Molecular Biology Commons^™

Characterization Of Fusarium Species And Alternaria Alternata, And Their Effects On Switchgrass Health And Chemical Components, Sara Beth Collins

Masters Theses

Panicum virgatum L., readily referred to as Switchgrass, is a perennial warm-season bunch grass, used as an alternative energy source for biofuel production. There is insufficient research on switchgrass pathogens, and is expected that an increase in disease pressure will result as more land is reserved for this perennial crop. The purpose of this research was to identify and characterize pathogenic Alternaria alternata and Fusarium species on Switchgrass (Panicum virgatum L.), and to evaluate their impact on stand establishment and plant health. Molecular identifications of fungal isolates obtained from infected commercial switchgrass seed yielded eight species (A. alternata, …

Rapid Molecular Detection And Population Genetics Of Pityophthorus Juglandis, A Vector Of Thousand Cankers Disease In Juglans Spp., Emel Oren

Masters Theses

Thousand Cankers Disease (TCD) is a disease complex involving the fungal pathogen Geosmithia morbida, an insect vector Pityophthorus juglandis, and the hosts, Juglans spp. and Pterocarya spp. Signs and symptoms of TCD include crown thinning due to branch dieback, yellowing and wilting of the leaves, appearance of epicormic shoots, numerous entrance/exit holes, gallery formation by P. juglandis, and the development of small, dark brown cankers underneath the bark. TCD originally described from western U.S., has now expanded to eastern U.S. and northwestern Italy. The disease complex is often difficult to diagnose due to the absence of symptoms …

Modifications Of Myofilament Structure And Function During Global Myocardial Ischemia, Mike K. Woodward

Masters Theses

Cardiac arrest is a prevalent condition with a poor prognosis, attributable in part to persistent myocardial dysfunction following resuscitation. The molecular basis of this dysfunction remains unclear. We induced cardiac arrest in a porcine model of acute sudden death and assessed the impact of ischemia and reperfusion on the molecular function of isolated cardiac contractile proteins. Cardiac arrest was electrically induced, left untreated for 12 min, and followed by a resuscitation protocol. With successful resuscitations, the heart was reperfused for 2 h (IR2) and the muscle harvested. In failed resuscitations, tissue samples were taken following the failed efforts (IDNR). Actin …

Characterizing The Inhibition Of Katanin Using Tubulin Carboxy-Terminal Tail Constructs, Corey E. Reed

Masters Theses

Understanding how the cellular cytoskeleton is maintained and regulated is important to elucidate the functions of many structures such as the mitotic spindle, cilia and flagella. Katanin p60, microtubule-severing enzymes from the ATPase associated with cellular activities (AAA+) family, has previously been shown in our lab to be inhibited by free tubulin as well as α- and β-tubulin carboxy-terminal tail (CTT) constructs. Here we investigate the inhibition ability of several different tubulin CTT sequences. We quantify the effect of the addition of these constructs on the severing and binding activity of katanin. We find that some constructs inhibit katanin better …

Investigating The Balance Between Estrogen Receptor Mediated Cell Proliferation And Genomic Surveillance, Margarita Brown

Masters Theses

Breast cancer is a leading cause of cancer in women and the second leading cause of cancer death. Lifetime exposure to estrogen contributes to this risk but high dose estrogen has been used to induce apoptosis as treatment for breast cancer. These opposing tumorigenic and anti-tumorigenic effects of estrogen may be regulated differently by the two Estrogen Receptors (ER), Estrogen Receptor alpha (ERα) and Estrogen Receptor beta (ERβ). Although the receptors share a 96% homology in their DNA binding domain, they are unique in the ligand-binding domain with 53% amino acid homology. Previous studies have shown that ERα drives cell …

Environmental Controls On The Diversity And Distribution Of Endosymbionts Associated With Phacoides Pectinatus (Bivalvia: Lucinidae) From Shallow Mangrove And Seagrass Sediments, St. Lucie County, Florida, Thomas Walters Doty

Masters Theses

Lucinid bivalves are capable of colonizing traditionally inhospitable shallow marine sediments due to metabolic functions of bacterial endosymbionts located within their gills. Because lucinids can often be the dominant sediment infauna, defining their roles in sediment and pore fluid geochemical cycling is necessary to address concerns related to changes in coastal biological diversity and to understanding the sensitivity of threatened coastal ecosystems over time. However, there has been limited research done to understand the diversity and distribution of many lucinid chemosymbiotic systems. Therefore, the goals of this thesis were to evaluate the distribution of Phacoides pectinatus and its endosymbiont communities …

