Browsing by Author "Widger, William R."
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Item A Genome-Wide Search for Tumor Suppressor MicroRNAs in Ovarian Cancer(2014-08) Hernandez Herrera, Anadulce 1982-; Gunaratne, Preethi H.; Flores, Elsa R.; Wang, Yuhong; Zhang, Xiaoliu Shaun; Widger, William R.Ovarian cancer is one of the most lethal cancers among women. The Cancer Genome Atlas (TCGA) is a collaborative effort, which seeks to characterize the complete set of molecular changes associated with cancer and provide a public resource that will allow the development of new therapies and better diagnostic tools for cancer. Much of the focus is on protein coding genes and our understanding of the contribution from non-coding RNAs is lagging behind. MicroRNAs are small non-coding RNAs that can bind and repress hundreds of gene targets to regulate gene networks. Therefore, defining and understanding the miRNA-regulated genes offer new insights that can be clinically applied for many of the disease. In order to identify new tumor suppressors for ovarian cancer and downstream targets that drive key aspects of this disease such as drug resistance and metastatic spread, 3 candidates were selected from the microRNA-mRNA bioinformatic analyses from the TCGA. A combination of molecular and functional studies confirmed that miR-29a that can regulate genes from the histone modifier and cell cycle pathways, inhibit proliferation and moderately increase cisplatin response in the p53-WT HEYA8; miR-509-3p which targets genes from the ECM/EMT networks, inhibits cell proliferation in p53-WT HEYA8 and p53-mut OVCAR8 and correlated with improved overall survival when analyzed by in situ hybridization in an independent cohort; miR-130b increases apoptosis by 3-fold in p53-mutant OVCAR8 and p53-wild-type HEYA8 and significantly induces TAp63 and BCL2L11 (BIM). Forced expression of TAp63 decreases cell viability by 60-80% and miR-130b-ABT-737 (BCL2L11-mimetics) combination increases apoptosis by 9-fold suggesting TAp63 and BIM are critical effectors of the tumor-suppressive mechanisms driven by miR-130b, and can be used to develop new therapeutic strategies that will target p53 WT and p53 mutant tumors.Item A Proteomic Signature of Dormancy in an Actinobacterium: Micrococcus luteus(2017) Mali, Sujina; Rangel, Jonathan; Mitchell, Morgan; Havis, Spencer; Bodunrin, Abiodun; Khan, Arshad; Widger, William R.; Bark, Steven J.This project was completed with contributions from Arshad Khan from the Department of Pathology and Laboratory Medicine, UT Health Science Center, Houston.Item A Proteomic Signature of Dormancy in the Actinobacterium: Micrococcus luteus(2018-05) Mali, Sujina 1987-; Bark, Steven J.; Schwartz, Robert J.; Widger, William R.; Willson, Richard C.Dormancy is a protective state in which diverse pathogenic and non-pathogenic bacteria curtail metabolic activity to survive external stresses, including antibiotics. Evidence suggests dormancy consists of a continuum of interrelated states including viable-but-non-culturable (VBNC) and persistence states that contribute to the antibiotic tolerance. Reactivation from latent infection are observed in many serious pathogens including Mycobacterium turberculosis, Staphylococcus, Streptococcus, and Borrelia bacteria. Despite the obvious threat presented by dormant bacteria, the protein mechanisms regulating these dormancy states are not well understood. We have studied VBNC dormancy in Micrococcus luteus NCTC 2665 by tandem mass spectrometry-based quantitative proteomics to uncover some of these mechanisms. M. luteus is a nonpathogenic actinobacterium exhibiting a uniquely well-defined and reproducible VBNC state induced by nutrient deprivation. Dormant M. luteus demonstrated a global loss of protein diversity accompanied by increased levels of eighteen proteins that are conserved across actinobacteria including M. tuberculosis. Four of these proteins have been previously associated with latent tuberculosis, but the other 14 proteins are novel protein targets for dormancy studies. We have developed rapid methods to quantitate dormancy-related proteins across growth phases by targeted proteomics. The proteins upregulated during dormancy implicate important roles for anaplerotic metabolism, redox and amino acid metabolism, ribosomal regulatory processes, and nucleoid associated proteins in dormancy. Our data show that M. luteus is a viable model system for dissecting the protein mechanisms underlying dormancy and we identified new protein targets for future studies on therapeutics active against dormant bacterial infections, which is a severe limitation of current antibiotics.Item ALGORITHMS AND DATA STRUCTURES TO DETECT ONCOVIRUSES IN HUMAN CANCER USING NEXT GENERATION SEQUENCING DATA(2012-12) Zhu, Rui 1980-; Fofanov, Yuriy; Widger, William R.; Tsekos, Nikolaos V.Evidence suggests human cancer can be induced by viruses. One way to test this hypothesis is to look for viral sequences in the human cancer genome. Next Generation