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Title: Characterization of the Pseudomonas aeruginosa NQR Complex, a Novel Form of Bacterial Proton Pump, and the Ubiquinone Binding Site
Creator: Raba, Daniel Alexander
Date: 2019
Description: The proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of...
Show moreThe proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of bacteria, including pathogenic species such as Vibrio cholera, Pseudomonas aeruginosa, Chlamydia trachomatis, as well as others. Residing in the cytoplasmic membrane of these bacteria, the enzyme couples the transfer of electrons to the pumping of cations across the cell membrane. In all previously studied homologues, the enzyme generates a sodium gradient through its pumping activity that can be utilized by the cell to power essential homeostatic processes. Furthermore, the electrochemical gradient generated by this enzyme has been shown to regulate the production of virulent factors and the efficacy of antibiotic extrusion and elimination. Although certain homologues have been investigated, particularly that of V. cholerae (Vc-NQR), the NQR homologues belonging to important pathogenic species have not been well studied. In the research detailed in this thesis, the first characterization of the NQR of P. aeruginosa (Pa-NQR) is described which identified this homologue as a new form of bacterial proton pump, differentiating it from all other studied homologues of NQR. Additionally, as part of this study our research group characterized the mechanism of inhibition of Pa-NQR by the molecule HQNO which is produced by P. aeruginosa and is known to be a strong inhibitor of Vc-NQR. Our results show that Pa-NQR possesses resistance to inhibition by this molecule compared to Vc-NQR, pinpointing residue F155 of subunit D as being important to resistance and the type of inhibition to be partial-mixed. Moreover, in further developing the understanding of the NQR of V. cholerae, we investigated the binding site of ubiquinone, the final electron acceptor of NQR’s electron transfer process, determining residues P185, L190, and F193 to be important for maintaining the structural composition of the ubiquinone pocket, ensuring efficient substrate binding and catalysis.
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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: Small Molecule Ligands and Antibody Drug Conjugates for Potential Application in Targeted Cancer Therapy and PET Imaging
Creator: Ren, Siyuan
Date: 2020
Description: Cancer has been a deadly disease threatening humans. Traditional treatment methods such as chemotherapy with non-specific targeting may cause...
Show moreCancer has been a deadly disease threatening humans. Traditional treatment methods such as chemotherapy with non-specific targeting may cause severe side effects to normal cells and tissues. Novel and better methods for treatment and imaging of cancer has been eagerly sought. We wanted to develop new small molecule inhibitors and antibody drug conjugates for targeted cancer therapy and imaging. Theranostic conjugates combining therapeutic entity and imaging agent are useful in treatment and diagnosis of cancer. We designed a theranostic conjugate containing iron chelating anti-tumor agent and optical imaging probe. Novel iron chelator CAB-NE3TA displayed significant anti-proliferation activity against several cancer cell lines and was conjugated to targeting antibody panitumumab (PAN). The therapeutic conjugate exhibited excellent anti-tumor efficacy and targeting ability to EGFR overexpressed on cancer cells such as skin cancer (A431). The theranostic conjugate CAB-NE3TA-PAN loaded with a near IR fluorescent dye provided promising anti-tumor efficacy and optical imaging in tumor-bearing mice. Tetrahydroisoquinoline (THIQ) analogues were synthesized and evaluated for cytotoxicity against different cancer cell lines. Our in vitro studies showed that the THIQ analogues exhibited anti-proliferative activities against multiple cancer cell lines. Cytotoxicity study revealed that anti-tumor activity of THIQ analogues are structure-dependent. Binding affinity between THIQ analogues and a potential target Tdp1 was determined. Positron emission tomography (PET) has been used in diagnosis of cancer. In this study, small molecules and chelators with potential donor groups were evaluated systematically for complexation with 89Zr for PET imaging. New ligands with different donors were evaluated for radiolabeling efficiency and complex stability with 89Zr. The theranostic conjugate (CAB-NE3TA-PAN-IR800) and small molecule THIQs and 89Zr-chelators showed encouraging results for potential applications in for therapy and imaging of cancer.
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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: Kinetic and Structural Characterization of the Vibrio cholerae Flavin Transferase ApbE
Creator: Fang, Xuan
Date: 2019
Description: Cholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the...