Promoting Extracellular Matrix Crosslinking In Synthetic Hydrogels, Marcos M. Manganare

Masters Theses

The extracellular matrix (ECM) provides mechanical and biochemical support to tissues and cells. It is crucial for cell attachment, differentiation, and migration, as well as for ailment-associated processes such as angiogenesis, metastases and cancer development. An approach to study these phenomena is through emulation of the ECM by synthetic gels constructed of natural polymers, such as collagen and fibronectin, or simple but tunable materials such as poly(ethylene glycol) (PEG) crosslinked with short peptide sequences susceptible to digestion by metalloproteases and cell-binding domains. Our lab uses PEG gels to study cell behavior in three dimensions (3D). Although this system fosters cell …

Rnai Validation Of Resistance Genes And Their Interactions In The Highly Ddt-Resistant 91-R Strain Of Drosophila Melanogaster, Kyle Gellatly

Masters Theses

4,4’-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as …

The Evolution Of Thermotolerance A Characterization Of A Directionally Evolved Cyanobacterium, Nathen Emil Bopp

Masters Theses

Chaperone proteins are essential components in the maintenance and turnover of the proteome. Many chaperones play integral functions in the folding and unfolding of cellular substrates under many conditions, including heat stress. Most chaperones can be characterized into two categories; the typical ATP dependent chaperones and the ATP independent chaperones. One ATP independent chaperone class it the Small Heat Shock Proteins (sHSPs), which as molecular life vests and are thought to protect misfolding proteins from irreversible aggregation. One such organism, the cyanobacterium Synechocystis sp. PCC 6803, is an excellent model for the study and understanding of these proteins and their …

Manipulating Adipose Tissue Fatty Acid Oxidation To Reduce Fatness In Broiler Chickens, Tania Emmanuelle Torchon

Masters Theses

Compared to rodents, broiler chickens, those reared for meat, are an attractive model for studies of adipose biology, and obesity development in children. The broiler chicken lacks the gene for uncoupling protein 1, the hallmark for brown adipose tissue making them a useful model to study lipid metabolism in white adipocytes. Two studies were performed to investigate if white adipose tissue had the metabolic ability for fatty acid oxidation (FAO), and to investigate the effects of dietary fatty acids on abdominal fat development of young broiler chickens as a model for childhood obesity. In study one, chickens were fasted for …

Thermocycle-Regulated Wall Regulator Interacting Bhlh Encodes A Protein That Interacts With Secondary-Cell-Wall-Associated Transcription Factors, Ian P. Whitney

Masters Theses

Lignocellulosic biomass is one of the most abundant raw materials on earth that can be utilized to created carbon-neutral biofuels as a replacement for conventional fossil fuels. In order to create ideal energy crops, the regulation and deposition of cell wall polysaccharides must first be fully understood. Improved understanding of cell wall regulation will enable selection of traits that can optimize biofuel feedstocks. Herein, I utilize the grass model system Brachypodium distachyon in order to understand the transcriptional regulation of secondary cell wall deposition. Gene expression profiling was used to elucidate transcription factors that regulate secondary cell wall biosynthesis. Through …

Localization Of Chemoreceptors In Azospirillum Brasilense., Anastasia Aksenova

Masters Theses

In order to ensure their survival, bacteria must sense and adapt to a variety of environmental signals. Motile bacteria are able to orient their movement in a chemical gradient by chemotaxis. During chemotaxis, environmental signals are detected by chemotaxis receptors and are propagated via a signal transduction cascade to affect bacterial motility. In a model organism Escherichia coli, chemotaxis receptors, also called MCPs (for methyl-accepting chemotaxis proteins) sense changes in concentration gradients by making temporal comparisons about the chemical composition of their surroundings. Decreased attractant concentration or increased repellant concentration results in conformational changes in the MCPs that culminate …

A Step Towards Understanding Of The Molecular Basis Of Ligand Promiscuity In The Aminoglycoside Modifying Enzymes, Sherin R. Raval

Masters Theses

Aminoglycosides have proven very useful in the treatment of infections; lately their effectiveness has been greatly reduced due to increasing resistance. Among many known mechanisms of resistance to aminoglycosides, enzymatic modification is the most prevailing. More than 14 aminoglycoside -N3-acetyltransferases- a class of aminoglycoside modifying enzymes, are known today. This study focuses on a pair of acetyl transferases: The aminoglycoside-N3- acetyltransferase IIIb (AAC-IIIb) and the aminoglycoside-N3- acetyltransferase IIa (AAC-IIa). AAC-IIa and AAC-IIIb are very similar in their amino acid sequence and structure – yet they have a strong difference in their substrate selectivity, kinetic …

