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High-Precision Tracking With Non-Blinking Quantum Dots Resolves Nanoscale Vertical Displacement, Kyle Marchuk, Yijun Guo, Wei Sun, Javier Vela, Ning Fang Mar 2012

High-Precision Tracking With Non-Blinking Quantum Dots Resolves Nanoscale Vertical Displacement, Kyle Marchuk, Yijun Guo, Wei Sun, Javier Vela, Ning Fang

Chemistry Publications

Novel non-blinking quantum dots (NBQDs) were utilized in three-dimensional super-localization, high-precision tracking applications under an automated scanning-angle total internal reflection fluorescence microscope (SA-TIRFM). NBQDs were randomly attached to stationary microtubules along the radial axis under gliding assay conditions. By automatically scanning through a wide range of incident angles with different evanescent-field layer thicknesses, the fluorescence intensity decay curves were obtained. Fit with theoretical decay functions, the absolute vertical positions were determined with sub-10-nm localization precision. The emission intensity profile of the NBQDs attached to kinesin-propelled microtubules was used to resolve the self-rotation of gliding microtubules within a small vertical distance ...


Conformational Selection And Substrate Binding Regulate The Monomer/Dimer Equilibrium Of The C-Terminal Domain Of Escherichia Coli Enzyme I, Vincenzo Venditti, G. Marius Clore Jan 2012

Conformational Selection And Substrate Binding Regulate The Monomer/Dimer Equilibrium Of The C-Terminal Domain Of Escherichia Coli Enzyme I, Vincenzo Venditti, G. Marius Clore

Vincenzo Venditti

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by the binding of phosphoenolpyruvate (PEP) to the C-terminal domain (EIC) of enzyme I (EI), a highly conserved protein that is common to all sugar branches of the PTS. EIC exists in a dynamic monomer/dimer equilibrium that is modulated by ligand binding and is thought to regulate the overall PTS. Isolation of EIC has proven challenging, and conformational dynamics within the EIC domain during the catalytic cycle are still largely unknown. Here, we ...


Understanding Protein Motions By Computational Modeling And Statistical Approaches, Lei Yang Jan 2008

Understanding Protein Motions By Computational Modeling And Statistical Approaches, Lei Yang

Retrospective Theses and Dissertations

Because of its appealing simplicity, the elastic network model (ENM) has been widely accepted and applied to study many molecular motion problems, such as the molecular mechanisms of chaperonin GroEL-GroES function, allosteric changes in hemoglobin, ribosome motions, motor-protein motions, and conformational changes in general. In this dissertation, the ENM is employed to study various protein dynamics problems, and its validity is also examined by comparing with experimental data. First, we apply principal component analysis (PCA) to identify the essential protein motions from multiple structures (X-ray, NMR and MD) of the HIV-1 protease. We find significant similarities between the first few ...


Relatedness Of Haemophilus Parasuis Strains And Their Proteins' Possible Roles As Virulence Factors, Emilie Susanna Noel Zehr Jan 2008

Relatedness Of Haemophilus Parasuis Strains And Their Proteins' Possible Roles As Virulence Factors, Emilie Susanna Noel Zehr

Retrospective Theses and Dissertations

Haemophilus parasuis, the causative agent of Glasser's disease, is considered to be a remerging pathogen of swine in high-health status herds. Several molecular typing methods have been suggested as alternatives to serotyping, which failed to identify up to 30% of field isolates. This study compared random amplification of polymorphic DNA (RAPD) profiles and protein profiles of H. parasuis reference strains and field isolates as methods for identifying and grouping virulent and avirulent isolates. DNA and protein profiles of 15 reference strains and 31 field isolates of H. parasuis, analyzed with two computer-based similarity analyses, revealed unique and reproducible DNA ...