Show moreCholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the antibiotic-resistance of its causative agent, V. cholerae. The resistance is mainly supported by a transmembrane electrochemical gradient of Na+ produced by the respiratory complex Na+-NQR coupled with an internal electron transfer pathway. The assembly and function of Na+-NQR is fulfilled by ApbE, the only known flavin transferase which covalently attaches two FMN molecules to the complex as part of its electron transport chain. Hence, ApbE is closely associated with the cause of antibiotic resistance. Because it does not have any human homologues, ApbE becomes an excellent drug target. In this work, we have investigated the physical properties of the enzyme and clarified its substrate specificity and pH dependence. For instance, our experiments indicate that divalent cations are essential for ApbE function, and that the selectivity depends largely on the size and the coordination sphere of the cation. Our data also show that ApbE regulation by pH, ADP and potassium is an important mechanism that enhances the adaptation, survival and colonization of V. cholerae in the small intestine. Moreover, pH dependence, mutagenesis, and steady-state kinetic studies have led us to identify the conserved His257 as a residue with dual roles: substrate binding and catalysis. Furthermore, bi-substrate kinetic studies have also revealed that ApbE follows a random Bi Bi mechanism. Together with structural studies, we propose a reaction mechanism where His257 functions as a base, shedding light into the understanding of the ApbE family.
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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: Stimulation-Responsive Materials for the Treatment of Disordered Tissues
Creator: Clutter II, Elwin Dean
Date: 2022
Description: Microenvironments offer physical and chemical cues to cells that affect their behavior. These cues can be bioactive chemicals such as drugs...
Show moreMicroenvironments offer physical and chemical cues to cells that affect their behavior. These cues can be bioactive chemicals such as drugs and cytokines, matrix cues like stiffness and composition, and electrical signals as seen in membrane potentials and epithelial wound healing. Controlling these cues can drive cell behavior toward apoptosis, proliferation, or changes in protein expression. In this thesis research, three components were used to model changes in cell behavior: the near infrared dye indocyanine green (ICG), silk fibroin (SF) from Bombyx mori silkworm cocoons, and carbon nanotubes (CNT). The mechanism of ICG photobleaching was studied for the efficacy to kill neuroblastoma cancer cells. ICG was twice as effective at killing neuroblastoma than fibroblast cells. Albumin stabilized monomeric ICG to enhance photobleaching by improving light interaction, and photodegradation of ICG into α,β-unsaturated aldehydes led to significant reduction of proliferation in neuroblastoma cells by targeting cell signaling components such as protein transcription factors. SF-ICG composite materials were developed into films and electrospun fibers. These composite materials were examined as light-activated wound coverings to control bleeding in hemorrhage, using ICG’s photothermal effect. ICG added an absorbance peak to SF at 805 nm. Irradiation decreased this peak, produced a new absorbance peak at 352 nm, and an increased fluorescence peak around 490 nm, showing photochemical changes that may be useful in sensor design. Increased heat production from irradiation of SF-ICG occurred in the films with 0.2% w/w ICG in SF during 1 min irradiation, whereas SF-ICG fibers required improvement of processing by ethanol vapor treatment (EVT) to reduce loss of ICG during preparation. EVT improved ICG retention in SF fibers during sterilization with 70% ethanol solution thus improving heat generation in the SF-ICG fibers. Heat evolved from SF-ICG 0.2% film solidified bovine blood within 42 s, with visible changes after the first 6 s. SF electrospun fibers were investigated to optimize silk preparation to reduce diameters and increase alignment to mimic local native cell environments with and without CNT to add conductivity for enhancement of electrical stimulation. CNT reduced SF fiber diameters below 1% and effected alignment differently as the concentration increased. Longer degumming times decreased SF fiber diameters and alignment of the same concentration. The optimized conditions were 1 h degumming time with 1.5 ml/h flow rate at a concentration of 100 mg/ml spinning solution with 0.25% CNT to form fibers with 1.36 (± 0.09) µm diameter and 0.31 (± 0.01) a.u. alignment. Additionally, SF fibers were used as a long-term cell growth scaffold to compare with decellularized native tissue. Decellularized tissue decreased fibroblast mRNA expression of collagen type 1, lysyl oxidase-like 1 and matrix metalloproteinase 9, while all other genes expression was the same as cells on plastic. The SF fiber scaffold reduced fibroblast expression of collagen type 3 compared to growth on plastic, and both collagen types 1 and 3 increased over growth time on SF. Immunofluorescence staining showed both collagens newly deposited on the SF scaffold and improved over time. In conclusion, proteins stabilized ICG monomers to improve light interaction, efficacy of photobleaching, and heat evolution to induce apoptosis in neuroblastoma and agglutination of blood in SF composites. Irradiation of ICG enhanced changes in optical properties and may have sensor applications. The decrease of genes may be due to each scaffold having lower stiffness compared to plastic. ICG and SF are both materials with applications as biological scaffolds in pelvic organ prolapse and treatment of neuroblastoma and hemorrhage.