Expression And Purification Of Human Lysosomal Β-Galactosidase From Pichia Pastoris, Sarah E. Tarullo

Masters Theses

Lysosomal storage diseases are genetically inherited diseases caused by the dysfunction of lysosomal enzymes. In a normal cell, lysosomal enzymes cleave specific macromolecules as they are transported to the lysosome. However, in diseased cells, these lysosomal enzymes are either absent or malfunctioning, causing macromolecular substrates to accumulate, becoming toxic to the cell. Over fifty lysosomal storage diseases have been identified, collectively occurring in one out of 7,700 live births. We investigated the lysosomal enzyme β-galactosidase (β-gal). In order to study the biochemistry and enzymology of this protein a robust expression system was needed. The GLB1 gene has been inserted into …

Sequence Analysis Of Maize Yellow Stripe3 Candidate Genes, Dennis B. Depaolo

Masters Theses

The work presented here focuses on the molecular mechanism of phytosiderophore secretion in graminaceous plants. In maize, yellow stripe3 (ys3) is a mutant that is deficient in its ability to secrete iron-chelating compounds of the mugineic acid family known as phytosiderophores. Phytosiderophores are specific to grasses and are used for the acquisition of iron. Genetic linkage mapping of the ys3 locus lead to a region of interest on chromosome 3 defined by marker UMC1773. The sequence of eleven candidate genes (GRMZM2G390345, GRMZM2G390374, GRMZM2G342821, GRMZM5G800764, GRMZM2G502560, GRMZM5G849435, GRMZM2G105766, GRMZM5G876835, GRMZM2G036976, GRMZM2G502563, miR167g) revealed several small deletions …

Designing A Pore-Forming Toxin Cytolysin A (Clya) Specific To Target Cancer Cells, Alzira Rocheteau Avelino

Masters Theses

Cytolysin A (ClyA) is a member of a class of proteins called pore-forming toxins (PFTs). ClyA is secreted by Gram-negative bacteria, and it attacks a number of mammalian cells by inserting into and forming channels within the cell membrane (Oscarsson J et al., 1999). It has been suggested that ClyA binds to cholesterol (Oscarsson J et al., 1999) and thus can insert into the membranes of many different cell types of eukaryotic origin. In our studies we propose to engineer a ClyA protein that can only attack a small subset of cell types. We propose to engineer ClyA that can …

Functions Of Mitoneet: What The Protein-Protein Interactions Reveal, Fran Hirschfelder

Masters Theses

MitoNEET is a [2Fe-2S] cluster protein found on the outer mitochondrial membrane. First discovered as a binding site for pioglitazone, an anti-diabetic drug, it was the first protein described containing a CDGSH iron-sulfur domain (CISD). However, the function(s) of mitoNEET in both normal cellular physiology and the physiology in type-2 diabetes remains unclear. The proposed functions of mitoNEET, include cluster transfer, electron transport and oxidative respiration. This thesis describes the use of proteomic pull-down techniques to identify possible protein binding partners of mitoNEET from both mouse and human liver cell lysates. In order to accomplish this, the expression and purification …

Dephosphorylation Of Iqg1 By Cdc14 Temporally Regulates Actin Ring Formation, Daniel Patrick Miller

Masters Theses

"Cytokinesis is the final step in cell division when the cell separates the cytoplasm by contracting a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential scaffolding protein in budding yeast (S. cerevisiae) required for actin recruitment to, and contraction of, the actomyosin ring. Actin is recruited by the calponin homology domain (CHD) in anaphase after Iqg1 is localized to the bud neck. Consensus sites for the cyclin-dependent kinase (CDK) Cdc28 were identified flanking the CHD. This led us to the hypothesis that phosphorylation of Iqg1 by Cdc28 negatively regulates actin …

Demonstration Of A Targeted Proteome Characterization Approach For Examining Specific Metabolic Pathways In Complex Bacterial Systems, Adam Justin Martin

Masters Theses

Multiple Reaction Monitoring (MRM) is a powerful tandem mass spectrometry (MS/MS) tool frequently implemented in proteomic studies to provide targeted analysis of proteins and peptides. The selectivity that MRM delivers is so strong that it provides the quadrupole mass spectrometers (QQQ), on which it is commonly employed, with pertinence to proteomic studies that they would otherwise lack for their relatively low resolution. Additionally, this increased level of selectivity is sufficient enough to supplant complicated fractionation techniques, additional dimensions of chromatography, and 24 hour long MS/MS experiments in simplistic biological samples. But there is a deficiency of evidence to determine the …