Remodeling Of Higher Order Chromatin Structure By The Jil-1 Histone H3 Kinase In Drosophila, Huai Deng Jan 2008

Remodeling Of Higher Order Chromatin Structure By The Jil-1 Histone H3 Kinase In Drosophila, Huai Deng

Retrospective Theses and Dissertations

The higher order organization of chromatin within the cell nucleus plays an essential role in epigenetic regulation of gene transcription. In Drosophila, the JIL-1 histone H3S10 kinase has been identified as a major regulator of chromatin structure. JIL-1 associates with chromosomes throughout the cell cycle, and predominantly localizes to less-condensed interband regions on the salivary gland polytene chromosome. In males, JIL-1 is upregulated on the male X chromosome and is associated with MSL dosage compensation complex. Absence of JIL-1 results in loss of interphase H3S10 phosphorylation as well as lethality and multiple defects including severely disrupted polytene chromosome morphology.;Further ...


Molecular Interactions Of The Mammalian Intermediate Filament Protein Synemin With Cytoskeletal Proteins Present In Adhesion Sites, Ning Sun Jan 2008

Molecular Interactions Of The Mammalian Intermediate Filament Protein Synemin With Cytoskeletal Proteins Present In Adhesion Sites, Ning Sun

Retrospective Theses and Dissertations

The type VI intermediate filament (IF) protein synemin is a very large, unique member of the IF protein superfamily. Synemin associates with the major type III IF protein desmin and/or vimentin forming heteropolymeric intermediate filaments (IFs) within mammalian muscle cells. Previous studies in our lab showed that avian synemin interacts with vinculin and alpha-actinin, both of which are cytoskeletal proteins present in adhesion sites such as the focal adhesions within cells. Thus, synemin may link the heteropolymeric IFs to adhesion sites within mammalian muscle cells or within some non-muscle cells that express synemin. At least two isoforms of synemin ...


Molecular, Genetic, Physiological And Biochemical Studies Of 3-Methylcrotonyl-Coa Carboxylase And Biotin Carboxyl Carrier Protein-Like Proteins In Arabidopsis Thaliana, Geng Ding Jan 2008

Molecular, Genetic, Physiological And Biochemical Studies Of 3-Methylcrotonyl-Coa Carboxylase And Biotin Carboxyl Carrier Protein-Like Proteins In Arabidopsis Thaliana, Geng Ding

Retrospective Theses and Dissertations

Biotin-containing enzymes are involved in metabolic reactions that are essential for normal cellular functions in all organisms. In plants, four biotin-containing enzymes have been identified to date: homomeric acetyl-CoA carboxylase, heteromeric acetyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase (MCCase), and geranoyl-CoA carboxylase. This dissertation contains two studies that are both part of the biotin metabolic network. The first study is the functional reverse genetic analysis of MCCase in Arabidopsis. MCCase is a nuclear-encoded, mitochondrial-localized enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism in mitochondria, and it may also play a role in the catabolism of isoprenoids and in ...


Functional Analysis Of The C-Terminal Domain In The Drosophila Jil-1 Histone H3 Kinase, Xiaomin Bao Jan 2008

Functional Analysis Of The C-Terminal Domain In The Drosophila Jil-1 Histone H3 Kinase, Xiaomin Bao

Retrospective Theses and Dissertations

Epigenetic regulation is an important process utilized by biological systems to control gene expression and organize development, with histone modification enzymes among the most important components in this regulatory network. The predominant interphase H3 Serine10 kinase in Drosophila is JIL-1, which plays an important role in regulating chromosome structure and modulating gene expression. The full-length JIL-1 protein is composed of four domains including a N-terminal domain (NTD), two kinase domains (KDI and KDII), and a unique C-terminal domain (CTD). In order to study the biological importance of these individual domains, a domain analysis research approach was taken and different functions ...


Dissection Of The Mitotic And Nuclear Functions Of Chromator, A Nuclear-Derived Spindle Matrix Component In Drosophila, Yun Ding Jan 2008

Dissection Of The Mitotic And Nuclear Functions Of Chromator, A Nuclear-Derived Spindle Matrix Component In Drosophila, Yun Ding

Retrospective Theses and Dissertations

A spindle matrix has long been proposed to serve as a stationary or elastic molecular matrix substrate for the organization and activities of microtubules and motors, based on the consideration of a mechanical and functional support for the stabilization of microtubule spindle during force generation from mitotic motors. Recently, the identification of four Drosophila proteins, Skeletor, Megator, EAST and Chromator has provided molecular evidence for the existence of this macromolecular matrix structure during mitosis. All of these four proteins have been shown to interact with each other within a protein complex and redistribute from the nucleus at interphase to form ...


