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Physiology And Biochemistry Of Cold-Hardy Table Grapevines, Annasamy S. Chandrakala

Master's Theses and Capstones

Grapes are grown worldwide to produce wine, grape juice and are also popular as fresh table grapes or dried raisins. Due to their nutritional value and importance in the multibillion-dollar wine industry, grapes are considered the most commercially important berry crop. Grape production has primarily concentrated on European wine grapes, Vitis vinifera, in the dry, hot Mediterranean and Central Asian climates. V. vinifera is not cold tolerant enough to endure winter temperatures below -15°C. The introduction of several interspecific hybrids (of both wine and table grape) cultivars in the 20th century and selection of a training system has helped propel …

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 …

Technique For Normalization Of Cross-Linked Peptide Ion Intensity To Elucidate Enzymatic Conformational Changes, Donna Hogan

Master's Theses and Capstones

Cross-linking coupled to Liquid Chromatography/Mass Spectrometry (CXMS) has become an invaluable technique for examining protein-protein interactions and monitoring enzymatic conformational changes. Thus far, comparative quantitation of cross-linked peptides measured by their mass spectrometry (MS) ion intensity is employed to deduce residue cross-linking propensity and spatial proximity. Using two distinct conformations of a structurally well-characterized model protein, we examined the correlation of cross-linked peptide MS signals and distances, but were not able to observe any obvious correlation. Conceivably, several physiochemical factors can affect residue reactivities thus MS signal intensity of the corresponding cross-linked peptides. For NHS ester chemical cross-linkers, the NHS …

Bink Domain Functional Characterization In The Regulation Of Bioluminescence In Vibrio Fischeri, Ian Ster

Master's Theses and Capstones

Prokaryotes encode a remarkable ability to adapt to niches by sensing environmental cues through signal transduction systems (STSs). Typical STS proteins interact through a phosphorylation relay between histidine (His) and aspartate (Asp) residues within modular domains on sensory kinase and response regulator (RR) proteins to elicit cellular responses. A single point mutation in the sensor kinase BinK (BinK1 R537C) conferred an outstanding ability for the non-native V. fischeri strain MJ11 to successfully colonize Euprymna scolopes by affecting multiple symbiotic phenotypes including luminescence activation. However, the role of BinK in luminescence, the interacting partners, and functional mechanism are unknown. We hypothesized …

Molecular Dynamics Studies Of Nucleic Acids And Ribonucleoprotein Complexes, Tyler James Mulligan

Master's Theses and Capstones

Molecular simulations of protein-nucleic acid complexes, as well as the HIV-1 Trans Activation Response Element (TAR) RNA molecule, were conducted. First, three different molecular dynamics techniques were studied on the molecule HIV-1 TAR RNA. The three techniques studied were classical molecular dynamics, steered molecular dynamics (SMD), and metadynamics. The classic molecular dynamics simulations were used to equilibrate the HIV-1 TAR RNA system, as well as every other system studied in this thesis. The SMD technique was used in order to observe the breaking force of the nucleotide interactions within TAR. This breaking force averaged to about 100pN. The metadynamics technique …

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. …

Effect Of In Vivo Polybrominated Diphenyl Ether (Pbde) Treatment On Hepatic Glyceroneogenesis And Lipid Metabolism, Kylie Rose Cowens

Master's Theses and Capstones

Polybrominated diphenyl ethers (PBDEs) are flame-retardant chemicals that contaminate the environment. Through ingestion and inhalation, these chemicals get into the human body, where they affect the liver by suppressing the metabolic enzyme phosphoenolpyruvate carboxykinase (PEPCK), which is partially responsible for glyceride-glycerol production via glyceroneogenesis. This study investigated the effects of PBDE-induced hepatic PEPCK suppression on glyceroneogenesis, and the associated perturbations in liver lipid metabolism. Twenty-eight male, weanling Wistar rats were treated daily with 14 mg/kg body weight PBDE mixture, DE-71 (TRT, n=14) or corn oil vehicle (CON, n=14) for 28 days. After a 48-hour fast, rats were sacrificed and blood …