Studies On Solo Working Mechanism In The Meiosis Of Drosophila Melanogaster, Qian Ma

Masters Theses

In eukaryotes, sister chromatids are closely aligned due to cohesion, a process essential for chromosome pairing and segregation during both mitosis and meiosis. A conserved cohesin complex in a ring structure is composed of four subunits, including each of these four members or their homologs, SMC1, SMC3, SCC1/RAD21/REC8, and SCC3/SA. Up to now, no REC8 homolog has been identified in the meiosis of Drosophila. SOLO is a meiotic protein required for accurate chromosome segregation, centromere cohesion, and cohesin complex localization in Drosophila meiosis. In addition, SOLO is required for synapsis and recombination in Drosophila female meiosis.

In this study, …

Characterization Of A Glycosyphosphatidylinositol Anchor Transamidase In Arabidopsis Thaliana And The Function Of Gpi Anchored Proteins In Stomatal Development, Mark Gerald Ronald Bundy

Masters Theses

In plants stomata play a vital role for survival by allowing the gas exchange of CO₂ [carbon dioxide] and water vapor to occur. A stoma is a central pore flanked by two kidney shaped guard cells and in wild type there is at least one pavement cell between each stoma. The ERECTA (ER) gene family consisting of ER, ERL1, ERL2 is involved in regulation of stomata development, where a triple mutant of er erl1 erl2 displays an increased stomata index and clusters of stomata that disobey the one cell spacing rule. To better understand the pathway of stomata development, …

Bioconfinement Of A Putatively Sterile Nicotiana Hybrid And Development Of Tools For Assessing Gene Flow, John Hollis Rice

Masters Theses

Production of transgenic crops in open field environments is an ongoing concern of due to the potential for gene flow. New transgenic crops, such as plant-made-pharmaceuticals may generate additional concerns about effects of adventitious transgenes. Use of a bioconfinement strategy may alleviate any consequences by preventing gene flow. The following chapters discuss previous and current research on gene flow, testing of a Nicotiana hybrid system for bioconfinement efficiency, and development of methods for transgene detection. The candidate ‘platform plant’ that was tested is a Nicotiana hybrid (Nicotiana tabacum ‘TN 90’ × Nicotiana glauca) previously identified to be sexually …

Single-Nucleotide Polymorphisms Associated With Performance Traits In Beef Cattle Grazing Endophyte-Infected Tall Fescue, Bryan Christopher Bastin

Masters Theses

Tall fescue (Lolium arundinaceum Schreb.) is the most prevalent forage in the Midsouth United States due in part to the presence of the endophytic fungus Neotyphodium coenophialum. The fungus, while conferring hardiness to tall fescue, contributes to decreased production efficiency in cow-calf operations. A previous genome-wide association study was performed using the Illumina 50k bovine SNP chip. Twenty-four SNP were found to be associated (P < 0.05) with adjusted birth weight and adjusted 205-day weights of calves from 48 beef cows at Ames Plantation. The first objective was to validate each SNP by testing associations with several additional phenotypes. Custom Taqman genotyping assays (Applied Biosystems, Foster City, CA) were subsequently designed to genotype each SNP in beef cattle located at Tennessee Tech University (n = 654), to validate associations in a large, independent herd. The results yielded 15 associations that were significant (P < 0.05) with 6 phenotypes linked to those affected by fescue toxicosis. The second objective investigated the link between fescue toxicosis and the XK, Kell blood group complex subunit-related, member 4 …

Molecular Mechanisms Involved In Anhydrobiosis Of Insect Cells, Diyagama Arachchi Ralalage Dilini Sewwandi Samarajeewa

Masters Theses

Animals possessing tolerance to extreme water stress are termed anhydrobiotes. Many desiccation tolerant organisms respond to water stress by intracellular accumulation of selected sugars such as trehalose and larger macromolecules such as Late Embryogenesis Abundant (LEA) proteins and thereby maintain the cell viability. Evidence indicates that the presence of trehalose and Late Embryogenesis Abundant (LEA) proteins may work synergistically to confer cellular protection during drying in eukaryotic cells. We evaluated any increase in cellular desiccation tolerance by expressing different LEA proteins in a non-desiccation tolerant insect cell line Drosophila melanogaster (fruit fly) (Kc167 cells) in the presence of trehalose.

