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Biophysical Properties Of An Antifreeze Protein And The Effects Of Ionic Liquids On The Model Protein Gb1, Korth Wade Elliott

Doctoral Dissertations

Antifreeze proteins (AFPs) are a unique class of protein characterized by their ability to depress the freezing point of water sufficient to prevent the formation of ice crystals by adsorbing to the surface of ice crystals. This unique ability allows organisms (e.g. plants, fish, insects, etc.) which live in extremely cold climates to survive. Because of these proteins’ ability to prevent and slow the rate of ice crystal formation, they have great potential in the application of cryopreservation in medicine, agriculture and food science. Antifreeze proteins have been known for over five decades, however, their exact mechanism of action is …

Dysregulation Of O-Linked Β-N-Acetylglucosamine (O-Glcnac) Cycling Supports Tumorigenicity Of Cancers Of The Female Reproductive Tract, Nicole Jaskiewicz

Doctoral Dissertations

Hyper-O-GlcNAcylation of proteins is a subsequent artifact of metabolic disorder and is indicative of many cancers, including cancers of the female reproductive tract. While the incidence of most cancer types has been declining in the U.S., endometrial and cervical cancer remain among the most common cancers diagnosed in women. Diabetic women have a 2-3 fold increased risk of developing endometrial cancer, and tend to have more aggressive cases of cervical cancer, however, the molecular aspects of these risks are not fully understood. This study investigated the alteration of cellular O-GlcNAcylation of proteins as the potential mechanistic connection between diabetes and …

Survey Of The Charge Properties Of Phospholipids Using Nanodiscs And Membrane-Confined Electrophoresis, Cheng Her

Doctoral Dissertations

Phospholipids (PL) are a major, diverse constituent of cell membranes. PL diversity arises from the nature of the fatty acid chains, as well as the head group structure. The head group charge is thought to contribute to both the strength, and specificity of protein-membrane interactions. Furthermore, the divalent cations Ca2+ and Mg2+, have been shown to be essential for optimal binding for some of these interactions. Because it has been difficult to measure membrane charge, it has been impossible to quantitate the role charge plays in these interactions. However, Nanodiscs provide a stable, planar membrane bilayer suitable for biophysical studies. …

The Application Of Analytical Ultracentrifugation With Fluorescence Detection System To The Study Of Macromolecular Complexes In Biological Systems, Wen Xi

Doctoral Dissertations

Using the novel technique of analytical ultracentrifugation with fluorescent detection (AU-FDS), I have conducted the analysis of the properties of two types of intracellular macromolecular complexes: the translational mRNP complex and the intermediate soluble aggregates present in Huntington’s disease. With AU-FDS it is possible to differentiate a broad size range of soluble molecules from complex mixtures and determine the size and abundance of each individual complex based on its sedimentation rate under a centrifugal field.

In the first part of my thesis, the characteristics of the translational repressor SBP1 was determined by analyzing the mRNP complexes it was associated with. …

Identification And Stoichiometric Analysis Of The Monosomal Translational Complex, Xin Wang

Doctoral Dissertations

The identification of the components involved in translational complexes has relied primarily on in vitro studies. Determining which proteins associate together in these complexes, under what conditions they do so, and how the composition of the complexes change under different conditions have became the key issues of in vivo studies. After a one-step affinity purification, using a novel technique of analytical ultracentrifugation with a fluorescence detection system (AU-FDS) I have identified a 77S monosomal translational complex in the yeast Saccharomyces cerevisiae. Major components of the 77S complex include the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G1/eIF4G2 …

The Identification And Characterization Of The Interaction Between Upf1 And Pab1 During Nonsense-Mediated Decay & The Identification Of Novel Protein Complexes Associated With Translation Termination Factor Erf1, Roy Richardson