Sequencing (NGS) technology sequences the whole human genome in a short period of time. This opens a door for a systematic analysis of the human genome and a thorough search for oncogenic viral sequences in cancer. However, a huge amount of sequencing reads generated by NGS poses a great challenge on the computational part of data analysis in terms of computing speed and memory usage. Data structures such as hash and tree are widely implemented to improve the performance of computing algorithms. Here, I described both data structures that have been developed in our center and compared their performance. Hash out performed tree when mapping the reads to a small reference sequence database. Subsequently, real human cancer data were analyzed by using the hash-based mapper and different oncoviral sequences were found in different cancers.Item ATP and polyphosphoinositide metabolism in normal and diabetic peripheral nerve(1988) Chu, Hsi; Eichberg, Joseph E.; Bartel, Allen H.; Hazelwood, Robert L.; Widger, William R.Polyphosphoinositides (PPI) play an important role in tissue activity and PPI metabolism is altered in experimental diabetic neuropathy. Previous studies have shown that 32P incorporation is significantly increased into phosphatidylinositol-4,5-bisphosphate (PIP2) in sciatic nerve from streptozotocin-diabetic rats. It was also found that uptake of 32P into PIP2 in the proximal portion was more than 80% higher than in the distal portion of sciatic nerve and the altered metabolism of this lipid was predominantly associated with the distal portion in the diabetic states. The aims of the present study were to compare the metabolic characteristics of ATP and PIP2 and to investigate whether alterations in the precursor ATP pool could account for the changes in PIP2 labeling in diabetic nerve. [...]Item Characterization of Novel Liver X Receptor Ligands in Pancreatic Cancer(2020-12) Srivastava, Shivangi; Lin, Chin-Yo; Zhang, Weihua; Widger, William R.; Şen, Mehmet; Bailey, Jennifer M.Pancreatic ductal adenocarcinoma (PDAC) is the predominant form of pancreatic cancer with a high mortality rate due to the lack of early detection and effective treatment options for advanced diseases. Efforts to directly target mutant KRAS found in >94% of PDACs have not been successful due to structural constraints. An alternative strategy to directly targeting KRAS is to identify and target druggable receptors involved in dysregulated cancer hallmarks such as metabolic reprogramming downstream of KRAS dysregulation. Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-modulated transcription factors and are involved in the regulation of genes which function in key cancer-related processes, including cholesterol transport, lipid and glucose metabolism, and inflammatory and immune responses. Modulation of LXRs via small molecule ligands has emerged as a promising approach in cancer therapeutics. Analysis of transcriptomic data from PDAC clinical samples reveals upregulation of LXR and its target genes in tumors. In a screen of a focused library of drug-like small molecules predicted to dock in the ligand-binding pocket of LXRβ, we identified two novel LXR ligands with more potent anti-tumor activity than current LXR agonists used in our published studies. Characterization of the two lead compounds (GAC0001E5 and GAC0003A4) indicates that they function as LXR inverse agonists which inhibit their transcriptional activity. Prolonged treatments with novel ligands further revealed their function as LXR “degraders” which significantly reduced LXR protein levels in all three PDAC cell lines tested. Using metabolomics approach, we discovered that GAC0001E5 inhibits glutamine anaplerosis and induces oxidative stress in PDAC cells. Furthermore, we revealed that high extracellular cystine concentration drives the antiproliferative effect of GAC0001E5 in PDAC cells. These findings support the idea of inhibiting LXR activity using inverse agonists and degraders for the treatment of advanced pancreatic cancer. This study also highlights a novel role for LXR in regulating glutamine metabolism and glutathione metabolism in pancreatic cancer. These novel LXR ligands can further be used to advance basic research on ligand design, allosteric mechanisms, and LXR functions in other cancer models.Item Cloning, characterization, and expression of genes encoding Pseudomonas putida salicylate hydroxylase and Pseudomonas cepacia salicylate hydroxylase and m-hydroxybenzoate-6-hydroxylase(1988) Kim, Youngsoo, 1950-; Tu, Shiao-Chun; Eichberg, Joseph E.; Gray, Horace B., Jr.; Widger, William R.A Pseudomonas cepacia DNA fragment containing the gene encoding a functional salicylate hydroxylase was cloned into pRO 2317 and expressed in P. aeruginosa PAO. The P. cepacia DNA fragment in the clone is 7.5 kb in size and its physical map has been constructed. The P. aeruginosa PAO containing the clone can grow at the expense of salicylate as the sole carbon source whereas the parental P . aeruginosa PAO