Transgenic …

Designing Protein Expression And Purification Systems For Recombinant Z Alpha1-Antitrypsin Using The Methylotrophic Yeast, Pichia Pastoris, Monique Jenaie Lemieux

Masters Theses

It is well established that improper protein folding often leads to the formation of aggregates whose consequences are cellular impairment and cell death. One example of this is the aggregation of the mutant Z Alpha1-Antitrypsin protein, which results in blocking of its secretion due to inclusion body formation. This can contribute not only to the development of chronic obstructive pulmonary disease but also to hepatitis, cirrhosis and hepatocellular carcinoma. Current treatments are principally limited to intravenous Alpha1-Antitrypsin therapy and organ transplantation. In the scientific community though, it is widely thought that more effective forms of treatments lie within the polymerization …

Angiotensinogen Gene Silencing Reduces Lipid Accumulation And Inflammation In Cultured 3t3-L1 Adipocytes, Wenting Xin Carroll

Masters Theses

Obesity is characterized by metabolic complications which are related to several life-threatening diseases. Dysregulated inflammatory adipokines secretion from adipose tissue is believed as the major contributor to obesity-associated local and systemic inflammation, insulin resistance, and other metabolic dysfunctions.

Numerous studies in our lab and others pointed to the role of local adipose tissue renin-angiotensin system (RAS) in the pathogenesis of obesity, inflammation and insulin resistance. We hypothesized that adipocytes-derived angiotensinogen (Agt) played a critical role in adipogenesis and/or lipogenesis as well as adipose inflammation. To test, we established 3T3-L1 preadipocytes stably transfected with Agt-shRNA or scrambled sequence (Sc-shRNA). Transfected preadipocytes …

Genetic Basis Of Flocculation In Azospirillum Brasilense., Priyanka Satish Mishra

Masters Theses

Azospirillum brasilense is a class of rhizobacteria capable of nitrogen fixation, root colonization and hence promoting host plant growth. The bacteria posses cell interaction behaviors like clumping and flocculation that contribute the survival of the organism in nutrient limited conditions. Change in the cell surface adhesive properties allows the cells to progress from free swimming to clumping and finally flocculation. Less is known about the genetic regulation of these processes with flcA being the only transcriptional regulator known so far to directly control flocculation. Recent evidence suggesting that Che1, a chemotaxis like signal transduction pathway controls the cell behavior clumping …

Elucidating The Effect Of Silver On Ethylene Signaling In Arabidopsis Thaliana, Brittany Kathleen Mcdaniel

Masters Theses

Ethylene, a gaseous plant hormone, is involved in numerous plant developmental processes such as seed germination, senescence, and fruit ripening. In Arabidopsis thaliana, ethylene is perceived by a family of five membrane-bound receptors, which upon binding ethylene trigger downstream effects. At the receptor level, it is known that the coordination of a copper ion is necessary for ethylene to bind, resulting in a conformational change of the receptor and the initiation of the ethylene signal transduction pathway. Interestingly, silver ions are also able to support binding of ethylene but ethylene responses are blocked in the presence of silver. When …

Examining The Roles Of Pstoc75 Potra Domains In Chloroplast Protein Import, Richard Franklin Simmerman

Masters Theses

During chloroplast formation via endosymbiosis most of the plastid genome was transferred to the host nuclear genome. Genomic and proteomic analysis suggests that >95% of the original plastid proteome is now encoded in the nucleus, and these now cytosolically fabricated proteins require a post-translational transport pathway back into the organelle. This process is not well understood, yet it has been shown to involve translocons at the outer and inner envelope of the chloroplast membranes (TOC & TIC). These translocons interact with a cleavable N-terminal extension of between 20 and 100 residues on chloroplast-bound precursor proteins known as the transit-peptide. Precursor …

The Effects Of Bisphenol A On Adipose Tissue Development, Metabolism, And Endocrine Function And The Role It May Play In The Development Of Obesity, Brantley Nelson Wyatt

Masters Theses

While diet and sedentary lifestyle remain important factors in the development of obesity, recent findings have shown the possible involvement of environmental obesogens, chemicals that can disrupt homeostatic energy balance and increase adiposity. Bisphenol A (BPA) is a compound used in the manufacturing of plastics as a hardening agent and is ubiquitous in the environment due to its widespread use. BPA has been shown to be an endocrine disruptor through its ability to mimic estrogen, which is now known to play important roles in adipose tissue growth and metabolism. In fact, a small but compelling number of studies have shown …