Doctoral Dissertations

There are still many protein interactions that occur during translation termination that are poorly understood. One of the important termination pathways still under investigation is nonsense-mediated decay (NMD), which rapidly degrades mRNAs that contain a premature stop codon (PTC). I identified that the interaction between Upfl, which is required for NMD, and PAB1 occurs via the RRM1 domain of PAB1 in the yeast Saccharomyces cerevisiae. Determining the role of this interaction during NMD was performed with pulse-chase assays using a PGKlpG mRNA. These assays revealed that the interaction between Upfl and PAB1 is required for a shift from distributive to …

Regulation Of The Catalytic And Allosteric Properties Of Photoreceptor Phosphodiesterase (Pde6) By The Glutamic Acid-Rich Protein-2 (Garp2), Wei Yao

Doctoral Dissertations

The photoreceptor phosphodiesterase (PDE6) must be precisely regulated to control the sensitivity, amplitude, and kinetics of the photoresponse during excitation, termination and adaptation to light stimulation in rod and cone photoreceptors. The central hypothesis of this thesis is that one PDE6 binding partner, the glutamic acid-rich protein (GARP2), may regulate PDE6 to reduce its "dark noise", enhance its sensitivity and conserve metabolic energy during rod photoresponse saturation.

The first aim of this research is to better understand the unique biochemical and biophysical characteristics of GARP2 in order to reveal its functional attributes for regulating PDE6 during phototransduction in rod photoreceptors. …

Patterns Of Cytosine Methylation In The Genome Of Caenorhabditis Elegans, Kazufusa Okamoto

Doctoral Dissertations

Recent large-scale comparative analysis of cytosine DNA methylation across diverse eukaryotes suggest that early features of DNA methylation present in the last common ancestor of all eukaryotes some 1.6 to 1.8 billion years ago included the methylation of gene bodies and transposable elements (Zemach, McDaniel et al. 2010; Parfrey, Lahr et al. 2011). These potentially ancient patterns may reflect a primitive role of methylation in transcriptional fidelity and as a mechanism to protect the germ line from transposon, or repeat, mediated mutation. Because spurious transcription and mutation are hypothesized to be among the critical limiting factors to genome size, an …

Study Of The Rate And Spectrum Of Spontaneous Mutations, Way Sung

Doctoral Dissertations

Mutations are the initial force responsible for all aspects of genetic variation, and are a central part to evolution in all organisms. Yet despite its importance, the previously high cost that is associated with surveying mutations at a genome-wide scale has limited the understanding of the mutation process in eukaryotes. However, recent high-throughput sequencing technology has greatly reduced the cost of surveying mutations. By applying high-throughput sequencing to mutation accumulation experiments, we have begun to characterize the genome-wide mutation spectrum of eukaryotes.

Across all eukaryotes, we observe a biased rate of G/C-> A/T mutations that exceeds the number of A/T- …

Identifying Novel Proteins In Translation Complex By Using Analytical Ultracentrifugation With Fluorescent Detection System, Chongxu Zhang

Doctoral Dissertations

The primary components of the translation initiation complex have been identified by a variety of techniques. However, it is likely that all components of the translation initiation complex are still not fully discovered. Identifying new components should lead to a better understanding of the translation process and how it is regulated. Using mass spectrometric studies, we have identified 41 non-ribosomal proteins and non-translation initiation factors as possible components of the translation initiation complex. To determine which of these proteins are in the translation complex, we applied analytical ultracentrifugation with fluorescent detection system (AU-FDS) to detect this complex. Following a one-step …

Modeling Congenital Disorders Of Glycosylation In Caenorhabditis Elegans: Genetic Influences And Structural Consequences Of N-Linked Glycosylation, Weston Booth Struwe

Doctoral Dissertations

The attachment of oligosaccharides to the amide nitrogen of asparagine side chains in proteins is a fundamental process occurring in all metazoans. This process, known as N-glycosylation, is complex and is achieved by the precise interactions of various cellular components. The initial stage of N-glycosylation occurs in the endolasmic reticulum and is preserved among eukaryotes. Glycans are further developed in the Golgi and the structural complexity depends greatly on the animal species, tissue and developmental stage. Oligosaccharides are unique biomolecules because unlike DNA or proteins, no primary sequence exists nor is its' synthesis template driven. A major goal of glycobiologist …