can grow at the expense of catechol but not salicylate as the sole carbon source because of its deficiency of salicylate hydroxylase. Salicylate hydroxylase activities were detected in crude lysates prepared from cells of P. aeruginosa PAO containing the clone. The cloned salicylate hydroxylase was also identified by antibodies against purified P. cepacia salicylate hydroxylase. The gene for P. cepacia m-hydroxybenzoate-6-hydroxylase was cloned into pRO 1727 and expressed in P. aeruginosa PAO. The P. cepacia DNA fragment in the clone is 1.8 kb in size and its physical map has been constructed. The P. aeruginosa PAO containing the clone can be grown on m-hydroxybenzoate as the sole carbon source. However, the parental P. aeruginosa PAO can grow at the expense of gentisate but not m- hydroxybenzoate as the sole carbon source because of its deficiency of in-hydroxybenzoate-6-hydroxylase. Crude lysates prepared from cells of P. aeruginosa PAO containing the clone exhibited m-hydroxybenzoate-6-hydroxylase activities. The cloned enzyme was further detected by immunoprecipitation using polyclonal antibodies against purified P. cepacia m- hydroxybenzoate-6-hydroxylase. The gene for P. putida salicylate hydroxylase was subcloned into pRO 2317 and expressed in P. aeruginosa PAO. The two salicylate hydroxylases from P. putida and P. cepacia did not exhibit any homologies with respect to DNA cross hybridization and immunoblot with polyclonal antibodies against P. cepacia salicylate hydroxylase.Item Cluster Ion Beam Induced Nano Metallic Ripple Structure for Localized Surface Plasmon Resonance (LSPR) Based Biosensor and Bacterial Growth on Nano Ripple Glass Substrate under the Influence of Weak Magnetic Fields(2017-08) Saleem, Iram 1985-; Chu, Wei-Kan; Masood, Samina S.; Widger, William R.; Gunaratne, Gemunu H.; Cai, DongThis thesis is comprised of two sections. First section describes the development of a simple and cost-effective scheme for bio-sensing, described as the detection of various biological elements inside a system or a body (that can be harmful or dangerous) with the help of specific biological receptor units. Gold nano-ripple pattern is prepared using a gas cluster ion beam (GCIB) system. When the nano-ripple surface is exposed to electromagnetic light, it shows localized surface plasmon resonance (LSPR) effect. Because of this plasmonic behavior, interaction of the targeted biomolecules and the receptors on the surface, sends optical signals that is processed to determine the presence of the specific biomolecule in the body. This LSPR nano-ripple gold biosensor was used to detect antibody-antigen reaction of rabbit X-DENTT antibody and DENTT blocking peptide (antigen) using adsorbate-induced LSPR-wavelength shift from the nano-ripple gold surface and its dependence on the antigen concentration. This approach does not require any chemical processes for its design and has the prominent advantage of possibility of large surface area coverage and applicability to different starting materials. These biosensors have monolayer scale sensitivity and high selectivity. The nano-ripple biosensor can be further developed to obtain a real time analytical detection mechanism. The second section presents the experimental results based on the effect of weak magnetic field on the growth of bacteria on glass nano structures. Bacteria is found to adhere more on nano-ripple pattern and larger bacterial colonies were observed in comparison to the plain glass surface. Moreover, magnetic field affects the growth by reducing the size of colonies. In another experiment, we examined the effects of different magnetic field configurations, including static (homogeneous and non-homogeneous) and time-varying magnetic fields, on various species of bacteria on a plain glass surface based on their growth rates. Magnetic field suppresses the growth of bacteria and slowest growth rate was observed in bacteria treated with time-varying magnetic field.Item Computational Approaches to Detect Pathogens in the Presence of Complex Backgrounds(2012-12) Rojas, Mark 1973-; Fofanov, Yuriy; Widger, William R.; Chapman, Barbara M.; Tsekos, Nikolaos V.; Shah, Shishir KiritFast and accurate identification of pathogenic microorganisms in complex clinical and environmental samples is essential for the prevention and treatment of infectious diseases. The most sensitive and accurate detection approaches are based on the examination of the nucleic acid composition of the sample in order to identify the presence of pathogens DNA and/or RNA. A large spectrum of nucleic acid-based tests (such as PCR, RT-PCR, and oligonucleotide microarrays) is designed to examine a sample for the presence of pre-defined genomic signatures: short pathogen-specific DNA and/or RNA fragments. Identification of such signatures however, represents significant computational challenges. To be pathogen specific, each signature (or combination of signatures) must be present (conserved) across all strains of the pathogen, and