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Title: STRUCTURE AND DYNAMICS OF MODIFIED NUCLEOSOMES UNDER EPIGENETIC REGULATION
Creator: Kohestani, Havva
Date: 2022
Description: Epigenetic regulations are critical in inducing heritable phenotype changes in biological systems without alternating their core genetic DNA...
Show moreEpigenetic regulations are critical in inducing heritable phenotype changes in biological systems without alternating their core genetic DNA sequences. In vivo, reversible epigenetic mechanisms engage various molecular structures from RNAs to larger proteins. The present thesis investigates the influence of epigenetic regulatory factors such as histone protein variants and small non-coding RNAs on the dynamics and structure of nucleosome core particles. Our results show that a histone substitution is an efficient tool in increasing or decreasing the exposure of DNA to post-translational modification (PTMs) factors or larger molecular assembly elements. Substitution of canonical H2A with H2A.B alters DNA-dimer interface resulting in increased breathing and accessibility of DNA. Replacement of canonical H3 with CENP-A variant impacts the overall core-DNA dynamics with flexibility of DNA entry/ exit sites and more rigid tetramer structure. Histone substitution also affects the micro to macro level molecular communication in the nucleosome system. The long-range correlated motions are weakened in H2A.B compared to canonical NCP. We observed a reduction in effective long-range DNA-DNA and DNA-core allosteric pathways in CENP-A NCP compared to canonical and Widom NCPs. Non-coding RNAs increase the tendency of the H3 tail histones to interact with DNA and induce the structural changes in the initial ideal B-DNA of NCP. Overall, the interaction of epigenetic regulatory factors in the form of protein or nucleic acids shifts the energetic and structural properties of the original nucleosome system. As a result, the chromatin structure is prepared to generate the proper biological response throughout spermatogenesis, chromosome segregation, or PTMs assembly.
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Title: COMPREHENSIVE ANALYSIS OF EXON SKIPPING EDITS WITHIN DYSTROPHIN D20:24 REGIONS
Creator: Niu, Xin
Date: 2020
Description: Exon skipping is a disease modifying therapy that operates at the RNA level. In this strategy, oligonucleotide analog drugs are used to...
Show moreExon skipping is a disease modifying therapy that operates at the RNA level. In this strategy, oligonucleotide analog drugs are used to specifically mask specific exons and prevent them from being included in the mature mRNA. Exon skipping can also be used to restore protein expression in cases where a genetic frameshift mutation has occurred, and this how it is applied to Duchenne muscular dystrophy, DMD. DMD most commonly arises as a result of large exonic deletions that juxtapose flanking exons of incompatible reading frame, which abolishes dystrophin protein expression. This loss leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is to restore the reading frame by skipping an exon adjacent to the patient’s original. While restoring some protein expression is good, how removing some region from the middle of protein affects its structure and function is unclear. Complicating this in this case is that the dystrophin gene is very large, containing 79 exons. Many different underlying deletions are knowns, and exon skipping can be applied in many ways. It has previously been shown that many exon-skip edits result in structural perturbations of varying degrees. Very few studies are focused on the protein biophysical study and it is still basically unclear whether and how such editing can be done to minimize such perturbations. In order to provide the solid evidences which prove the significant variation among those cases (especially for the clinically relevant cases) and better understanding the general principles of “what makes a good edit”, we examine a systematic and comprehensive panel of possible exon edits in a region of the dystrophin protein. The domain D20:24 of dystrophin rod region are selected for its entirety which is separated by hinge region (mostly random coiled structure) and addition of other STRs will not disrupt the structure stability. Also D20:24 regions lie in the Hot Spot region II (HS2) which holds the most number of DMD patients. During the comprehensive scan, we identify for the first time, exon edits that appear to maintain structural stability similar to wild-type protein and those clinically relevant edits. Then we figure out the factors that appear to be correlated with the degree of structural perturbation, such as the number of cooperative protein domains, as well as how the edited exon structure interacts with the protein domain structure. Our study is the first systematic and comprehensive scan for an entire multiple STRs domain. This would help us understand the protein nature of various exon skipping edits and provide useful target for clinical treatment. Also the knowledge we learned may be applied to produce more sophisticated CRISPR edits in the future work.
