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Title: ROLES OF RESPIRATORY CHAIN ENZYMES IN BIOFILM FORMATION OF PSEUDOMONAS AERUGINOSA
Creator: DING, JIE
Date: 2019
Description: Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium, resistant to many antibiotics. It can cause several chronic infections such...
Show morePseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium, resistant to many antibiotics. It can cause several chronic infections such as lung, bloodstream, urinary tract, and surgical wound infections. This bacterium produces biofilms which confer resistance to hazardous environments. P. aeruginosa contains five stages of colony development, which are planktonic attachment, cell to cell adhesion, proliferation, maturation, and dispersion. After five stages, biofilms of P. aeruginosa are matured. The biofilm structure produced by P. aeruginosa is important for cell survival, providing protection and resistance to harsh environment and antibiotics. In this research, the biofilms formed by wildtype strain PA01 and mutated strains of PA14, including NDH-2, NQR F, and NUO I, were developed in LB medium and Artificial Urine Medium separately for 96 hours. After washing, collecting, and staining the biofilms, the analyses of measurement of OD562 showed that in LB medium, PA01 formed more biofilms than mutants while NUO I and NDH2 had less biofilms, although not significantly. In AUM the situation was different. PA01 formed least biofilms while NQR F formed largest biofilms than any other strains. Also, the NDH-2 formed more biofilms than NUO I in AUM. The deficiencies of enzymes loss in those strains result in growing biofilm concentrations. Because the difference was not significant, we can only say that the NQR and NADH dehydrogenases have important roles in biofilm formation.
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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: Inflammation induced changes in adipocytes
Creator: Kim, Kihwan
Date: 2019
Description: The significant features of Crohn’s Disease include creeping fat that covers more than 50% of both small and large intestine surfaces and high...
Show moreThe significant features of Crohn’s Disease include creeping fat that covers more than 50% of both small and large intestine surfaces and high level of inflammatory cytokines such as TNF-alpha and IL-6. However, the relationship between these two factors of Crohn’s Disease is still unknown. Therefore, verifying the relationship could contribute to understanding the cause of Crohn’s Disease. In this study, preadipocytes were used because they have a potential to grow as adipocytes which are developed as creeping fat. The objective of this study was to observe proliferation, differentiation, and chemotaxis of preadipocytes in inflammatory microenvironment. It was found that only TNF-alpha stimulates preadipocyte proliferation whereas IL-6 does not. However, both TNF-alpha and IL-6 inhibit differentiation of preadipocytes. Furthermore, preadipocytes did not have chemotactic responses towards both cytokines. Therefore this study concludes that these inflammatory microenvironments induce the preadipocytes proliferation in Crohn’s. However, they inhibit adipogenesis and recruitment of the preadipocytes in Crohn’s.
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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: 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: PURIFICATION AND ANALYSIS OF BAXΔ2 PROTEIN AGGREGATES FROM MAMMALIAN CELLS
Creator: Wang, Xiling
Date: 2020
Description: BaxΔ2 is a unique isoform of the proapoptotic protein Bax that does not target mitochondria. The proapoptotic function of BaxΔ2 is through...
Show moreBaxΔ2 is a unique isoform of the proapoptotic protein Bax that does not target mitochondria. The proapoptotic function of BaxΔ2 is through forming cytotoxic aggregates in the cytosol. The cytotoxicity of BaxΔ2 is known as associated with the BH3 killing domain and the C-terminus, which recruits caspase 8. BaxΔ2 proteins without C-terminal form large cytosolic protein aggregates unable to induce caspase 8-dependent cell death. Since abnormal cytosolic protein aggregates often contain complexes of proteins that involved in many diseases, we would like to purify BaxΔ2 aggregates and examine their components. In this study, we expressed GFP-tagged BaxΔ2(Δ6) in the Bax-negative HCT116 cell line and purified the aggregates via different digestion processes. We found that most aggregates were trapped into a DNA pellet after cell lysis. Digestion with DNase could release the aggregates, which were susceptible to detergent solvent. The yield of purification is very low and needed improvement. The results from Western Blot showed that, in addition to BaxΔ2 proteins, stress granule protein TIAR was also potentially in the aggregates. Identification of the components inside aggregates will help us to understand the mechanism of BaxΔ2 cytotoxicity.
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Title: IDENTIFICATION OF BAX∆2 FRAMESHIFTING REGION VIA DUAL LUCIFERASE ANALYSIS
Creator: Reiner, Katherine
Date: 2020
Description: The antitumor protein Bax is susceptible to microsatellite instability (MSI) mutations that alter its open reading frame by changing Baxs’...