absent in all other organisms including its close neighbors, and must have assay specific biochemical and thermodynamic properties, such as binding energy, melting temperature, and nucleotide composition. All available signature design algorithms rely on heuristics and are known to miss cases when potential signatures are (explicitly or with small number of mismatches) also present in host (human) and/or non-pathogen microorganisms causing false positive outcomes. Even greater challenge for the design of biochemical platform specific genomic signatures (probes and primers) is that each type of instrument uses different biochemical protocols to detect signatures which also have to be included in the consideration during the signatures design process. To address these challenges we have developed novel algorithms and data structures able to bring all possible subsequences located in given pathogen genome into signatures design process. Moreover, the developed algorithms make it possible to consider mismatches (insertions, deletions, and substitutions for all positions and combinations) into the design process. We also have developed the concept of ultra-specific genomic islands: genomic regions in which every subsequence is several mismatches away from the closest subsequence which may appear in a host genome and/or non-pathogenic near-neighbors of targeted pathogen. This concept allows to improve the quality and flexibility (genomic islands can be used to identify thermodynamically acceptable signatures) of the design of biochemical platform specific detection tests. Developed approach was successfully used to design a variety of tests for Category A, B, and C, pathogens including the 2009 H1N1 Influenza outbreak originated in Mexico.Item Computational Techniques Applied to RNA Structures and Bacterial Genomes(2019-08) Tran, Quyen Duc 1984-; Fox, George E.; Williams, Loren Dean; Briggs, James M.; Widger, William R.Modern molecular biology studies increasingly require customized computation support. This is especially true for studies associated with atomic resolution structures and/or NextGen sequencing. In this dissertation, three separate studies are described in which modern computational methodologies play a key role. The first case is a model system that seeks to understand how complexity might arise in a prebiotic RNA World in which RNA polymerization through an RNA replicase was not possible. The experimental system exposes an initial 20-mer seed RNA to 180 min of continuous cycles of synthesis and degradation. The resulting pools of RNA were characterized by NextGen sequencing. The seed RNA rapidly disappeared and was replaced by an increasing number and variety of both larger and smaller variants. The analysis made it possible to characterize the extent and manner in which sequence space was explored. The RNA products lacked large numbers of point mutations but instead incorporate additions and subtractions of fragments of the original RNAs. The system demonstrates that, if such equilibrium were established in a prebiotic world, it would result in significant exploration of RNA sequence space and likely increased complexity. No matter how much complexity can be generated by an RNA World alone, the ability to synthesize peptides would be a momentous discovery. In modern organisms, the PTC, where peptide bonds are fashioned, also serves as an entrance to an exit tunnel so that a growing peptide can leave. In essence, the PTC exit cavity is an evolved RNA nano-pore that accommodates the termini of the A and P-site tRNAs, which carry the activated amino acid or the nascent peptide, respectively. Is this pore unique? In examining various atomic resolution structures, it became clear that such pores can arise readily from the packing of RNA or RNA-like molecules as they increased in size. This hypothesis is supported by our finding of at least five additional pores in the 23S rRNA that range 1.0-1.5 nm in size. Examination of other RNAs identified 11 other RNA pores. Structurally, the PTC and other 23S rRNA pores are very similar, i.e. both are created from the packing of helices, but RNA bases in the latter do not protrude into the pore. Instead, these pores are lined by negatively charged backbone atoms. While the additional 23S rRNA pores seem to lack catalytic function, they illustrate that random RNAs would not have to be very large to create a variety of pores and voids. Thus, pores of the PTC type may have occurred with some frequency in an RNA World, thereby allowing the rapid emergence of primitive translation machinery. Finally, a detailed examination of structural conservation in a ribosomal RNA was undertaken. 