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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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Title: UTILIZING BACTERIAL INTERACTIONS TO CONTROL PATHOGENIC BIOFILM FORMATION
Creator: Fang, Kuili
Date: 2020
Description: Many chronic infections involve bacterial biofilms, which are difficult to eliminate using conventional antibiotic treatments. Biofilm...
Show moreMany chronic infections involve bacterial biofilms, which are difficult to eliminate using conventional antibiotic treatments. Biofilm formation is a result of dynamic intra- or inter-species interactions. However, the nature of molecular interactions between bacteria in multi-species biofilms are not well understood compared to those in mono-species biofilms. The first project (Chapter 3) investigated the ability of probiotic Escherichia coli Nissle 1917 (EcN) to outcompete the biofilm formation of pathogens including enterohemorrhagic E. coli (EHEC), Pseudomonas aeruginosa, Staphylococcus aureus, and S. epidermidis. When dual-species biofilms were formed, EcN inhibited the EHEC biofilm population by 14-fold compared to EHEC mono-species biofilms. This figure was 1,100-fold for S. aureus and 8,300-fold for S. epidermidis; however, EcN did not inhibit P. aeruginosa biofilms. In contrast, commensal E. coli did not exhibit any inhibitory effect toward other bacterial biofilms. We identified that EcN secretes DegP, a bifunctional (protease and chaperone) periplasmic protein, outside the cells and controls other biofilms. Although three E. coli strains tested in this study expressed degP, only the EcN strain secreted DegP outside the cells. The deletion of degP disabled the activity of EcN in inhibiting EHEC biofilms, and purified DegP directly repressed EHEC biofilm formation. Hence, probiotic E. coli outcompetes pathogenic biofilms via extracellular DegP activity during dual-species biofilm formation. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a pathogen causing the outbreaks of hemorrhagic colitis. Conventional antibiotics treatment is not recommended for EHEC infection as antibiotics trigger Shiga toxin production of EHEC and aggravate hemolytic-uremic syndrome. EHEC biofilm formation is closely associated with its virulence expression. Previously, we identified that probiotic E. coli Nissle 1917 (EcN) secretes DegP resulting in the inhibition of EHEC biofilm formation in a dual culture. DegP is a serine protease exhibiting both proteolytic and chaperone functions and binds to outer membrane proteins (OMPs) of target cells. However, the extracellular function of DegP is not clear. We hypothesized that binding of DegP to OMPs of EHEC might inhibit EHEC biofilm formation by affecting the adhesion ability or changing biofilm-related gene regulations of EHEC. We constructed EHEC mutants lacking ompA, ompC, or ompF individually and in combination and assessed their biofilm formation in the presence of DegP-secreting EcN in the co-culture or by adding purified DegP. It was found that both ompA and ompC double deletion decreased EHEC single species biofilm, and also caused that DegP inhibited more EHEC biofilm (about 25 fold inhibition) than DegP inhibited EHEC wt biofilm (about 10 fold), indicating that OmpA and OmpC are more related to EHEC biofilm than OmpF, and OmpA and OmpC might deplete DegP inhibitory functions. On the other hand, DegP S210A, a DegP mutant lacking protease function, inhibited EHEC wt biofilm, indicating that DegP’s biofilm inhibition function is not from its protease activity. Additionally, EHEC transcription profiles in the presence of DegP showed that DegP up-regulated expressions of cellulose production related genes (csgD and bcsA) and motility related genes (flhD, qseB), which were all involved in EHEC biofilm inhibition, and down-regulated Shiga toxin 2 virulence gene (stx2). Besdies, DegP promoted EHEC cellulose production and motility, which is consistent with transcription profile, and Shiga toxin 2 production will be further tested. This study reveals a new function of DegP secreted by EcN in controlling biofilms and leads us to develop an alternative strategy to control biofilm-related infections. Foodborne pathogen Listeria monocytogenes biofilm formation renders these cells highly resistant to current sanitation methods, and probiotics may be a promising approach to the efficient inhibition of Listeria biofilms. In the Chapter 5 study, three Leuconostoc mesenteroides strains of lactic acid bacteria isolated from kimchi were shown to be effective probiotics for inhibiting