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 …

Detection Of Trehalose In Porphyra Extracts Using Mass Spectrometry And Gas Chromatography-Mass Spectrometry, Ying Huo

Master's Theses and Capstones

It is known that trehalose helps many plants to survive under desiccated environment26. This property of trehalose has also been proposed to explain why Porphyra could survive desiccation when left on dry land for a long period of time25. However, after extensive study25, effort at identifying trehalose in Porphyra has been unsuccessful, leaving the question of whether trehalose really exists in Porphyra unanswered. In my thesis research, in collaboration with Dr. Anita Klein, I set out to use ion-trap mass spectrometry and gas chromatography coupled with mass spectrometry to identify and quantify the amount of trehalose in Porphyra extracts. Using …

The Effect Of In Vivo Pbde Treatment On Hepatic Phosphoenolpyruvate Carboxykinase (Pepck) Enzyme Kinetics In Male Wistar Rats, Jessica T. Nash

Master's Theses and Capstones

Polybrominated diphenyl ethers (PBDEs) are synthetic flame-retardant chemicals that enter the environment and mammalian body and may disrupt glucose metabolism. This study investigated the effect of PBDEs on a key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). Forty-eight male Wistar rats were gavaged with corn oil or corn oil containing 14 mg/kg DE-71 for 3, 14 or 28 days (N = 8 per group). At each time point, fasting plasma glucose, insulin and C-peptide were measured and liver PEPCK enzyme activity was assayed. PBDEs significantly decreased PEPCK Vmax (mumol/min/g liver weight) at 3 days by 26%; this reduction persisted through 28 days. …

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 …

Truncated Mortalin In Animal Cancer, Katrina K. Olson

Master's Theses and Capstones

Hematopoetic neoplasia or clam hemocyte cancer (a leukemia-like disease) has been studied in a number of bivalve molluscs for the last 20 years. Recent molecular studies of the hemocytes of the soft shell clam, Mya arenaria, have demonstrated an interaction between p53 and mortalin, the mitochondrial Hsp70. The former protein is intimately involved in the initiation of cell-cycle arrest, apoptosis, DNA repair, and cell differentiation. In cancerous clams, wild-type p53 is sequestered in hemocyte cytoplasm by mortalin and cannot be translocated to the nucleus. This is critical because although p53 is functions properly, it is unable to enter the nucleus …

Investigating Pheromone Variability In A Terrestrial Woodland Salamander, Adam James Chowinard

Master's Theses and Capstones

The communication system of Plethodontid salamanders relies heavily upon chemical signaling. Pheromone molecules convey a tremendous amount of information to conspecifics, and facilitate a wide variety of the essential social functions of these animals. Much of the information contained within the pheromone mixture is variable from one individual to the next, such as the ability to recognize specific individuals, the size of the sender, and the relative "quality' of the signal. In order for variable information to be conveyed through chemical signals, the corresponding variability must be contained within the pheromone mixture itself. This variability may exist as a gradient …

Regulation Of Photoreceptor Phosphodiesterase By Prenyl Binding Protein And By Other Interacting Proteins In Photoreceptor Cells, Hannah J. Gitschier

Master's Theses and Capstones

Photoreceptor phosphodiesterase (PDE6) is a central component of the visual pathway. While initial PDE6 activation following light stimulation of photoreceptors is well understood, regulation of PDE6 during recovery and light adaptation may require additional components that interact with PDE6. Two approaches were taken to characterize these potential interactions. The first explored the interaction of PDE6 with prenyl binding protein (PrBP/delta) and demonstrated that changes in membrane localization of PDE6 may occur during light adaptation as a result of association with PrBP/delta. The second approach characterized the PDE6 "interactome" by a mass spectrometric identification of 75 proteins that co-purify with PDE6 …

Development Of Microwave-Assisted Methods To Aid In Carbohydrate Analysis, Stephanie Maniatis

Master's Theses and Capstones

This study involved development of three high-throughput chemical techniques to assist in carbohydrate analysis. The methods utilized both microwave radiation and a volatile organic base to achieve short reaction times and high product yield. Two methods, one reductive and one non-reductive, released intact O-glycans for structural analysis, and one method aided in determination of the site of O-glycosylation on the peptide.