5S ribosomal RNA is thought to assist communication between the PTC region of the ribosome and the decoding center on the small ribosomal subunit. This is facilitated by changes in the orientation of 5S rRNA. The canonical 5S rRNA secondary structure includes 30 non-standard base-base interactions. The initial expectation was that these interactions would change as the ribosome went through its synthesis cycle. Forty atomic resolution structures of 5S rRNAs from both different organisms and different stages in the translation cycle were examined. The set of non-standard base pairs continues to be conserved in essentially all of the structures. This strongly suggests that the 5S rRNA behaves as a rod with structural changes originating from the backbone.Item Decoding the Sporulation Process in Bacillus Subtilis(2017-05) Kiehler, Brittany Elaine 1987-; Fujita, Masaya; Widger, William R.; Cooper, Timothy F.; Conrad, Jacinta C.Bacillus subtilis undergoes the process of sporulation under nutrient poor conditions. The initiation of sporulation begins with the autophosphorylation of KinA, the major sporulation kinase. While it is widely accepted that an as-yet-unknown starvation signal(s) triggers the autophosphorylation through interaction with the N-terminal PAS-domain of KinA, there has been no definitive identification of such a signal. Here, I suggest that the autokinase activity of KinA is dependent on tetrameric formation of the kinase which occurs upon reaching a certain threshold. The following autophosphorylation, a phosphorelay occurs in a complex version of a two-component response regulator system ending with the phosphorylation of Spo0A, the master regulator of sporulation. A certain threshold of Spo0A~P must be reached to enter into sporulation and Spo0A~P levels have been found to increase in the mother cell compartment even after septation. I investigated the control of Spo0A~P on sporulation specific genes by use of a Spo0A inhibitor. I found that expression of a competitive Spo0A inhibitor within the mother-cell compartment resulted in a decrease in activity of specific sporulation reporter genes. This effect was greatly seen at the late stage reporter (PgerE). RNA-Seq analysis of the late stage reporter under sporulation conditions reported several significant Spo0A-controlled genes during sporulation. Notably, genes (ldh and lctP) involved in overflow metabolism and fermentation were significantly upregulated. Finally, I performed preliminary studies on three known Spo0A-controlled genes spoIID, spoIIE, and divIVA. The levels of SpoIID within the cell were found to be moderated by a triple AND gate controlling the expression of spoIID. SpoIIE and DivIVA protein expression was observed by fluorescent microscopy in the presence of the Spo0A inhibitor to determine the effect of the inhibitor on protein localization. My initial results showed abnormal localization patterns for each. These studies may lead to a greater understanding of the regulations of control by Spo0A during sporulation.Item Delineation of bacterial luciferase aldehyde site by bifunctional labeling reagents and by chemical modification(1988) Paquatte, Olivier; Tu, Shiao-Chun; Cox, James R., Jr.; Fox, George E.; Eichberg, Joseph E.; Widger, William R.Previously we have established that an essential cysteinyl group on the [alpha] subunit is at the aldehyde site of ([alpha beta]) dimeric Vibrio harveyi luciferase. Three isomeric bifunctional reagents have been used to further delineate the luciferase aldehyde site. These probes differ in their relative positions of and distances between the two functional groups active in chemical and photochemical labelings, respectively. Each of the probes can effectively and reversibly inactivate luciferase by forming a disulfide linkage primarily to the essential cysteinyl residue. Upon subsequent photolysis, a diazoacetate arm in each probe was activated for photochemical labeling of amino acid residues within reach. After reductive regeneration of the essential cysteinyl residue, 0.35-0.40 probe per dimeric luciferase was found to have been photochemically incorporated, correlating well with the degree of irreversible enzyme inactivation. [...]Item Differential expression of the glyceraldehyde-3-phosphate dehydrogenase gene family in the nematode Caenorhabditis elegans(1988) Huang, Xinyun; Hecht, Ralph M.; Tu, Shiao-Chun; Nelson, Daniel A.; Widger, William R.; Wagner, Michael J.The differential expressions of the glyceraldehyde-3- phosphate dehydrogenase (GAPDH) gene family and their mRNA localizations in the nematode Caenorhabditis elegans were studied. Two tandemly linked direct-repeat GAPDH genes, gpd- 2 and gpd-3 , were isolated and sequenced. Compared with the two identical previously cloned GAPDH genes gpd-1 and gpd-4, gpd-2 and gpd-3 have slightly different structures regarding the locations of introns and the encoded amino acids. Northern blot analysis of total RNAs isolated from worms at different developmental stages shows that gpd-1 and gpd-4 are coordinately expressed and their transcripts are enriched in embryos which are predominantly composed of non-muscle cells. gpd-2 and gpd-3 are also coordinately transcribed, but show greater post-embryonic expression. Results of in situ hybridization with biotinylated asymmetrical DNA probes indicate that