Biochemical And Genetic Analyses Of Physical Associations Among Zea Mays Starch Biosynthetic Enzymes, Tracie Ann Hennen-Bierwagen Jan 2008

Biochemical And Genetic Analyses Of Physical Associations Among Zea Mays Starch Biosynthetic Enzymes, Tracie Ann Hennen-Bierwagen

Retrospective Theses and Dissertations

Mutations affecting specific starch biosynthetic enzymes commonly have pleiotropic effects on other enzymes in the same metabolic pathway. Such genetic evidence indicates functional relationships between components of the starch biosynthetic system including starch synthases (SS), starch branching enzymes (BE), and starch debranching enzymes (DBE), however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/or DBEs associate physically with each other in multisubunit complexes. To test this hypothesis, this study sought to identify stable associations between SSI, SSIIa, SSIII, SBEI, SBEIIa, and SBEIIb from maize amyloplasts. Three separate detection methods, yeast ...


Development Of An Rna Aptamer To Pd173955n Through Selex, Samir Mehanovic Jan 2008

Development Of An Rna Aptamer To Pd173955n Through Selex, Samir Mehanovic

Retrospective Theses and Dissertations

With all the technology available to us, the causes of cancers are still puzzling researchers all over the world. Everyday different approaches are being tested to help cure various cancers. With the development of molecular biology, we are able to understand cancer more at molecular level, which helps us look at different methods to treat cancer patients.;The molecular basis for leukemia cancers are understood to a level where we are able to design treatments that specifically target proteins responsible for the disease. One of those proteins is the Bcr-Abl tyrosine kinase which is mostly responsible for chronic myelogenous leukemia ...


The Structure And Mechanism Of Saicar Synthetase, Nathaniel Daum Ginder Jan 2008

The Structure And Mechanism Of Saicar Synthetase, Nathaniel Daum Ginder

Retrospective Theses and Dissertations

SAICAR synthetase catalyzes the eighth step in bacterial de novo purine nucleotide biosynthesis: ATP + L-aspartate + CAIR → ADP + Pi + SAICAR Enzymes involved in nucleotide synthesis are common targets for chemotherapeutic and antimicrobial drugs. They work by reducing the rate of growth of rapidly proliferating cells, thus preventing tumor growth and microbial fecundity. L-Alanosine, an L-aspartate analogue that acts as a substrate for SAICAR synthetase, is a natural product with antiviral and antitumor activities. The product of the reaction is a potent inhibitor of adenylosuccinate synthetase and adenylosuccinate lyase and is responsible for L-alanosine toxicity. L-Alanosine may be effective as a chemotherapeutic ...


Genetic Regulation Of The Healthfulness Of Beef Fatty Acid Composition, Shu Zhang Jan 2008

Genetic Regulation Of The Healthfulness Of Beef Fatty Acid Composition, Shu Zhang

Retrospective Theses and Dissertations

The effects of genetic factors, such as breed and single nucleotide polymorphisms (SNPs), on beef fatty acid composition were investigated in the current study. The first objective was to identify single nucleotide polymorphisms (SNPs) in the thioesterase (TE) domain of bovine fatty acid synthase (FASN) gene and evaluate the extent to which they were associated with beef fatty acid composition. The four exons in FASN that encode for TE were sequenced, and three SNPs, AF285607:g.17924A>G, g.18663T>C, and g.18727C>T, were identified. Purebred Angus bulls (n = 331) were classified into three genotype groups, AA (n ...