Gonadotropin Releasing Hormone In Agnathans, Scott Ira Kavanaugh

Doctoral Dissertations

Hagfish and lampreys are the only two representatives of Agnathans among extant vertebrates. The regulatory hypothalamic neurohormone, gonadotropin-releasing hormone (GnRH), regulates reproduction in all vertebrates through the hypothalamic-pituitary-gonadal axis. Most vertebrates have at least two forms of GnRH in the brain. In hagfish the primary amino acid structure of GnRH has not been identified as yet, however, indirect methods have shown an immunoreactive GnRH or a GnRH-like peptide in the brain of hagfish. In addition concentrations of brain immunoreactive-GnRH have been correlated with reproductive stages in the Atlantic hagfish ( Myxine glutinosa). Therefore the objective of the first study was …

Understanding Polyamine Metabolism Through Transgenic Manipulation In Poplar Suspension Cultures, Sridev Mohapatra

Doctoral Dissertations

Polyamines are low molecular weight aliphatic amines that are obligatory requirements for cell survival and growth. The commonly occurring polyamines in plants are putrescine, spermidine, and spermine. Suspension cultures of poplar (Populus nigra x maximowiczii), transformed with a mouse ornithine decarboxylase gene (under the control of a 2X 35S CaMV promoter) were used to study the impact of up-regulation of putrescine biosynthesis (and consequent enhanced catabolism) on several aspects of cellular metabolism. The transgenic cells were compared with a control cell line that was transformed with the beta-glucuronidase (GUS) gene.

It was observed that enhanced putrescine metabolism resulted in: (i) …

Uncovering Unique N-Linked Glycan Structural Isomers In Cancer Via Ms(N) Disassembly, Justin Michael Prien

Doctoral Dissertations

Molecular glycosylation is involved in key developmental roles including control of cell differentiation, innate immunity and signal transduction. Equally as demonstrable are numerous aspects of tumor development, from cellular proliferation to angiogenesis and metastasis. This sensitivity to cellular pathological is reflected in the diversity of glycoforms presented on glycoproteins. In spite of the potential utility of using molecular glycosylation as potential diagnostic and therapeutic agents little has been accomplished in bringing these capabilities to the clinic. Reasoning for this omission must relate to the paucity of analytical protocols to understand the full details of glycan structure. Analytical advancement in ion …

Development And Application Of Methods For Analysis Of Fungal Glycosphingolipid Structure And Functional Interactions, Emma Adhiambo Arigi

Doctoral Dissertations

Fungal lectins from the mushrooms Polyporus squamosus and Coprinus cinereus have an affinity for beta-galactosides, with extended binding sites for the non-reducing terminal trisaccharide sequence NeuAcalpha2→6Galbeta1→4Glc/GlcNAc, and substituted beta-galactosides resembling mammalian blood group determinants, respectively. In considering the possibility that glycosylinositol phosphorylceramides (GIPCs) could be endogenous ligands of such fungal lectins, structural characterization of glycosphingolipids (GSLs) from the fruiting bodies of these mushrooms was performed. The extraction, isolation and purification of the lipids, and their subsequent characterization by nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography-mass spectrometry (GC-MS) was performed. One monohexosylceramide (CMH) and three major GIPCs were …

Structural Analysis Of Cytokine Signaling Modulators, Cristina Ileana Silvescu

Doctoral Dissertations

The N-linked glycans attached to cytokine receptors modulate signal transduction by interacting with galectin-3 and generating a cell surface lattice. This opposes constitutive endocytosis and reduces the thresholds for cytokine signaling. The preferred ligands for galectin-3 are the poly-N-acetyl lactosaminylated tetra-antennary glycans which are synthesized by Mgat 5 and are present at high levels in tumor cells. A null mutation in Mgat 5 inhibits lattice formation and cancer progression in cells with an oncogenic background. GlcNAc feeding reestablishes lattice formation, cytokine signaling and tumorigenesis.

