonstrated to have all vital characteristics of an ideal photosensitizer, however produced low amounts of the reactive oxygen species of interest due to the chemical instability of a carbon-halogen bond present in the molecule. Various subsequent halogenations (bis-bromo and bis-chloro) completed in order to remedy this instability revealed specific regioselectivity in regards to the dipyrrolonaphthyridinedione parent that are exhibited upon substituents effects by the substrate, electronic effects exhibited by the reagents of interest, and overall photophysical characterization of the molecules.
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Title: AN IMPROVED VALIDATED METHOD FOR THE DETERMINATION OF SHORT-CHAIN FATTY ACIDS IN HUMAN FECAL SAMPLES BY GC-FID
Creator: Freeman, Morganne M
Date: 2022
Description: Short-chain fatty acids (SCFAs) are metabolites produced by the gut microbiota through the fermentation of non-digestible carbohydrates....
Show moreShort-chain fatty acids (SCFAs) are metabolites produced by the gut microbiota through the fermentation of non-digestible carbohydrates. Recent studies suggest that gut microbiota composition, diet and metabolic status play an important role in the production of SCFAs. Current methods for the analysis of SCFAs are complex and inconsistent between research studies. The primary objective of this study was to develop a simplified method for standardized SCFA analysis in human fecal samples by gas chromatography with flame ionization detection (GC-FID). A secondary objective was to apply the method to fecal samples from a previous randomized, crossover clinical trial comparing participants with pre-diabetes mellitus and insulin resistance (IR-group, n=20) to a metabolically healthy reference group (R-group, n=9) after daily consumption of a red raspberry smoothie (RRB, 1 cup fresh-weight equivalent) with or without fructo-oligosaccharide (RRB + FOS, 1 cup RRB + 8g FOS) over a 4-week intervention period. Extraction parameters, including solvent selection and water content of the sample, were investigated before finalizing the method. Freeze-dried fecal samples (0.5 g) were suspended in 5 mL of milli-Q water, vortexed and centrifuged at 3,214 x g for 10 minutes. The supernatant was transferred to a clean tube, acidified with 5.0 M HCl and centrifuged again at 12,857 x g for 5 minutes. The resulting supernatant was transferred to a GC vial for analysis by GC-FID. Linear regression data for standards at concentrations 5-2000 ppm ranged from 0.99994-0.99998. Limit of detection (LOD) ranged from 0.02-0.23 µg/mL. Limit of quantification (LOQ) ranged from 0.08-0.78 µg/mL. The validated method was then applied to fecal samples collected from a previously conducted study. Nine SCFAs were identified and quantified (acetic, propionic, iso-butyric, butyric, iso-valeric, valeric, 4-methyl valeric, hexanoic and heptanoic acids). Statistical analysis (Student’s t-test, ANCOVA) was performed on PC-SAS 9.4 (SAS Institute). Acetic acid was significantly lower in the IR-group compared to the R-group before starting intervention (baseline, Week 0, IR v R-group, p=0.014). Intervention analysis comparing RRB to RRB + FOS at 4 weeks (WK4) showed a significant difference in 4-methyl valeric acid (p = 0.040) in the R-group. Trends of decreased SCFA content after 4-weeks of RRB and RRB + FOS compared to baseline were observed in both groups, though changes were not significantly different between dietary interventions at 4 weeks (p>0.05). Metabolic status and dietary intervention are discussed in relation to their impact on SCFA content in fecal samples and mechanisms of biological use as a metabolite. Limitations of the study include sample size and using only feces and not other biological samples for SCFAs analysis, which may be considered for future research.
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Title: ELECTROSPUN SILKWORM SILK FIBROIN - INDOCYANINE GREEN BIOCOMPOSITE FIBERS: FABRICATION, CHARACTERIZATION AND APPLICATION TOWARDS HEMORRHAGE CONTROL
Creator: Siddiqua, Ayesha
Date: 2022
Description: Silk fibroin (SF), a structural protein found in the Bombyx mori cocoons has gained attention in several biomedical applications as tissue...