Studies Of Intermediates And Regulation In Snare-Mediated Membrane Fusion, Xiaobing Lu Jan 2008

Studies Of Intermediates And Regulation In Snare-Mediated Membrane Fusion, Xiaobing Lu

Retrospective Theses and Dissertations

At the synapse, neurotransmitters are released via Ca2+-triggered exocytotic fusion of synaptic vesicles with the presynaptic plasma membrane. The whole process is controlled by various proteins. SNAREs have been recognized as the key components that drive membrane fusion. In addition, many other proteins, such as Munc18/nSec1, Mun13, synaptotagmin, complexin, etc. are characterized to regulate synaptic transmission temporally and spatially.;The in vitro bulk fluorescence assay was applied to examine the kinetics of membrane fusion of liposomes mediated by recombinant neuronal SNAREs and led to the demonstration of hemifusion as an intermediate in the pathway. In order to monitor ...


Use Of Fourier Transform Infrared (Ftir) Spectroscopy To Determine The Health-Promoting Index (Hpi) Of Cow's Milk, Wendy Leanne Thomas Jan 2008

Use Of Fourier Transform Infrared (Ftir) Spectroscopy To Determine The Health-Promoting Index (Hpi) Of Cow's Milk, Wendy Leanne Thomas

Retrospective Theses and Dissertations

Fourier transform infrared (FTIR) spectroscopy steadily has been gaining popularity for the analysis of milk components including total fat, protein, lactose, and ketones. To date, there have been few descriptions of the use of FTIR spectroscopy for the determination of milk fatty acid composition.;The first objective of this thesis was to develop a method utilizing FTIR spectroscopy for prediction of the health-promoting index (HPI) of milk. The HPI is known to affect the relative softness of butter (Bobe et al., 2003); so, this model could be used to predict which cows' milk could be designated for generation of niche ...


Functional Analysis Of Paralogous Genes In Acetyl Coenzyme A Metabolism, Joel D. Schmidt Jan 2008

Functional Analysis Of Paralogous Genes In Acetyl Coenzyme A Metabolism, Joel D. Schmidt

Retrospective Theses and Dissertations

No abstract provided.


The Locations And Signaling Of H-Ras On Endosomes And Plasma Membrane, Xing Wang Jan 2008

The Locations And Signaling Of H-Ras On Endosomes And Plasma Membrane, Xing Wang

Retrospective Theses and Dissertations

No abstract provided.


Development Of Ss Dna Aptamers For C-Myc:Max By Selex (Systematic Evolution Of Ligands By Exponential Enrichment), Ying Liu Jan 2007

Development Of Ss Dna Aptamers For C-Myc:Max By Selex (Systematic Evolution Of Ligands By Exponential Enrichment), Ying Liu

Retrospective Theses and Dissertations

Cancer, a complicated disease, results from various causes including abnormal overexpression of oncogene products. The c-myc gene was found as the cellular homolog of v-myc oncogene. Its deregulated expression can result in a wide range of tumors in human and mouse. c-Myc, as a transcription factor, requires the Max protein to form a heterodimer that binds to its target DNA sequence, CACGTG, termed an E box, to drive some cancer-related downstream gene expression. Here a strategy of developing DNA aptamer(s) using SELEX (Systemic Evolution of ligands by Exponential Enrichment) was performed to isolate single-stranded DNA molecules that could recognize ...


Studies Of Protein Designability Using Reduced Models, Myron Peto Jan 2007

Studies Of Protein Designability Using Reduced Models, Myron Peto

Retrospective Theses and Dissertations

One the most important problems in computational structural biology is protein designability, that is, why protein sequences are not random strings of amino acids but instead show regular patterns that encode protein structures. Many previous studies that have attempted to solve the problem have relied upon reduced models of proteins. In particular, the 2D square and the 3D cubic lattices together with reduced amino acid alphabets have been examined extensively and have lead to interesting results that shed some light on evolutionary relationship among proteins. Here, additionally to the 2D square lattice, we study the 2D triangular and 3D face ...


The Roles Of Lcn2 In The Inflammatory Lung And Liver, Yinghua Liu Jan 2007

The Roles Of Lcn2 In The Inflammatory Lung And Liver, Yinghua Liu

Retrospective Theses and Dissertations

Uterocalin (Lcn2; also called 24p3, SIP24, or siderocalin and abbreviated as Utc) is an acute phase protein, for which the physiological function remains to be determined. Basal Utc expression is highest in the uterus, mammary gland and lung, which are three tissues with direct exposure to the diverse bacterial flora of the external environment. The demonstrated ability to bind siderophores is one means by which Utc can provide protection against pathogenic bacteria. However, its specificity for siderophores limits the ability of Utc to ward off pathogens. Epithelial tissues that interact with the external environment are exposed to many different nonpathogenic ...