A MALDI-TOF analysis was employed to identify the N-glycans involved in modulating signal transduction in wild …

Regulation Of Rod Photoreceptor Phosphodiesterase (Pde6) By The Glutamic Acid -Rich Protein 2 (Garp2), Dana C. Pentia

Doctoral Dissertations

Rod photoreceptor phosphodiesterase (PDE6), the central enzyme of visual transduction in vertebrate photoreceptors, associates with the disk membranes of the rod outer segment (ROS) of the photoreceptor cell. This association insures the high efficiency of activation by the G-protein, transducin, and the precise control of its inactivation. In addition to binding to transducin during visual excitation, PDE6 is hypothesized to be regulated by other interacting proteins.

The first aim of this research was to isolate and identify the proteins that interact with PDE6 during various stages of the visual signaling pathway. We evaluated methods for solubilizing PDE6 and its binding …

Analysis Of The Role Of Poly(A) -Binding Protein (Pab1) In The Mrna Degradation Process In Yeast, Gang Yao

Doctoral Dissertations

The mRNA deadenylation process influences multiple aspects of protein synthesis and is known to be the major factor controlling mRNA decay rates. My data demonstrates that yeast PAB1 plays both positive and negative roles in controlling deadenylation, and I have identified particular regions of PAB1 involved in controlling different aspects of the mRNA degradative process. I have found that yeast PAB1 does not play a simple, obstructionist role in regulating CCR4 deadenylation. Instead, PAB1-PAB1 protein interactions, as mediated by the PAB1 proline-rich region (P domain) and the RRM1 domain, are required for the CCR4 deadenylase activity. The P and RRM1 …

Function, Expression And Evolution Of The *Src Family Kinases In Caenorhabditis Elegans, Chery Angel Whipple

Doctoral Dissertations

The vertebrate proto-oncogene, Src, is the prototype of a family of membrane-associated protein tyrosine kinases involved in cell signaling pathways that control cell growth, development, and differentiation. Activation of Src by mutation or overexpression has been implicated in a range of cancers, particularly breast and colon cancer. Inappropriate activation of Src has pronounced oncogenic effects on cell morphology, adhesion, and motility. Despite its implications in cancer, the normal biological role of Src is not well understood.

I used the nematode C. elegans to investigate the expression, function, and evolution of two src genes (src-1 and src-2) utilizing a combination of …

Characterization Of The Major Extrapallial Fluid Protein Of The Mollusc, Mytilus Edulis: Implications For Function, Yan Yin

Doctoral Dissertations

The major protein component of the extrapallial (EP) fluid of the mollusc Mytilus edulis, named the EP protein, has been previously isolated and partially characterized. It has been proposed to play an important role in shell mineralization because of its intriguing property of Ca2+ -binding induced self-assembly.

This dissertation reports on the characterization of the primary structure of the EP protein by RT-PCR and cDNA sequencing methods. The EP protein is comprised of 213 amino acids post cleavage of a signal peptide of 23 amino acids, and is rich in His, Glu and Asp residues. The consensus site of N-glycosylation …

Applications Of Fluorescence Detected Sedimentation I Studying The Thermodynamic Impact Of Valence In Non-Ideal Solutions Ii High Affinity Interactions And Biological Media, Rachel R. Kroe

Doctoral Dissertations

The recent addition of the fluorescence detection optical system (FDS) to the currently available absorbance and interference optical systems on the analytical ultracentrifuge has greatly expanded the wide application of this technique. The new FDS is able to detect very low concentrations of fluorophore allowing the study of high affinity interactions (Kd ∼ pM) not previously accessible with the absorbance optics. The types of high affinity interactions that can be explored include drug-protein interactions, lipid-receptor interactions, DNA-protein interactions and protein-protein interactions. Selective labeling of one or both of the macromolecules can allow elegant dissection of complex assemblies. The only limiting …