Show moreSilk fibroin (SF), a structural protein found in the Bombyx mori cocoons has gained attention in several biomedical applications as tissue engineering scaffolds and wound dressings owing to its properties such as biocompatibility, water vapor permeability and biodegradability. Indocyanine Green (ICG) is an FDA approved tricarbocyanine dye used in medical diagnostics due to its unique photothermal and fluorescent properties. Electrospinning is a highly efficient, easy, and inexpensive technique used to generate nanometer to micrometer thick fibers. In this study, SF and ICG were co-spun to generate flexible microfibers with high surface area to volume ratios. Pure silk, SF-ICG (0.1%) and SF-ICG (0.4%) were chosen for the purpose of this study. Since, as-spun fibers are unstable in aqueous solutions, post treatment methods were explored to enhance the durability of the fibers and to minimize ICG leaching. It was found that ethanol vapor treatment (EVT) not only induced β-sheet formation in SF but also improved the SF-ICG interaction thereby reducing ICG leaching from the composite fibers. Ethanol vapor treated SF-ICG fibers showed less ICG leaching than liquid ethanol treated (LET) SF-ICG fibers indicating the efficacy of the EVT. The increase in SF solution viscosity with ICG concentration suggested a strong silk-ICG interaction which was further confirmed by DSC. The 1h water uptake and the three-day mass loss experiments indicated that the fibers are stable and highly absorbent material. Heat evolution was evaluated by measuring the temperature change in water of a fixed volume after irradiation with a 500 mW, 808 nm diode laser. The heat evolved by the flat fiber scaffolds was higher than the 3D fiber balls, indicating improved light penetration in the former. Pure silk produced negligible heat and it was used as a control. With 14.9 W/cm2 irradiation, the post-treated SF-ICG (0.4%) 3D fibrous ball of 2-3 mg dry weight, solidified a drop of bovine blood in 40 s. In contrast, a single layer fiber matrix required 3 min. to achieve the same clotting effect. Fibers folded into flat scaffolds were able to solidify a blood drop in 25 s. Pure silk fibers in all the cases showed negligible change after irradiation. The results suggest that a larger contact area of fibers is desirable for faster blood clotting, and EVT prompted better ICG retention in SF fibers. Based on the above results, SF-ICG (0.4%) fibers were utilized in a device developed to mimic blood flowing at a rate of 0.5 mL/h through a damaged blood vessel. It was found that irradiation of SF-ICG locally placed at the “damage” region effectively stopped “bleeding” whereas irradiated pure silk was unable to control the blood flow, which demonstrated the success of our SF-ICG fibers towards hemorrhage control.
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Title: AMPLIFICATION AND PURIFICATION OF RECOMBINANT PRO-DEATH BAXΔ2 PROTEINS FOR STRUCTURE ANALYSIS
Creator: Zhou, Yi
Date: 2020
Description: BaxΔ2 is an isoform of the pro-apoptotic Bax family of proteins, which is an important anti-cancer protein. BaxΔ2 behaves differently from...
Show moreBaxΔ2 is an isoform of the pro-apoptotic Bax family of proteins, which is an important anti-cancer protein. BaxΔ2 behaves differently from Baxα to induce apoptosis. The current computationally predicted model of BaxΔ2 is based on known Baxα structure, which is considered biased. Therefore, the elucidation of the BaxΔ2 crystal structure is critical. The goal of this project was to obtain a sufficient amount of purified recombinant Bax∆2 protein for crystallization. We cloned full-length BaxΔ2 fused with a poly-histidine tag on either N-terminus (His-Bax∆2) or C-terminus (Bax∆2-His) into an inducible bacterial expression vector. We found that His-Bax∆2 proteins were expressed better than Bax∆2-His, which totally inhibit host growth. However, the protein concentration of His-Bax∆2 was still too low to be detected by Coomassie blue staining. To increase His-Bax∆2 expression and avoid cytotoxicity, we further tested different bacterial host cells and applied the chaperone system. However, all attempts could not overcome Bax∆2 cytotoxicity and the protein expression levels were not high enough to be feasible for further large-scale purification. The mechanism underlying how Bax∆2 inhibits bacterial growth is still a mystery because Bax∆2 eukaryotic targets (mitochondria and caspases) do not exist in bacteria. Further experiments are required to explore the mechanism of Bax∆2 cytotoxicity in bacteria, so as to finally optimize and elevate the BaxΔ2 protein yields.
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