Show moreThe antitumor protein Bax is susceptible to microsatellite instability (MSI) mutations that alter its open reading frame by changing Baxs’ microsatellite of eight guanines (G8) to seven guanines (G7). This mutation results in a frameshift that is corrected by alternative splicing, making Bax∆2. Evidence shows that non-MSI mutated full length Bax∆2 (Bax∆2 G8) can be found in tissue. However, the extra guanine in Bax∆2 should result in premature termination of protein synthesis. Therefore, we believe that Bax∆2 is capable of +1 frameshifting to correct the out of frame sequence caused by splicing. The dual luciferase assay system is a useful tool for measuring frameshifting and in this study, we cloned full length Bax∆2 G8 into a dual luciferase vector to analyze frameshifting. Using this method, we found that the full length Bax∆2 G8 sequence has 3.5% frameshifting activity. To further determine whether the frameshifting occurs in or near the G8 microsatellite, we focused on several truncated constructs containing the first three exons. The results from dual luciferase assay showed that frameshifting activity was high in the constructs containing the G8 microsatellite but diminished when the G8 microsatellite region was removed. Surprisingly, constructs containing exon 4 and 5, which are away from the predicted frameshifting region, also showed frameshifting activity. One possibility to explain these results is that mRNA structures, which are critical to frameshifting, could be altered by construct truncation and consequently lead to artificial frameshifting. Thus, using truncated constructs may not be a viable option for testing frameshifting activity. To maintain mRNA integrity, point mutations within the full sequence, could be a better option to identify the frameshifting site.
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Title: EXPERIMENTAL AND STRUCTURAL STUDIES OF FDA APPROVED EXON SKIPPING TREATMENT DRUGS
Creator: Zhang, Jingwen
Date: 2021
Description: DMD is an X-chromosome related genetic disease caused by loss of dystrophin protein expression, and which impacts 1 in 5000 boys born in the...
Show moreDMD is an X-chromosome related genetic disease caused by loss of dystrophin protein expression, and which impacts 1 in 5000 boys born in the world. The usual cause of this at the genetic level is a frame shift due to internal deletions of one or more exons that results in a change of the reading frame. This results in loss of expression of the protein encoded by this gene, dystrophin, which in turn leads to the disease phenotype. Exon skipping is a therapy for DMD which restores dystrophin pre-mRNA reading frame to produce a modified dystrophin. This is done by antisense oligonucleotides, AONs, which disturb the process of exon splicing and exclude targeted exons near the patient’s defect which restore the correct reading frame in the pre-mRNA transcript. In 2016, the first AON was approved for clinical use targeting exon 51, called eteplirsen. This provided the first disease modifying therapy for DMD, but it was only relevant to ~6% of patients who had defects that were correctable by skipping this specific exon. In 2020 two more AONs targeting exon 53 were developed, viltolarsen and golodirsen, providing benefit to an additional 5% of patients, and in 2021 casimersen targeting exon 45 was approved.However, this raises an interesting issue, in that for some patients, with an exon 52 deletion, skipping exon 51 or skipping exon 53 could both restore the reading frame. Which approved exon skipping treatment is better and the differences between them are still unknown. This is the aim of this study: to help patients figure out which AON can have a consequence of less long-term health problems like cardiomyopathy and longer life and get more precise treatment. We selected three exon skipped edits – two that represent exon 53 skipping repair of an underlying Δe52 defect and one targeting exon 51 skipping repair of a Δe52 defect. We then used a panel of biophysical and biochemical including dynamic light scattering, circular dichroism Spectroscopy, thermal denaturation, and protease K challenge to investigate the biophysical characteristics of these different exon skipped edits. From our results we found that Δe51-52 has the more structure (i.e., is less perturbed), compared to e52-53, as assessed by CD or by proteinase K challenge, but it also has lower thermal stability, with a low Tm=48C transition that begins to unfold at the physiological relevant temperature of 37C. On the other hand, e52-53 has less helical structure, but what structure did form had unfolding transitions in the normal range for wild type STRs, Tm> 60C; but this edit also had more non-helical structure. So, the total experimental results of these three edits are very complex, which may be due to the fact that these edits span the normally unfolded H3 region.
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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: DEVELOPING FUSION BACTERIOCINS FOR ERADICATING PSEUDOMONAS AERUGINOSA BIOFILMS
Creator: An, Sungjun
Date: 2022
Description: The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality in cystic fibrosis patients and...
Show moreThe opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals. Due to its remarkable ability to resist antibiotics, eradicating P. aeruginosa has become increasingly difficult. As previously reported, we have successfully engineered a colicin-secretion system that kills target biofilm cells rapidly and selectively in multispecies biofilms as well as demonstrated the potential of using live microorganisms engineered to produce antimicrobial colicin protein to treat biofilm-associated infections. In this study,we constructed a fusion colicin-pyocin that could target P. aeruginosa by DNase activity of colicin E2. The newly engineered bacteriocin-secretion system upon the shift in target, maintained biofilm inhibition capacity. Both during biofilm formation and after its development, the system was able to suppress the P. aeruginosa biofilm. This result opened up the possibility that it could be used for novel live biotherapeutics. A further study was conducted to overcome the challenge of requiring an exogenous inducer. We applied the concept of Quorum-Sensing signal that recognize autoinducer as a trigger of fusion colicin-pyocin producing genetic circuit so that it automates the production and secretion of fusion colicin-pyocin as soon as the genetic circuit senses the target population growing. This study demonstrated that combining the domains of colicin and pyocin could broaden the genetic circuit target range, maintaining strain specificity, while employing the QS system could remove the fundamental problem of diffusion or degradation of extra compounds as they approach engineered cells.