On The Neuroendocrine Regulation Of Reproduction: Functional Characterization And Kinetic Studies Of The Lamprey Gonadotropin -Releasing Hormone Receptor And Cloning And Analysis Of The Cdna Encoding Lamprey Gonadotropin-Releasing Hormone-Iii, Matthew Ren Silver

Doctoral Dissertations

The vertebrate hypothalamic-pituitary-gonadal (HPG) axis is regulated by gonadotropin-releasing hormone (GnRH), a decapeptide that is produced and released from the hypothalamus. At the anterior pituitary, GnRH action is mediated through high affinity binding with the GnRH receptor, a rhodopsin-like seven transmembrane G-protein coupled receptor (GPCR). Interest in the evolution of reproductive physiology has led scientists to study the lamprey, a member of the oldest extant class of vertebrates, the agnathans. The studies presented herein contribute to the field of reproductive neuroendocrinology through developing our understanding of ancestral, or ancestral-like characteristics and mechanisms of the HPG axis. This dissertation is divided …

Hydrodynamic Behavior And Thermal Stability Of A Pegylated Protein: Studies With Hen Egg Lysozyme, Yatin R. Gokarn

Doctoral Dissertations

We studied the effect of covalent attachment of polyethylene glycol (PEGylation) on the hydrodynamic behavior and thermal stability of a model protein---Hen Egg Lysozyme (HEL). HEL was modified with a linear, 20-kD, PEG to produce mono (PEG1-HEL), di (PEG2-HEL), and triPEGylated (PEG3-HEL) species.

The hydrodynamic properties of HEL were altered upon PEGylation. A decrease in sedimentation (s) and diffusion (D) coefficients was observed for all three PEG-HEL molecules in comparison to HEL (1.81 s). Despite differences in molecular weights of the PEG-HEL molecules (∼34, 55 and 80 kD), their s values were very close (1.0--1.1 s). Significant hydrodynamic non-ideality was …

Mechanism Of Activation Of Photoreceptor Phosphodiesterase By Transducin And By Low Molecular Weight Proteins, Angela W. Norton

Doctoral Dissertations

Rod photoreceptor phosphodiesterase (PDE) is the central enzyme of visual transduction. PDE is a heterodimer (Palphabeta) with two associated inhibitory gamma subunits, and is bound to the disk membrane by isoprenyl groups. Each catalytic subunit contains a catalytic site and a high-affinity cGMP binding site. In this research, two independent mechanisms of PDE regulation are addressed. The first part examines transducin activation of PDE, focusing on the interactions between the gamma subunits and Palphabeta. The second part examines the 17 kDa delta protein believed to regulate bovine PDE. We found that transducin activation of PDE induces heterogeneity at the active …

Identification Of Suppressors Of Spt10 That Affect Adh2 Expression In The Yeast Saccharomyces Cerevisiae, Bradley David Anderson

Doctoral Dissertations

Suppression of the effects of an spt10 mutation on ADH2 expression is a phenotype shared by a small number of genes involved in the regulation of gene expression. All six known suppressors of spt10 are either components of the CCR4-NOT Complex (CCR4, CAF1, NOT4), or have been shown to interact with the complex ( DBF2, SRB9, SRB10). This indicated that spt10 suppression may be characteristic of CCR4-like gene regulation. In this work, I conducted a screen for additional suppressors of spt10 at ADH2. The screen identified no less than ten complementation groups, including ADH2 and two known suppressors of spt10, …

Transition Metal Complexes Of Novel Ligands Derived From The Cis,Cis-1,3,5-Triaminocyclohexane (Tach) Framework: Structural And Biological Studies, Gyungse Park