Characterization Of Rns2, An S-Like Rnase In Arabidopsis Thaliana Suggests Role In Phosphate Recycling, Saralyn Ohanian Jan 2007

Characterization Of Rns2, An S-Like Rnase In Arabidopsis Thaliana Suggests Role In Phosphate Recycling, Saralyn Ohanian

Retrospective Theses and Dissertations

Ribonucleases play important biological roles within all organisms but many have not been well characterized. The family of T2 RNases is well-conserved throughout the kingdoms of life and yet their properties and functions are not well understood. RNS2 is one of five such RNases found in Arabidopsis, and preliminary evidence shows that it may be involved in phosphate recycling and localized to an intracellular compartment. Using rns2 knock-out mutant plants, I showed that RNS2 has an effect on flowering time during times of phosphate starvation. I also showed that RNS2 is localized to the ER, more specifically, to ER-bodies. To ...


Production Of 3-Hydroxypropionate From Biomass, Netra Rajguru Agarkar Jan 2007

Production Of 3-Hydroxypropionate From Biomass, Netra Rajguru Agarkar

Retrospective Theses and Dissertations

Biorenewable technology is a developing field of science that researches alternative sources for petroleum products like fuels, plastics, paints, etc. The DoE biomass program has identified the 12 top chemicals that can be produced from biomass and further processed to replace many petroleum products; 3-hydroxypropionate (3HP) is a top 12 chemical that can be used to produce plastics, paints, tires, and other consumer products. This thesis describes the attempts to produce 3HP from glucose in E. coli and from syngas in R. rubrum. Malonyl CoA reductase from C. aurantiacus and R. castenholzii was cloned and expressed in E. coli and ...


Methanobactin And The Membrane-Associated Methane Monooxygenase In Methanotrophy: A Tale Of Two Proteins, Dongwon Choi Jan 2007

Methanobactin And The Membrane-Associated Methane Monooxygenase In Methanotrophy: A Tale Of Two Proteins, Dongwon Choi

Retrospective Theses and Dissertations

No abstract provided.


Analysis Of Potential Interaction Of Spindle Matrix Proteins And The Motor Protein Ncd In Drosophila, Lei Zhu Jan 2007

Analysis Of Potential Interaction Of Spindle Matrix Proteins And The Motor Protein Ncd In Drosophila, Lei Zhu

Retrospective Theses and Dissertations

The microtubule-based mitotic spindle is dynamic rather than static, as polymerization and depolymerization of microtubules occur in sequence. Motor proteins including a minus end directed Ncd are associated with the microtubule-based spindle, and have long been proposed to generate force in microtubules by crossbridging and sliding microtubules relative to adjacent microtubules.;However, the force exerted by motor proteins is only part of the force exerted in the spindle. A stationary "spindle matrix" structure has been proposed to provide a strut on which motor proteins and microtubules interact during force generation. So far at least four molecular components of the spindle ...


Characterization Of Three Spindle Matrix Proteins, Megator, East And Asator, In Drosophila, Hongying Qi Jan 2007

Characterization Of Three Spindle Matrix Proteins, Megator, East And Asator, In Drosophila, Hongying Qi

Retrospective Theses and Dissertations

Two previously identified proteins Skeletor and Chromator provide the molecular evidence for the existence of a spindle matrix complex in Drosophila. The spindle matrix has been proposed to be a macromolecular complex, which plays a role in organization and stabilization of the mitotic spindle as well as in providing structural support for counterbalancing force production.;In this dissertation, I present the identification and characterization of three more putative spindle matrix candidate proteins, Megator, EAST and Asator. Megator, an ortholog of the mammalian TPR protein, contains a large coiled-coil domain at its NH2-terminus and an acidic, non-structural COOH-terminus. Immunohistochemistry studies show ...