gpd-1 and gpd-4 mRNAs are present in all cells in small amounts. In contrast, gpd-2 and gpd-3 mRNAs are specifically localized within the actin-containing zones of body-wall muscle cells. These results suggest that gpd-1 and gpd-4 encode for the embryo-enriched minor isoenzyme GAPDH-I, while gpd-2 and gpd-3 encode for the body-wall muscle specific isoenzyme GAPDH-2. In addition, the actin genes act-1 and act-3 were shown to encode body-wall specific mRNAs that were also localized to the thin myofilaments.Item Effect of Repeatable Regions on Ability to Estimate Copy Number Variation in Human Genome by High Throughput Sequencing(2012-12) Golovko, Georgiy 1983-; Fofanov, Yuriy; Widger, William R.; Ordonez, Carlos; Tsekos, Nikolaos V.; Shah, Shishir KiritGenomic differences (mutations) in humans are profoundly influenced by their distinction as either germ line (inherited) or somatic (developed over one’s life span). Such mutations can vary from a single nucleotide insertion, deletion, or substitution in a gene to a complete duplication or deletion of a large amount of genomic material ranging from thousands of nucleotides to an entire chromosome ultimately referred to as Copy Number Variations (CNV). While a large number of genomic variations have no significant influence on the overall quality of life, certain types of variations in a human genome called abnormalities are known to be associated with genetic disorders including cancer, autism, schizophrenia, just to name a few. Recent advancements in DNA sequencing technologies have made it possible to utilize High Throughput Sequencing (HTS) to identify and detect CNVs. The focus of this research is the development of computational methods used to address the challenges of analyzing high throughput DNA sequence data for quality assessment in relatively large genomes (e.g. human genome) to detect copy number variations and including the data representation. An evolutionary programming approach has been developed to use the set of novel algorithms and data structures introduced in this dissertation for the purpose of efficiently and accurately mapping genomic reads to one or more reference genomes. I have developed computational tools that make it possible to identify the undesirable effects of repetitive regions in the human genome with the ability to identify CNVs and propose a novel approach to reduce their influence on genomic analysis.Item Electrophysiological Analysis and Characterization of Two Microbial Channels: The PapC Usher of Upec and the NSP4 Viroporin of Rotavirus(2016-08) Pham, Thieng Thanh 1987-; Delcour, Anne; Widger, William R.; Christie, Peter J.; Ziburkus, JokubasMicrobes, such as bacteria and viruses, utilize channel proteins that play an important role in the infection process, and thus are considered virulence factors. With the rise in antibiotic resistance and despite the existence of vaccines, there is a need for alternative drug therapies, and these virulence factors can be used as new drug targets. In this work, we examined the molecular properties of a bacterial pore-forming protein (PapC) and a viral ion channel (NSP4) using electrophysiological techniques. The PapC usher is produced by uropathogenic E. coli, the causative agent of urinary tract infections, and catalyzes the assembly and surface expression of P pili used to mediate host cell attachment. At rest, the PapC channel is closed by a plug domain that is displaced to allow the passage of the growing pilus. We showed that key residues involved in allosteric and electrostatic networks within the channel govern gating of the usher pore. In particular, a region localized to the periplasmic base of PapC appears to stabilize the usher in a closed configuration. In addition, we showed that the complex of the first pilus subunit associated with its chaperone activates the usher, and partially engages within the channel. Altogether, our work has provided mechanistic insight into usher gating and a basis for alternative drug design. The second part of our work is centered on the non-structural protein 4 (NSP4) of rotavirus. The expression of NSP4 alone was previously shown to increase cytoplasmic calcium, by releasing calcium stores from the endoplasmic reticulum, and thus NSP4 has been proposed to be a calcium channel. To test this hypothesis, we investigated the electrophysiological properties of the putative, channel-forming viroporin domain (VPD) of NSP4. We demonstrated that NSP4 VPD is indeed a bona fide ion channel, capable of conducting potassium, calcium and barium ions. This property sets it apart from most other viroporins studied to date. The channel is not strictly selective to calcium ions, but appears sensitive to inhibition by classical calcium channel inhibitors, such as cadmium and nickel ions. Altogether, our work established the foundations for future characterization of NSP4 properties using electrophysiology.Item Enhancement Of Mass Spectrometry And Affinity Purification Methodologies Used To Acquire Novel