Biochemistry Commons^™

The Staphylococcus Pseudintermedius Adhesin Spsd Contains A Central Fibronectin-Binding Domain, Andrea S. Bordt

Dissertations & Theses (Open Access)

Staphylococcus pseudintermedius is a Gram-positive bacterium significant because of its ability to cause costly and difficult to treat veterinary infections worldwide. It exhibits several similarities to Staphylococcus aureus, however, very little is known about its surface adhesins. Surface adhesins in S. aureus are significant contributors to pathogenesis. S. pseudintermedius encodes the surface protein SpsD, which contains characteristics of the microbial surface components recognizing adhesive matrix molecules family and confers attachment of the heterologous host Lactococcus lactis to fibronectin. This work has identified a centrally-located fibronectin binding domain in SpsD which binds the 30 kDa N-terminal domain of fibronectin with …

Iron-Regulated Cyanobacterial Predominance And Siderophore Production In Oligotrophic Freshwater Lakes, Ryan J. Sorichetti

Electronic Thesis and Dissertation Repository

The frequency and intensity of cyanobacterial blooms (cyanoblooms) is increasing globally. Contrary to existing phosphorus (P) and nitrogen (N) paradigms describing cyanobloom proliferation in eutrophic (nutrient-rich) freshwater lakes, many of the recent cyanobloom reports pertain to oligotrophic (nutrient-poor) freshwater lakes with no prior history of cyanobloom occurrence. There exists a critical research need to re-visit existing conceptual models, identify regulating factors currently unaccounted for and improve our ability to effectively detect and measure cyanobacterial toxins (cyanotoxins) in lakes. Iron (Fe) is required in nearly all pathways of cyanobacterial macronutrient use, though its direct role in regulating cyanobacterial biomass is not …

Characterization Of Caxck31, A Bacterial Calcium/Proton Antiporter, Marc Robert Ridilla

Open Access Dissertations

To better understand a class of transporters known as Calcium/Cation Antiporters (CaCAs), the bacterial calcium/proton antiporter CAXCK31 was purified and characterized. New methods were developed for its heterologous overexpression and purification. These methods help to define stress responses to toxic membrane overproduction in E. coli and may be broadly applicable to studies of membrane proteins. The results from a variety of biochemical and biophysical experiments demonstrated that CAXCK31 exists as a dimer in the membrane and can be purified in the dimeric state. The methods used include chemical cross-linking, FRET, and SEC-MALS. In addition, various transport properties of CAXCK31, including …

The Role Of Metallothionein In Zinc Homeostasis, Kelly L. Summers

Electronic Thesis and Dissertation Repository

The structure of the unique metal-binding protein, metallothionein (MT), consists of two metal-thiolate-clustered binding domains; the β-domain binds up to three divalent metals and the α-domain binds four. The mechanisms through which the metals are bound and arranged into domains, as well as the function of MT in metal ion homeostasis, remains largely unknown. By utilizing electrospray ionization mass spectrometry (ESI MS) to identify each species, and by comparing the data with simulations, MT 1a was found to bind Zn²⁺ non-cooperatively. Through a competition experiment between MT and its individual domain peptides, MT was proposed to bind Zn^{2+ …}

Identification Of Cyclophilin Gene Family In Soybean And Characterization Of Gmcyp1, Hemanta Raj Mainali

Electronic Thesis and Dissertation Repository

I identified members of the Cyclophilin (CYP) gene family in soybean (Glycine max) and characterized the GmCYP1, one of the members of soybean CYP. CYPs belong to the immunophilin superfamily with peptidyl-prolyl cis-trans isomerase (PPIase) activity. PPIase catalyzes the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptides. After extensive data mining, I identified 62 different CYP genes in soybean (GmCYP1 to GmCYP62), of which 8 are multi-domain proteins and 54 are single domain proteins. At least 25% of the GmCYP genes are expressed in soybean. GmCYP1 …

Regulation Of The Target Of Rapamycin Signaling Pathway In Saccharomyces Cerevisiae, Tammy Pracheil

University of New Orleans Theses and Dissertations

An integrative, biochemical, genetic, and molecular biology approach utilizing gene manipulation, gene knock outs, plasmid based protein expression, and in vivo protein localization of fluorescence tagged proteins was employed to determine the function of an essential protein, Lst8, in TORC1 and TORC2 signaling and a previously uncharacterized complex, the Far3-7-8-9-10-11 complex (Far complex) in the budding yeast, Saccharomyces cerevisiae. Mutations in SAC7 and FAR11 suppressed lethality of both lst8 and tor2-21 mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2.

