Biochemistry, Biophysics, and Structural Biology Commons^™

Structural And Biochemical Studies Of The Carboxyltransferase Domain From Pyruvate Carboxylase, Adam David Lietzan

Dissertations (1934 -)

Pyruvate carboxylase (PC; E.C. 6.4.1.1), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated, and then translocate to the carboxyltransferase (CT) domain where the carboxyl group is transferred from biotin to pyruvate. Kinetic analyses of PC have suggested that the spatially distinct reactions, which occur in the active sites of the BC and CT domains, are well coordinated. To gain insights into the molecular events necessary for coordinating catalysis in the CT domain, …

New Strategies To Reveal Protein Candidates In Protein-Protein Interactome Study, Meng-Chieh Chen

Open Access Theses

Comprehensive protein-protein interaction network analysis can help reveal protein functions in a system-wide manner. A reliable knowledgebase of interaction networks is not only important for selecting the candidates for drug therapies, but also for evaluating the disease risk. In current interaction databases, 322579 interactions comprised of 56460 proteins have been reported (statistical analysis from APID: Agile Protein Interaction DataAnalyzer; http://bioinfow.dep.usal.es/apid/index.htm). The huge datasets are contributed mainly by yeast -two -hybrid (Y2H) screening and affinity-purification followed by mass spectrometry (AP-MS). High false positive rates and failing to cover certain interaction categories are the limitations of these two methods. Here, we developed …

Hierarchical Cell Fluid Extracellular Matrix Interaction In Cell Microenvironment, Soham Ghosh

Open Access Dissertations

Hierarchical structural interactions between components of cell microenvironment, the extracellular matrix (ECM), cytoplasm, nucleus and fluid, are important phenomena that decide cell level physiological process and tissue engineering applications. One of those tissue engineering modalities is freezing of biomaterials, important in a wide variety of biomedical applications including cryopreservation and cryosurgeries. In order to design these applications, freezing-induced changes of the cells and tissues and corresponding biophysical mechanisms need to be well understood. Although the effects of freezing on cells in suspension have been extensively studied, the intracellular mechanics of cells embedded in the extracellular matrix (ECM) during freezing are …

Intranuclear Strain Measured By Iterative Warping In Cells Under Mechanical And Osmotic Stress, Jonathan T Henderson

Open Access Dissertations

The nucleus is a membrane bound organelle and regulation center for gene expression in the cell. Mechanical forces transfer to the nucleus directly and indirectly through specific cellular cytoskeletal structures and pathways. There is increasing evidence that the transferred forces to the nucleus orchestrate gene expression activity. Methods to characterize nuclear mechanics typically study isolated cells or cells embedded in 3D gel matrices. Often report only aspect ratio and volume changes, measures that oversimplify the inherent complexity of internal strain patterns. This presents technical challenges to simultaneously observe small scale nuclear mechanics and gene expression levels inside the nuclei of …

Towards A Paradigm Shift In The Modeling Of Soil Organic Carbon Decomposition For Earth System Models, Yujie He

Open Access Dissertations

Soils are the largest terrestrial carbon pools and contain approximately 2200 Pg of carbon. Thus, the dynamics of soil carbon plays an important role in the global carbon cycle and climate system. Earth System Models are used to project future interactions between terrestrial ecosystem carbon dynamics and climate. However, these models often predict a wide range of soil carbon responses and their formulations have lagged behind recent soil science advances, omitting key biogeochemical mechanisms. In contrast, recent mechanistically-based biogeochemical models that explicitly account for microbial biomass pools and enzyme kinetics that catalyze soil carbon decomposition produce notably different results and …

Quantum Mechanics In Complex Systems, Ross Douglas Hoehn

Open Access Dissertations

This document should be considered in its separation; there are three distinct topics contained within and three distinct chapters within the body of works. In a similar fashion, this abstract should be considered in three parts. Firstly, we explored the existence of multiply-charged atomic ions by having developed a new set of dimensional scaling equations as well as a series of relativistic augmentations to the standard dimensional scaling procedure and to the self-consistent field calculations. Secondly, we propose a novel method of predicting drug efficacy in hopes to facilitate the discovery of new small molecule therapeutics by modeling the agonist-protein …

The Modification Of Brucine Derivatives As Chiral Ligands And Its Application In The Asymmetric Synthesis, Jian-Yuan Li