Listeria biofilm formation. Biofilms of two L. monocytogenes serotypes, 1/2a (ATCC15313) and 4b (ATCC19115), in dual-species culture with each probiotic strain were decreased by more than 40-fold as compared with single-species Listeria biofilms; for instance, a reduction from 5.4 times 10^6 CFU/cm2 L. monocytogenes ATCC19115 in single-species biofilms to 1.1 times 10^5 CFU/cm2 in dual-species biofilms. Most likely, one of the Leuconostoc strains, L. mesenteroides W51, led to the highest Listeria biofilm inhibition without affecting the growth of L. monocytogenes. The cell-free supernatant from the L. mesenteroides W51 culture containing large protein molecules (> 30 kDa) also inhibited Listeria biofilms. These data indicate that Leuconostoc probiotics can be used to repress L. monocytogenes biofilm contamination on surfaces at food processing facilities.
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Title: The role of fibrillar collagen in tissue function
Creator: Ma, Yin
Date: 2020
Description: Fibrillar collagen plays an important role in maintaining soft tissue integrity and providing chemical and physical cues for cell fate...
Show moreFibrillar collagen plays an important role in maintaining soft tissue integrity and providing chemical and physical cues for cell fate decisions. Collagen remodeling, which alternates the amount, distribution, and biomechanics of collagen, primarily type I (COLI) and type III (COLIII), can change tissue properties. This process is essential not only in biological developments but also in pathological processes. Thus, it is meaningful to understand the correlation between collagen remodeling and tissue dysfunction and investigate the cells' response to fibrous protein matrices. However, current studies in biochemical analysis of collagen and biomechanical study of tissues were carried out at different scales. So it is hard to correlate the data to draw solid conclusions. In this thesis research, we used two collagen disorder associated pathological conditions, pelvic organ prolapse (POP) and micropapillary serous carcinoma (MPSC) of the fallopian tube, as models to unravel the correlation between tissue dysfunctions and the impaired microenvironment relevant to the composition, nanostructure, and biomechanics of a collagen fibril. In the case of POP, we found the collagen fibers in tissues of POP patients were less abundant but stiffer than those of non-POP individuals, implying a loose and fragile matrix that is weakly integrated with other components of the connective tissue to provide adequate support of the pelvic organs. On the other hand, the collagen D-period, the characteristic banding feature which signals the proper assembly of collagen molecules, decreased in POP tissues. We surmised that the molecular level changes of collagen in POP were accountable for the weak matrix mechanics, verified by a systematic in vitro study. We also examined the collagen matrix alternation in MPSC of the fallopian tube, which is thought to cause ovarian cancer via metastasis. Since cancer metastasis is often related to collagen remodeling, we examined the collagen matrix alternation in this disease. We observed the heterogeneous distribution of COLI and COLIII in the papillae of the tumor tissue. Noticeably, COLI was accumulated at the papillae tip, whereas COLIII was dominant at the papillae base. We also observed the absence of collagen matrix between the micropapillary tip and the fibrosis base. Such an uneven collagen distribution implies that the matrix exerted distinctive forces on the tumor cells to regulate their behaviors, including cell migration, directional growth, and shedding from the primary tumor to initiate metastasis. These conclusions have been supported by the results of our in vitro experiments. In investigating the effect of the microenvironment on cell behavior, we established and validated an AFM-based method to collect and quantitatively analyze the mRNA samples from targeted live cells at the single-cell level. This method overcomes issues, such as severe cell damage or even cell death, the capability of time-dependent and in situ analyses, in current methods. The application of the method in studying heterogeneous gene expression in single cells and the interaction between cancer cells and cancer-associated fibroblasts was demonstrated. We also demonstrated that this method can be potentially used to quantitatively analyze the gene expression level changes in a targeted cell in response to the microenvironment.
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