Protein Identifications(2020-08) Mitchell, Morgan; Bark, Steven J.; Widger, William R.; Cai, Chengzhi; Schwartz, Robert J.Mass spectrometry (MS)-based proteomics has become a primary tool to study proteins underlying infection disorders, antibiotic resistance mechanisms, and identify significant new targets for drug discovery. Mass spectrometers must be capable of discriminating molecules of interest while maintaining a high level of robustness, sensitivity, and measurement accuracy. These specifications require a multi-faceted approach combining mass spectrometry and high performance analytical and biochemical separations. The first study enhanced performance for a common mass spectrometer design. Significant variability in this instrument impaired consistent accurate mass measurements and severely reduced molecular identification. Spectral averaging provided a simple and highly effective strategy to mitigate these instrument limitations. The second study improved consistency and reproducibility in Affinity Purification Mass Spectrometry (APMS), a powerful method to study proteins and protein interaction complexes. An optimized APMS method was developed through systematic evaluation of the most common resins and protein tag strategies, demonstrating that APMS experiments must consider the effect of solid-phase resin on the entire experimental design. These improved methods were applied in two biological experiments. The first experiment applied optimized APMS to isolate phosphoinositide 3-kinase (PI3K) within pancreatic cancer BxPC-3 cells treated with different tyrosine kinase inhibitors targeting the epidermal growth factor receptor (EGFR). This experiment revealed a signaling processes within the EGFR/PI3K/Akt and ERK pathways as a mechanism of EGFR inhibitor response and provide new insight into the molecular basis tyrosine kinase inhibition and potential resistance mechanisms. The second experiment utilized liquid-chromatography mass spectrometry (LC-MS) to study dormancy in Micrococcus luteus (M. luteus), a close relative of Mycobacterium tuberculosis (M. tuberculosis). Eighteen proteins upregulated in dormancy were identified, four of which linked to M. tuberculosis latency, and the remaining 14 novel. These proteins are future targets for molecular and therapeutic studies. These studies demonstrate improved methods for mass spectrometry-based proteomics and apply these methods to important molecular and drug-discovery systems, thereby progressing the field of systems biology.Item Genetically Reprogramming Immune Modulatory Activity in Cancer Cells using TALE and CRISPR/Cas9 Technologies(2014-08) Anthony-Gonda, Kim 1980-; Zhang, Xiaoliu Shaun; Briggs, James M.; Widger, William R.; Otteson, Deborah C.The advent of TALE and CRISPR/Cas9 technologies has ushered in an era of new genome-engineering tools to precisely manipulate the human genome. These tools hold great promise to treat and cure complex human diseases, such as cancer. In the past few decades, cancer immunotherapy has emerged as an attractive cancer therapy that harnesses the natural ability of the immune system to treat malignancy. Major approaches in cancer immunotherapy include exogenously introducing an immune stimulatory gene into tumor cells for use as a tumor vaccine and manipulation of immunosuppressive mechanisms. However, current strategies to do so lack advanced techniques to manipulate the immune system in a precise way; and thus, compromises therapeutic efficacy or has unforeseen side effects. In this dissertation, we provide strong evidence for the ability of TALE and CRISPR/Cas9 technologies to reprogram endogenous expression of immune modulatory genes implicated in cancer immunotherapy. Overall, TALE-based genetic activators potently transactivated two immune stimulatory genes, IL-2 and GM-CSF, in which these genes are otherwise completely silenced in epithelial cancers. Furthermore, robust gene activation was linked to chromatin remodeling of silent cytokine gene promoters. To further enhance TALE potency, we engineered a novel multimerized TALE activator and demonstrated that it was highly efficient in transactivation of the IL-2 gene in a context-specific manner. Moreover comparative studies exploring CRISPR/Cas9 as genetic activators clearly identified TALE technology as a more efficacious strategy. We also present evidence that TALE and CRISPR/Cas9 technologies can be adapted as genetic repressors to regulate immune modulatory genes. For this purpose, we developed a collection of TALE and CRISPR/Cas9 genetic repressors and interrogated their functions on the IL-2 and CMV gene promoters as proof of principle. We clearly demonstrated that TALE and CRISPR/Cas9 repressors potently disrupted IL-2 gene activity when targeted to strong cis-regulatory elements. Moreover, we demonstrated that TALE-based repressors regulated genes downstream of their primary target. In conclusion, we have developed a novel collection of TALE and CRISPR/Cas9 genetic tools to translate our