Open Access Dissertations

The modification of brucine derivatives as chiral ligands and the use of a multifaceted chiral ligand, brucine diol, under different reaction conditions to produce various optical isomers is described. In Chapter 1, the generation of a number of brucine derivatives is described. Taking the advantage of brucine-diol's excellent molecular recognition capability for multiple organic functional groups, we focused on the synthetic modifications of brucine-diol and the synthesis of brucine N-oxide. We also produced various brucine derivatives with different functional moieties in good yields and selectivities. ^ In Chapter 2, we described the investigation of brucine N-oxide catalyzed Morita-Baylis-Hillman …

Theory For Diffusional Encounters In Heterogeneous Environments And Multivalent Electrolyte Screening Of Charged Interface, Ran Li

Open Access Dissertations

We develop a theory for encounter rates in a three-dimensional system of connected compartments. The model of connected compartments exhibits the length-scale dependent diffusion that is observed in many heterogeneous environments, such as porous catalysts and biological environments. We discovered a dimensionless number that is the dominant scaling variable and obtained, for the first time, an analytical expression for the encounter rate. The new theory generalizes the classic Smoluchowski diffusion limit to the case of heterogeneous environments. The new theory is tested using Brownian dynamics simulations.^ We also experimentally investigated the behavior of multivalent electrolyte near a charged solid-liquid interface. …

Structure-Functionality Relationship Of Collagen Scaffolds For Tissue Engineering, Seungman Park

Open Access Dissertations

Tissue engineering is a promising technology that enables scientists to create artificial organs or replace damaged tissues using animal cells and other components. For successful tissue regeneration, many factors should be taken into account, however, three components are most crucial: cell, scaffold, and soluble factor(s). In order to check the functionality after regeneration of desired tissues, various approaches have been attempted, depending on the physical, biological, and chemical properties of the tissues. Recently, the importance of the extracellular matrix (ECM) microstructure is being considered to be important in this regard. The ECM is closely associated with various functional properties of …

Understanding Preferred Leg Stiffness And Layered Control Strategies For Locomotion, Zhuohua H. Shen

Open Access Dissertations

Despite advancement in the field of robotics, current legged robots still cannot achieve the kind of locomotion stability animals and humans have. In order to develop legged robots with greater stability, we need to better understand general locomotion dynamics and control principles. Here we demonstrate that a mathematical modeling approach could greatly enable the discovery and understanding of general locomotion principles. ^ It is found that animal leg stiffness when scaled by its weight and leg length falls in a narrow region between 7 and 27. Rarely in biology does such a universal preference exist. It is not known completely …

Host Cell Death In Legionella Pneumophila Pathogenesis And Immunity, Wenhan Zhu

Open Access Dissertations

Legionella pneumophila is an intracellular pathogen that causes a severe, atypical pneumonia termed Legionnaires' disease. Upon entering the host cell, L. pneumophila resides in a membrane-bound vacuole, in which the bacterium evades lysosomal fusion and replicates. The establishment of the vacuole requires the Dot/Icm (Defect in organelle trafficking/ intracellular multiplication) transport system, which translocates a large number of substrates into host cells to re-orchestrate various cellular processes, such as intracellular trafficking, protein synthesis and host cell death pathways. Therefore, a key step in understanding the biology of Legionella is to dissect the mechanisms of action of the Dot/Icm substrates. By …

Transformation Of Biomass Carbohydrates By Transition Metal Catalysts, Christine M Bohn

Open Access Dissertations

By selectively removing functional groups from biomass derived carbohydrates, valuable platform chemicals can be generated from renewable sources. Through dehydration chemistry glucose can be upgraded into 5-(Hydroxymethyl)-2-furfuraldehyde (HMF) and levulinic acid. Iron (III) chloride hexahydrate has shown moderate activity to transform glucose into HMF and has also shown high yields and selectivity for the production of levulinic acid. Typically synthesized from acidic solutions made with mineral acids, levulinic acid has now been produced in high yields with a metal salt. The difference between maximizing production for HMF or levulinic acid from the same catalyst relies on the control of the …

Associated Behavioral, Genetic, And Gene Expression Variation With Alternative Life History Tactics In Salmonid Fishes, Ashley Chin-Baarstad

Open Access Dissertations

Individual differences in behavior can have potential fitness consequences and often reflect underlying genetic variation. My research focuses on three objectives related to individual level variation: 1) evaluating the innate behavioral variation within and between individuals, families, and progeny of different life-history types across time; 2) testing for differences in gene expression within the brain associated with this behavioral variation; and 3) using genetic polymorphisms to test for associations with ecotype, as well as population structure, in polymorphic populations. First, we evaluated the variation in a suite of ecologically relevant behaviors across time in juvenile progeny produced from crosses within …

A Role Of Yeast Adhesin Amyloids In Force-Dependent Adhesion And Biofilm Formation, Cho Xiao Juan Chan