Far11, a component of a six-member complex, was found to interact …

Validation Of Antibodies Used To Study Hypoxia Inducible Factors In Two Species Of Fundulus, Jenna D. Hill

University of New Orleans Theses and Dissertations

Hypoxia inducible factors (HIFs) are transcription factors and the master regulators of oxygen-dependent gene expression in animals. The focus of this thesis is the distribution of HIF protein in tissues of the fish Fundulus heteroclitus and F. grandis, two widespread species that occur in naturally hypoxic waters. Polyclonal antibodies against HIF-1α, HIF-2α, and HIF-3α were tested on proteins made in vitro and on extracts made from several tissues of normoxic and hypoxic fish. Antibodies against HIF-1α and 3α bound specifically to full length protein made in vitro, and produced bands on western blots of nuclear extracts of near …

Development And Application Of Mass Spectrometry-Based Proteomics To Generate And Navigate The Proteomes Of The Genus Populus, Paul Edward Abraham

Doctoral Dissertations

Historically, there has been tremendous synergy between biology and analytical technology, such that one drives the development of the other. Over the past two decades, their interrelatedness has catalyzed entirely new experimental approaches and unlocked new types of biological questions, as exemplified by the advancements of the field of mass spectrometry (MS)-based proteomics. MS-based proteomics, which provides a more complete measurement of all the proteins in a cell, has revolutionized a variety of scientific fields, ranging from characterizing proteins expressed by a microorganism to tracking cancer-related biomarkers. Though MS technology has advanced significantly, the analysis of complicated proteomes, such as …

The Use Of P53-Derived Stapled Peptides As Affinity Isolation Reagents, Kathryn Margaret Headley

Honors Capstone Projects - All

The transcription factor p53 can induce apoptosis and cell cycle arrest in response to cellular distress. Cancer cells often display increased cell survival. In most cases, this is due to a p53-related defect, such as mutation, deletion, degradation, or sequestration. HDM2 and HDMX are homologous proteins that regulate the function of p53, and their over-expression can lead to an ineffective p53 response. Various inhibitors, including hydrocarbon stabilized alpha-helices of p53 (SAH-p53s), have been developed to target HDM2 and HDMX and restore functionality to the p53 pathway. It has been recently found that SAH-p53 factors also elicit cell death responses in …

Characterization Of Dap2 (Defense-­‐Associated Aspartic Protease-­‐2) In Arabidopsis Thaliana And Determining Its Role In Plant Defense Responses, Kemardo Kitaro Henry

Honors Capstone Projects - All

Plants defend themselves from invading pathogens by coordinating activity of several plant defense-­‐associated genes. Defense-­‐Associated Aspartic Protease-­‐1 (DAP1) gene of Arabidopsis encodes for an atypical aspartic protease with a putative proteolytic activity. The DAP1 is a negative regulator of plant immunity and the transgenic plants ectopically expressing this genes support pathogen growth and pathogenicity. The Arabidopsis genome has another putative protese that has ~75% homology to DAP1 present in close proximity and therefore was named DAP2. Here, I describe the role of the DAP2 gene in plant defense responses. Two independent knockout dap2 mutants are found to be more susceptible …

Vitamin B12-Based Bioconjugate Probes For In Vitro And In Vivo Imaging, Anna Kahkoska

Honors Capstone Projects - All

Cancer is the second leading cause of death in the United States. This year, an estimated 577,190 Americans will die as a result of this family of diseases. Finding cancer at its most treatable stage gives patients the greatest chance of recovery; novel imaging agents that target primary and metastasized tumors offer hope for improved prognoses in the future. Based on the hypothesis that vitamin B12 (B12) and its association with specific transport proteins could offer selective access to cancer cell lines, a series of B12-based imaging agents were synthesized, characterized, and assayed for both in vitro and in vivo …

Novel Architecture Of Costal Cartilage And Implications In Chest Wall Deformities, Anthony J. Asmar