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Title: IDENTIFICATION OF THE RIBOFLAVIN BINDING SITE IN VIBRIO CHOLERAE ION PUMPING NQR COMPLEX
Creator: Lee, Chia-Hsing
Date: 2021
Description: NQR is a six-subunit complex that transfers electrons from NADH to ubiquinone, one of the essential enzymes in the bacterial respiratory chain...
Show moreNQR is a six-subunit complex that transfers electrons from NADH to ubiquinone, one of the essential enzymes in the bacterial respiratory chain of many pathogens such as Vibrio cholerae, Pseudomonas aeruginosa, Chlamydia trachomatis. Its electron transfer path requires three different flavin cofactors to facilitate: FAD, FMN, and riboflavin. The FMN in subunit B (FMNB) brings electrons to riboflavin and then transfers it to the final electron receptor UQ in subunit B, coupled with the Na+ pumping mechanism. NQR has a unique evolutionary history, and one of the pieces of evidence is that NQR has been reported as the only one flavoenzyme that uses riboflavin as its redox cofactor. However, the binding site of riboflavin has not been well understood. To gain insight into the electron transfer at this site in V.cholerae NQR, we generated mutants at the interface of subunits B, D, and E where the possible location of riboflavin is. To characterize these mutants, we assessed NQR properties with different approaches including enzyme kinetics and flavin radical profiling. We found that the mutagenesis surrounding the hydrophobic pocket disrupted the NQR activity, and cause the loss of neutral radical, but did not interfere with the binding affinity between the substrates and NQR. This study will help to understand electron transfer better in NQR and develop the drugs targeting the riboflavin binding site in the future.
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Title: LOW-COVERAGE GENOMES AS AN EFFECTIVE AND ECONOMICAL APPROACH FOR LEPIDOPTERAN MICROSATELLITE ISOLATION
Creator: Liang, Huijia
Date: 2020
Description: This study aimed to verify that whether a low-coverage genome can work as an effective approach to isolate Lepidopteran microsatellites. As...
Show moreThis study aimed to verify that whether a low-coverage genome can work as an effective approach to isolate Lepidopteran microsatellites. As microsatellites are useful tool to study population genetics, and there are many Lepidopteran agriculture pests which can cause huge economic damages every year, additionally, Lepidoptera have abundant similar flanking sequences making it difficult to develop reliable microsatellites. However, there are not enough published genomes of Lepidoptera species. If low-coverage Lepidopteran genomes can be used to isolate reliable microsatellites, the low-coverage genomes would be an effective and economical approach for microsatellites isolation, because low-coverage genome sequencing is much cheaper and less time-consuming than the published genome sequencing.
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Title: Utility of a Low-Coverage Genome Assembly for Discovery of Genes Associated with Pyrethroid Resistance in Smicronyx Fulvus LeConte
Creator: Markiv, Paulina Patrycja
Date: 2023
Description: Red sunflower seed weevil (RSSW) is a major insect pest of cultivated and wild common sunflowers in the Great Plains of North America. The...
Show moreRed sunflower seed weevil (RSSW) is a major insect pest of cultivated and wild common sunflowers in the Great Plains of North America. The extent of the sunflower damage due to RSSW infestation is too great for the natural sunflower defense mechanisms to protect the agriculture industry from losses. Pyrethroids are the only type of insecticide designated for the control of RSSW; however, instances of pyrethroid insecticide ineffectiveness against RSSW have been annually reported to entomologists at South Dakota State University since 2017. The biological bases of insecticide resistance are unknown but common mechanisms associated with pyrethroid resistance include general detoxification mechanism driven by cytochrome P450s (CYP450s) as well as mutations in the pyrethroid target, voltage-gated sodium channels (VGSCs). The goal of this study was to determine if the computational analysis of a low-coverage genome assembly is sufficient to identify and characterize genes associated with insecticide resistance which could contribute to pest control research efforts. By using a low-coverage genome assembly, RNA-Seq data, and bioinformatic tools, 40 complete and 33 partial gene models coding for CYP450 as well as a partial gene model coding for VGSC have been identified in the genome of RSSW. Twenty-seven mutation sites, previously associated with the pyrethroid resistance in other insects, have been identified in the VGSC gene of RSSW. The low-coverage genome proved to be a sufficient resource for preliminary studies of gene identification which could bring significant knowledge to subsequent research focusing on insecticide resistance and pest control.
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