conceptual model in vivo and explore its implication for cancer immunotherapy.Item INSIGHTS INTO THE BINDING AND CATALYTIC MECHANISMS OF BACILLUS THURINGIENSIS LACTONASE AND INTERACTIONS OF A PTEN-BINDING INHIBITORY PEPTIDE(2013-05) Charendoff, Marc Neil 1968-; Briggs, James M.; Willson, Richard C.; Bittner, Eric R.; Widger, William R.; Yeo, Hye-JeongThe lactonase enzyme (AiiA) produced by Bacillus thuringiensis (B. thuringiensis) serves to degrade autoinducer-1 (AI-1) signaling molecules in what is an evolved mechanism by which allows B. thuringiensis to better compete with other bacteria. Bioassays have been previously performed to determine whether the AI-1 aliphatic tail lengths have any effect on AiiA’s bioactivity; however, data to date are conflicting. To investigate these questions, multiple molecular dynamics simulations were performed across a family of seven acylated homoserine lactones (AHL) along with their associated intermediate and product states. Distance analyses and interaction energy analyses were performed to investigate current bioassay data. Our simulations are consistent with experimental studies showing that AiiA degrades AHLs in a tail length independent manner. Also, a proposed putative oxyanion hole function of Y194 toward the substrate is not observed in any of the reactant or product state simulation trajectories. However, Y194 does seem to show efficacy in stabilizing the intermediate state. Last, we argue through ionization state analyses, that proton-shuttling necessary for catalytic activity is possibly mediated by both water- and substrate-based, intra-molecular proton transfer. Based on this argument, an alternate catalytic mechanism is proposed. Drug dependence, or addiction ultimately described as the maladaptation of a neurochemical reward pathway that is normally used to reinforce behaviors that promote an organism’s survival and/or propagation (e.g., high calorie food seeking and sexual drive). These pathways are mediated via dopaminergic VTA-NAc neuronal networks. The molecular basis for this interaction has been shown to rely on a 5HT-2cR:PTEN interaction via the 3L4F-F1 fragment. The nature of the interaction is unknown. In this work a series of REMD runs are designed to generate candidate conformations that might exist under physiological conditions. These candidates are then subjected to exhaustive six-dimensional, coarse-grained docking simulations against the PTEN protein to produce a myriad of potential docking sites. From these dockings the top scoring poses are observed to point to a single binding site that in turn lend themselves to spectroscopic validation.Item Isolation and characterization of cDNA clones encoding the slow myosin alkali light chain(1988) Papadopoulos, Nickolas; Wells, Dan E.; Crow, Michael T.; Franklin, Luther E.; Widger, William R.The slow skeletal and cardiac myosin alkali light chains of many different species have identical electrophoretic and immunochemical properties, suggesting that they are encoded by similar or, perhaps, the same mRNAs. In this Thesis, full-length cDNA clones encoding the chicken slow skeletal muscle alkali light chain (MIC), SLC1s,have been isolated and sequenced. These cDNAs have been used to determine whether the mRNAs encoding it and its cardiac counterpart are in fact identical and represented in the genome as a single gene. The analysis of the deduced amino acid sequence of the slow alkali light chain cDNAs showed that there was strong homology with that of other alkali light chains and near identity to a published amino acid sequence of the cardiac isoform. Northern blotting, S1 nuclease protection analyses with probes to either the 5' or 3' regions of the cDNA, and primer extension assays of the 5' region all failed to detect any differences in the mRNAs encoding the adult slow, embryonic, and cardiac alkali myosin light chains. Furthermore, Southern blotting analyses of the chicken genome with probes that reacted with both the slow and cardiac MLC mRNAs showed only a single band of hybridization. The data presented here indicate that, despite their different tissue and temporal patterns of expression, the mRNAs encoding the slow skeletal and cardiac alkali light MLCs are identical and encoded by a single gene.Item Isolation, cloning and sequencing of a putative petD gene from Agmenellum quadruplicatum PR-6(1988) Rampersad, Joanne Nadia; Widger, William R.; Fox, George E.; Tu, Shiao-Chun; Jacobson, James W.Southern blots containing genomic Agmenellum quadruplicatum DNA digested with various restriction enzymes were screened for the petD gene using 1) a radiolabelled RNA probe from the petD gene from spinach and 2) a 21 base long end labelled oligomer with 32 fold degeneracy synthesized according to a highly conserved area of the subunit IV gene. The RNA probe did not hybridize to the blots apparently indicating not enough sequence similarity between the petD genes from the two sources. [...]
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