Biological Sciences Theses & Dissertations

Costal cartilage is a type of hyaline cartilage that forms rod-like structures that connect the ribs to the sternum. Deformation of costal cartilage is observed in the chest wall deformities, pectus excavatum and pectus carinatum. Pectus excavatum involves a sternal displacement causing a depression of the chest while pectus carinatum causes a protrusion of the chest. As costal cartilage is not a widely studied tissue, this leaves little knowledge into possible factors involved in the pathogenesis of pectus deformities. Costal cartilage in these deformities has been described as being weakened and may implicate proteoglycans which play an important role in …

Characterizing Cyclin J By Identifying Conserved Protein-Protein Interactions, Phillip Jacob Selman

Wayne State University Theses

Cyclins are proteins that bind to Cyclin-dependent kinases, or Cdks, through a conserved domain called the Cyclin Box. Many Cyclins regulate the cell cycle. A few Cyclins impact cellular processes outside of the cell cycle. Also, a few Cyclins have poorly understood functions.

Cyclin J is a member of the Cyclin superfamily of proteins. Cyclin J is conserved among all metazoans, but is presently not well understood. All the research done on Cyclin J has been done in Drosophila.

Its mRNA is present in the early embryo, then disappears, only to reappear in adult females. When probing protein extracts with …

Interaction Between Maltose Binding Protein And Escherichia Coli Maltose Transporter, Yan Huang

Open Access Theses

The ATP-binding cassette (ABC) transporter superfamily is one of the largest families of transport proteins. The ABC transporters are responsible for selective permeability of solute across membranes energized by ATP hydrolysis, which occurs in all domains of life. Maltose transporter is an ABC importer that mediates maltose/maltodextrin uptake in bacteria and archaea. It is identified as an essential virulence factor in pathogenic species of Streptococcis pyogenes and Vibrio cholera (1, 2). Escherichia coli maltose transporter is a well-characterized system with crystal structures and exclusive biochemical studies available. Knowledge of the E. coli maltose transport mechanism will lead to a better …

Plant-Pathogen Interactions Associated With Wasting Disease In The Tropical Seagrass Thalassia Testudinum., Kyle Loucks

UNF Graduate Theses and Dissertations

Lipopolysaccharide (LPS) was shown to serve as a strong elicitor of the early defense response in the subtropical seagrass Thalassia testudinum Banks ex König and was capable of inducing an oxidative burst identified at the single cell level. The formation of reactive oxygen species (ROS) included a diphenylene iodonium sensitive response, suggesting the involvement of an NADPH oxidase. A 900 bp fragment of this enzyme was sequenced and found to encode a NAD binding pocket domain with extensive homology to the Arabidopsis thaliana rbohF (respiratory burst oxidase homolog) gene. Pharmacological dissection of the early events preceding ROS emission revealed that …

Towards Elucidation Of A Viral Dna Packaging Motor, Chad T. Schwartz

Theses and Dissertations--Pharmacy

Previously, gp16, the ATPase protein of phi29 DNA packaging motor, was an enigma due to its tendency to form multiple oligomeric states. Recently we employed new methodologies to decipher both its stoichiometry and also the mechanism in which the protein functions to hydrolyze ATP and provide the driving force for DNA packaging. The oligomeric states were determined by biochemical and biophysical approaches. Contrary to many reported intriguing models of viral DNA packaging, it was found that phi29 DNA packaging motor permits the translocation of DNA unidirectionally and driven cooperatively by three rings of defined shape. The mechanism for the generation …

Y-Family Dna Polymerases : Mechanism Of Single-Base Deletion And Effect Of Pcna Interaction, Yifeng Wu

Legacy Theses & Dissertations (2009 - 2024)

Translesional synthesis (TLS) by specialized Y-family DNA polymerases is a DNA damage tolerance pathway to bypass DNA lesions that have not been repaired by other DNA repair mechanisms. Despite their valuable lesion bypass ability, the Y-family DNA polymerases display a much lower fidelity upon replicating undamaged DNA. Their activity therefore needs to be well regulated in the cell. In our first project, we examined how an archaeal Y-family DNA polymerase, Dpo4, makes single-base deletions during replication. Dpo4 belongs to the DinB subfamily which is known to make single-base deletions. Although previous studies suggested that it uses a dNTP-stabilized misalignment mechanism, …