Dissertations, Theses, and Capstone Projects

Candida albicans adhesins have amyloid-forming sequences (Ramsook et al. 2010, Otoo et al. 2008). Similarly, Tango and Waltz predicted that amyloid-forming sequences are also present in Saccharomyces cerevisiae flocculins, Flo1p and Flo11p. The cell surface of Flo1p- and Flo11p-expressing cells have ordered domains that are thioflavin T fluorescent and Congo red birefringent, two hallmarks of amyloids. Flo1p and Flo11p amyloids were important for activities of the flocculins including cell-to-cell adhesion, cell-to-substrate adhesion, and agar invasion. In addition, amyloid-perturbing dyes thioflavin S and Congo red inhibited aggregation, biofilm formation and agar invasion.

Force-induced formation and propagation of adhesion nanodomains in Als5p-expressing …

Design And Optimization Of A De Novo Protein Charge Separation Dyad, Andrew C. Mutter

Dissertations, Theses, and Capstone Projects

The ever-increasing demand for cheap, plentiful energy to fuel the needs of a growing population requires research into alternative clean energy. Solar irradiation has the potential to power the planet many times over; the challenge is efficient capture and conversion of this energy source. Nature has already solved this problem with photosynthesis, which harvests solar irradiation converting it to stored chemical energy and is the source of the energy for life. The goal of my dissertation is to use de novo designed protein to mimic the charge separation system in photosynthesis. A stable protein scaffold will be designed and used …

Fabrication And Characterization Of Sol-Gel Based Nanoparticles For Drug Delivery, Reeta Yadav

Dissertations, Theses, and Capstone Projects

Nanogels are cross linked polymeric sol-gel based nanoparticles that offer an interior network for incorporation and protection of biomolecules, exhibiting unique advantages for polymer based delivery systems. We have successfully synthesized stable sol-gel nanoparticles by means of [a] silicification reactions using cationic peptides like polylysine as gelating agents, and [b] lyophilization of sol-gels. Macromolecules such as Hemoglobin and Glucose Oxidase and small molecules such as Sodium Nitroprusside (SNP) and antibiotics were encapsulated within the nanogels. We have used transmission electron microscopy, dynamic light scattering, zeta potential analysis, and spectroscopy to perform a physicochemical characterization of the nanogels resulting from the …

Development Of Cell-Active Inhibitors And Activity-Based Probe Of Cysteine Cathepsins, Dibyendu Dana

Dissertations, Theses, and Capstone Projects

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. Still however, many of their cellular function remain poorly understood. Chemical biology approaches employing small molecules can be utilized for this purpose. Unfortunately small molecule probes that are cell-permeable and non-peptidyl in nature are scarcely available.

In this work, first a library of sulfonyloxiranes is synthesized. From this library, 2-(2-ethylphenylsulfonyl)oxirane is identified as a selective inhibitor of cysteine cathepsins. Cell-based study reveals that 2-(2-ethylphenylsulfonyl)oxirane is a cell-permeable, covalent, and irreversible inhibitor of cathepsin B with …

Identification And Characterization Of Protein Kinase C Substrates In Human Breast Cells, Xin Zhao

Dissertations, Theses, and Capstone Projects

Aberrations in PKC signaling can lead to the development of multiple human diseases and the most prominent association of PKC with disease has been in tumor growth and metastasis. PKC and its related pathways have been recognized as promising targets for blocking the malignancy of breast cancer cells. To better understand PKC-mediated pathway in breast cancer cells, it is important to identify the cellular substrates of PKC. The main focus of this work is to identify physiologically relevant cellular substrates of PKC in human breast cells and to characterize their roles in cancer-related phenotypes. The work to be described consists …

Venomic Characterization Of The Terebridae And Novel Terebrid Neuropeptides, Mary Elizabeth Wright

Dissertations, Theses, and Capstone Projects

Unravelling the complex mixture of neuropeptides produced by the terebrid venom duct holds the promise of discovering tomorrow's therapeutics. Cone snails have already demonstrated the potential found in the venom of these unusual marine organisms, through the commercial approval of drugs for pain and other indications. Terebrids, as the sister family to the cone snails, have been much less investigated, but have a species richness that makes them very attractive in the search for novel neuropeptides. The venomics research described in this work encompasses the first comprehensive characterization of the terebrid venom duct transcriptomes of two species, Cinguloterebra anilis and …

The Non-Canonical Hydroxylase Structure Of Yfcm Reveals A Metal Ion-Coordination Motif Required For Ef-P Hydroxylation, Kan Kobayashi, Assaf Katz, Andrei Rajkovic, Ryohei Ishii, Owen E. Branson, Michael A. Freitas, Ryuichiro Ishitani, Michael Ibba, Osamu Nureki