Optimal reaction conditions for each method were determined on standard glycoproteins. In comparison to common release methods, microwave-assisted reductive O-glycan release provided higher yields of O-glycans in every case. O-glycans were released rapidly and were able to be subsequently …

Characterization Of T24d14(Ok809) A Putative Alpha-1,2 Glucosyltransferase In C Elegans And Detailed Structural Analysis Of The Lipid Linked Oligosaccharide Pathway Via Msn, William Cobb Wiswall Jr.

Master's Theses and Capstones

The formation of glycoconjugates, lipids or proteins covalently linked to carbohydrate groups in biological systems, is a universal process in every form of life studied to date. The carbohydrate moiety of glycoconjugates range in complexity from single monomers to intricately branched structures containing over a dozen residues. This complexity is further compounded by the incorporation of several different monomer types with varying linkage positions and branching patterns. The number of possible carbohydrate structures is astronomical providing significant challenges to characterize, study, and catalog these molecules. Furthermore, assigning biological consequence can be a daunting task considering the numerous interactions caused by …

Design And Implementation Of A Statistical Analysis Tool For Two Biological States, Gang Lu

Master's Theses and Capstones

The major goal of research in this thesis is to design and implement a software tool (Q5+) that can easily, quickly and reliably search biomarkers by statistically analyzing mass spectrometry data from two different biological states. Q5+ implements most of the Q5 algorithm, a very good algorithm that is used for classifying mass spectrometry data (Lilien et al). Compared Q5 Q5+ improves the usability of Q5 by incorporating a Graphic User interface and Matrix Library. Results show that by running the same data, Q5+ and Q5 showed the equivalent classification ability. Q5+ also implements the Peak Screening feature, which can …

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 …

N-Linked Glycans Characterization And Biomarker Discovery By Sequential Mass Spectrometry, Hongqin (Jenny) Jiao

Master's Theses and Capstones

N-linked glycans from a well-studied glycoprotein, (hen albumin) were characterized using ion trap MS (MSn) without chromatographic separation. From the spectral profile, ten major ion compositions were selected for study, which resulted in the characterization of 37 isomeric structures, 16 of which were previously unreported. These findings were compared with previously reported studies using different analytical strategies: IMMS, MS/MS. MSn not only identifies but also structurally characterizes components in the absence of any adjunct instrumentation. Selected examples have been detailed.

A major challenge for adult stem cell (ASC) research is the lack of specific biomarkers for their identification and isolation. …

Regulation Of Polyamine Metabolism In Transgenic Poplar Cell Cultures, Smita Cherry

Master's Theses and Capstones

Polyamines (PA) are naturally occurring low molecular weight aliphatic amines found in all living organisms and essential for their growth and development. The present study uses the tool of transgenic manipulation to elucidate the regulation of PA pathway in poplar cells. This study was divided into two segments; the first segment provides insight into biochemistry of a putrescine overproducing cell line (HP) that overexpresses a mouse ornithine decarboxylase (mODC) under 35S-CaMV promoter, with respect to different treatments. The second segment focused on creation and biochemical characterization of a cell line (SOS1) that overexpresses ODC/SAMDC from Plasmodium falciparum controlled by the …

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) …

Development, Production, Purification, And Activity Of Recombinant Lamprey Gonadotropin In S2 Drosophila And Chinese Hamster Ovary Cell Lines, Geoffrey Bushold

Master's Theses and Capstones

The objective of this research was to develop methods for the production and purification of recombinant lamprey GTH beta. The overall goal was to produce an active purified recombinant lamprey GTH that could be used for immunological, physiological, and histological studies to further understand the role of the gonadotropin in the sea lamprey, Petromyzon marinus. Constructs with different combinations of tethered lamprey gonadotropin beta to human CG alpha and channel catfish gonadotropin alpha were transfected and expressed in stable cell lines of Drosophila (S2) cells and Chinese hamster ovary (CHO) cells. Cell lines were grown at a large scale and …

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 …