Doctoral Dissertations

Transition and Group IIB metal complexes of novel ligands derived from cis,cis-1,3,5-triaminocyclohexane (tach) were prepared for structural and biological activity study. Copper(II) complexes of N,N ',N″-trialkylated derivatives of tach [Cu(tach-R3)Cl2] (R = Me, Et, Pr, furanyl, and thiophenyl) were prepared from CuCl2 in MeOH or MeOH/Et2O solvent. The structures of [Cu(tach-R3)Cl2] in the solid and solution (aqueous and methanolic) states were extensively studied by UV-vis and EPR spectroscopies and were consistent to X-ray structure of [Cu(tach-Et 3)Br0.8Cl1.2] which reveals a slightly distorted square-based pyramidal geometry. Displacement of halide ions with water molecules in aqueous media was observed by the band …

Functional Characterization Of The Ccr4-Not Transcriptional Regulatory Complex, Vasudeo Badarinarayana

Doctoral Dissertations

The CCR4-NOT transcriptional regulatory complex affects expression of a number of genes both positively and negatively. This study demonstrates that the CCR4-NOT complex functionally and physically interacts with TBP and TAFs. Firstly, mutations in CCR4, NOT4, and NOT5 suppressed the his4-912 delta insertion by a mechanism similar to that observed for the defective TBP allele spt15-122. This mechanism appeared to involve stabilization of TBP binding to a specific non-consensus TATA sequence, CATAAA, in the his4-912 delta element. Secondly, using modified HIS3 promoter derivatives containing specific mutations within the TATA sequence, it was found that the NOT proteins were general repressors …

Targeted Gene Replacement To Mimic Familial Hypertrophic Cardiomyopathy In Caenorhabditis Elegans, Craig Alan Almeida

Doctoral Dissertations

The identification in invertebrates of genes homologous to human disease genes provides the possibility to broaden the spectrum of model organisms to include experimental invertebrates. The goal of this study is to test the possibility of using Caenorhabditis elegans as an animal model for the inherited human heart disorder familial hypertrophic cardiomyopathy. In 10-30% of the affected families that have been studied, this disease is caused by mutations in the $\beta$-cardiac myosin heavy chain (MHC) gene. The most common of these mutations results in the replacement of an evolutionarily conserved arginine residue (R403) with glutamine (R403Q). While this information has …

The Purification And Characterization Of An Extrapallial Fluid Protein From The Mollusc, Mytilus Edulis, Stephen Joseph Hattan

Doctoral Dissertations

Shell deposition within the bivalve mollusc Mytilus edulis is regulated by the macromolecules of the extrapallial fluid (EP fluid). These mineralization regulating macromolecules are thought to be responsible for the nucleation, growth regulation and growth cessation of the CaCO$\sb3$ mineral crystals that will inevitable constitute $\geq$95% of the mature shell.

This dissertation presents the results of the purification and characterization of the major EP fluid protein of the bivalve Mytilus edulis. The major EP fluid protein was determined to comprise 56% of the total fluid protein and to consist of 7.25% by weight carbohydrate. The protein was purified though a …

Investigation Of Pd(A)(20)Pd(T)(20) In The Analytical Electrophoresis Apparatus, John Oliver Wooll

Doctoral Dissertations

The analytical electrophoresis apparatus (AEA) is capable of generating and monitoring the electrophoretic migration of macroions. The oligonucleotide pd(A)$\sb{20}\cdot$pd(T)$\sb{20}$ was use as a model to compound to evaluate the range and validity of AEA measurements under a variety of electric fields and solvent conditions. A broad range of electric fields yield consistent, reproducible values. The charge determination from different procedures, steady state electrophoresis (SSE) and electrophoretic mobility, have not been consolidated into a consistent theory but advancements in the scope and understanding of the AEA's potential have been made. The apparent charge from the AEA measured electrophoretic mobility, $\mu$, of …