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

EF-P is a bacterial tRNA-mimic protein, which accelerates the ribosome-catalyzed polymerization of poly-prolines. In Escherichia coli, EF-P is post-translationally modified on a conserved lysine residue. The post-translational modification is performed in a two-step reaction involving the addition of a β-lysine moiety and the subsequent hydroxylation, catalyzed by PoxA and YfcM, respectively. The β-lysine moiety was previously shown to enhance the rate of poly-proline synthesis, but the role of the hydroxylation is poorly understood. We solved the crystal structure of YfcM and performed functional analyses to determine the hydroxylation mechanism. In addition, YfcM appears to be structurally distinct from any …

Large-Scale Identification Of Chemically Induced Mutations In Drosophila Melanogaster, Nele A Haelterman, Lichun Jiang, Yumei Li, Vafa Bayat, Hector Sandoval, Berrak Ugur, Kai Li Tan, Ke Zhang, Danqing Bei, Bo Xiong, Wu-Lin Charng, Theodore Busby, Adeel Jawaid, Gabriela David, Manish Jaiswal, Koen J T Venken, Shinya Yamamoto, Rui Chen, Hugo J Bellen

Faculty, Staff and Students Publications

Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of …

The Role Of The Striatal Neuropeptide Neurotensin In The Methamphetamine-Induced Neural Injury In Mice, Qingkun Liu

Dissertations, Theses, and Capstone Projects

Methamphetamine (METH) is a widely abused psychostimulant that induces neurotoxicity to several brain regions, including the striatum. Similar to dopamine (DA) in chemical structure, METH can be transported into DA pre-synaptic terminals, evoking the neurodegeneration in DA terminals and post-synaptic striatal neurons. Despite the critical role of DA in METH-induced neurodegeneration, no pharmaceutical therapeutics has been approved for these conditions. It is therefore essential to investigate the endogenous factors regulating the dopaminergic system. The neuropeptide neurotensin has emerged as a potential modulator of METH-induced striatal neurodegeneration mainly due to its intimate interactions with dopamine in the striatum.

In this study, …

Identification And Characterization Of A Functional L-Gulono-1,4-Lactone Oxidase In Arabidopsis Thaliana, Siddique Aboobucker

Student Theses and Dissertations

Vitamin C (ascorbate, AsA) is essential for plant and animal health. We cannot synthesize AsA on our own and therefore we must consume it from fruits and vegetables. Ascorbate plays multiple roles in plant physiology, and increasing AsA content in plants remains challenging. There are four biosynthetic AsA pathways in plants: the D-mannose/L-galactose, L-gulose, D-galacturonate, and myo-inositol routes. The AsA metabolic network is not completely understood. The inositol pathway has four enzyme-catalyzed steps, and our group has made significant progress on characterizing the first three enzymes. This work focuses on L-gulono-1,4-lactone oxidase (GulLO), the last enzyme in the inositol pathway …

A Distinct Tethering Step Is Vital For Vacuole Membrane Fusion, Michael Zick, William T. Wickner

Dartmouth Scholarship

Past experiments with reconstituted proteoliposomes, employing assays that infer membrane fusion from fluorescent lipid dequenching, have suggested that vacuolar SNAREs alone suffice to catalyze membrane fusion in vitro. While we could replicate these results, we detected very little fusion with the more rigorous assay of lumenal compartment mixing. Exploring the discrepancies between lipid-dequenching and content-mixing assays, we surprisingly found that the disposition of the fluorescent lipids with respect to SNAREs had a striking effect. Without other proteins, the association of SNAREs in trans causes lipid dequenching that cannot be ascribed to fusion or hemifusion. Tethering of the SNARE-bearing proteoliposomes was …

Mechanisms And Molecular Biology Of Major Tumor Suppressors, Brienne E. Engel

USF Tampa Graduate Theses and Dissertations

This dissertation is devoted to the study of the molecular biology of major tumor suppressors, defined as those that prevent the cellular processes identified as the hallmarks of cancer. Specifically, the major tumor suppressors pRb and STK11 are explored in the context of osteosarcoma and lung cancer, respectively.