Starch Function In Ornamental Tobacco Floral Nectary Development, Gang Ren Jan 2007

Starch Function In Ornamental Tobacco Floral Nectary Development, Gang Ren

Retrospective Theses and Dissertations

Morphological changes of floral nectary gland and subcellular transition of floral nectary cells of ornamental tobacco LxS8 were exclusively investigated in this study. Enlargement of the floral nectary gland of ornamental tobacco LxS8 that occurs during development is accompanied by a major accumulation of Periodic Acid-Schiff's staining (PAS-staining) starch grains in nectary amyloplasts. Quantification of starch purified from the nectary at various stages of development showed little starch at an early developmental stage, soon thereafter the amount of starch increased dramatically, reaching a peak approximately 24 hours prior to anthesis. After this time, the amount of starch declined dramatically ...


Effects Of Dietary Conjugated Linoleic Acid On European Corn Borer Survival, Growth, Fatty Acid Composition, And Fecundity, Lindsey Gereszek Jan 2007

Effects Of Dietary Conjugated Linoleic Acid On European Corn Borer Survival, Growth, Fatty Acid Composition, And Fecundity, Lindsey Gereszek

Retrospective Theses and Dissertations

No abstract provided.


Biophysical Characterization Of The 117 Amino Acids Long N-Terminal Segment Of D-Raf (Isoform A), Oren Tchaicheeyan Jan 2007

Biophysical Characterization Of The 117 Amino Acids Long N-Terminal Segment Of D-Raf (Isoform A), Oren Tchaicheeyan

Retrospective Theses and Dissertations

Drosophila Raf (D-Raf) is an orthologue of Human B-Raf. D-Raf may have two splice variants, a short (Isoform A, I-A) and a long one (Isoform B, I-B). Protocols for over-expression (E. Coli) and purification of 117 amino acids (aa) N-terminal segment (NTS) of D-Raf protein (I-A), both WT and Mutant (T60E) were established. These protein constructs were also biophysically characterized. Thrombin cleavage conditions were optimized, as monitored by 5 amino acid NTS sequencing, mass spectroscopy and SDS-PAGE electrophoresis. An attempt to purify the 160 aa D-Raf (I-B) NTS failed. NMR characterization in a 700 MHz magnet employing three experiments (HSQC ...


Allosteric Regulation Of Mammalian And Bacterial Fructose-1,6-Bisphosphatases, Justin Keith Hines Jan 2007

Allosteric Regulation Of Mammalian And Bacterial Fructose-1,6-Bisphosphatases, Justin Keith Hines

Retrospective Theses and Dissertations

Fructose-1,6-bisphosphatase (FBPase) is a critical regulatory enzyme in gluconeogenesis. In mammals, the enzyme is subject to metabolic regulation; the hormones insulin and glucagon reciprocally regulate the enzyme in the liver through the controlled production and degradation of the inhibitor fructose-2,6-bisphosphate (Fru-2,6-P2) and hence control hepatic glucose production. FBPase is nearly ubiquitous in living organisms and in the bacterium Escherichia coli , the homologous enzyme is imperative for growth on gluconeogenic substrates. How does this organism regulate FBPase without the use of exogenous hormones or Fru-2,6-P2? Investigations reported herein describe high resolution crystal structures of the enzyme from ...


Determinants Of Substrate Recognition And Mechanisms Of Subunit Exchange, Yang Zhou Jan 2007

Determinants Of Substrate Recognition And Mechanisms Of Subunit Exchange, Yang Zhou

Retrospective Theses and Dissertations

Adenylosuccinate synthetase (ADSS) offers a good opportunity to measure the effect of non-bonded interactions. Both IMP and 2'-deoxy-IMP are good substrates supporting identical binding affinities and maximal velocities; however, L-aspartate exhibits significant differences in binding affinities depending on which nucleotide is used as a substrate. Crystal structures of both complexes were identical except for the absence of the 2'-OH group in the dexoy-nucleotide complex. The decrease in energy (∼2 kcal/mole) in non-bonded interactions due to the loss of one atom explains the nearly 40-fold difference in the binding affinity of L-aspartate and an analog of L-aspartate called ...