RB1 was the first tumor suppressor gene discovered. Over four decades of work have revealed that the Rb protein (pRb) is a master regulator of biological pathways influencing virtually every aspect of intrinsic cell fate including cell growth, cell-cycle checkpoints, differentiation, senescence, self-renewal, replication, genomic stability and apoptosis. While these many processes …

Role And Regulation Of Snon/Skil And Plscr1 Located At 3q26.2 And 3q23, Respectively, In Ovarian Cancer Pathophysiology, Madhav Karthik Kodigepalli

USF Tampa Graduate Theses and Dissertations

Ovarian cancer is one of the most common causes of gynecological cancer related deaths in women. In 2014, the estimated number of deaths due to ovarian cancer is 14,270 with occurrence of over 22, 240 new cases (National Cancer Institute, http://seer.cancer.gov/statfacts/html/ovary.html). Despite improvement in treatment strategies, the 5-year survival rate is still below 50% mainly due to chemoresistance and relapse. Amplification of chromosomal region 3q26 is a common characteristic in various epithelial cancers including ovarian cancer. This region harbors various oncogenes including the TGFβ signaling mediators EVI1 and SnoN/SkiL, PKCι and PIK3CA amplified at 3q26.2 and 3q26.3, respectively, in ovarian …

Eukaryotic Gene Expression Patterns Of Microorganisms In The Amazon River Plume Parallel The Biogeochemistry Of Plume Waters, Brian L. Zielinski

USF Tampa Graduate Theses and Dissertations

Unraveling the microbiological processes that occur as water travels from a river's mouth into the ocean is critical to understanding the role of river plumes in global biogeochemical cycles. Metranscriptomics, the gene expression of a whole community of organisms, was utilized to examine six stations along the Amazon River Plume (ARP) in 2010 to test thehypothesis that there were measurable differences in gene expression for key biogeochemical genes along the ARP. This body of work focuses on methods developed to identify which genes are biogeochemically important for a particular environment along extreme salinity, nutrient and community gradients in the ARP, …

Trace Gas Emissions From Combustion Of Peat, Crop Residue, Domestic Biofuels, Grasses, And Other Fuels: Configuration And Fourier Transform Infrared (Ftir) Component Of The Fourth Fire Lab At Missoula Experiment (Flame-4), C. E. Stockwell, R. J. Yokelson, S. M. Kreidenweis, A. L. Robinson, P. J. Demott, R. C. Sullivan, J. Reardon, K. C. Ryan, D. W.T. Griffith, L. Stevens

Chemistry and Biochemistry Faculty Publications

During the fourth Fire Lab at Missoula Experiment (FLAME-4, October-November 2012) a large variety of regionally and globally significant biomass fuels was burned at the US Forest Service Fire Sciences Laboratory in Missoula, Montana. The particle emissions were characterized by an extensive suite of instrumentation that measured aerosol chemistry, size distribution, optical properties, and cloud-nucleating properties. The trace gas measurements included high-resolution mass spectrometry, one-and two-dimensional gas chromatography, and open-path Fourier transform infrared (OP-FTIR) spectroscopy. This paper summarizes the overall experimental design for FLAME-4-including the fuel properties, the nature of the burn simulations, and the instrumentation employed-and then focuses on …

Mistranslation Of The Genetic Code, Adil Moghal, Kyle Mohler, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

During mRNA decoding at the ribosome, deviations from stringent codon identity, or “mistranslation,” are generally deleterious and infrequent. Observations of organisms that decode some codons ambiguously, and the discovery of a compensatory increase in mistranslation frequency to combat environmental stress have changed the way we view “errors” in decoding. Modern tools for the study of the frequency and phenotypic effects of mistranslation can provide quantitative and sensitive measurements of decoding errors that were previously inaccessible. Mistranslation with non‐protein amino acids, in particular, is an enticing prospect for new drug therapies and the study of molecular evolution.

Identification And Quantification Of Gaseous Organic Compounds Emitted From Biomass Burning Using Two-Dimensional Gas Chromatography/Time-Of-Flight Mass Spectrometry, L. E. Hatch, W. Luo, J. F. Pankow, Robert Yokelson, C. Stockwell, K. C. Barsanti

Chemistry and Biochemistry Faculty Publications

The current understanding of secondary organic aerosol (SOA) formation within biomass burning (BB) plumes is limited by the incomplete identification and quantification of the non-methane organic compounds (NMOCs) emitted from such fires. Gaseous organic compounds were collected on sorbent cartridges during laboratory burns as part of the fourth Fire Lab at Missoula Experiment (FLAME-4), with analysis by two-dimensional gas chromatography/time-of-flight mass spectrometry (GC×GC/TOFMS). The sensitivity and resolving power of GC×GC/TOFMS allowed the acquisition of the most extensive data set of BB NMOCs to date, with measure ments for 722 positively or tentatively identified compounds. Estimated emission factors (EFs) are presented …