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Full-Text Articles in Molecular Biology

Investigating The Roles Of Fucosylation And Calcium Signaling In Melanoma Invasion, Tyler S. Keeley Nov 2018

Investigating The Roles Of Fucosylation And Calcium Signaling In Melanoma Invasion, Tyler S. Keeley

Graduate Theses and Dissertations

Melanoma is the deadliest form of skin cancer. Prognosis for early stage melanoma patients is excellent, and surgery is often curative for these patients. However, once patients have presented with invasive disease, the average 5-year survival rate drops significantly from over 90% to between 10 and 15%. Several therapies have been developed to target a commonly mutated oncogene BRAF, or its downstream effectors. Unfortunately, while these treatments show robust initial response, most patients relapse within a year. Moreover, therapy-resistant tumors are often more invasive and metastatic. Therefore, it is important to investigate the molecular mechanisms underlying melanoma invasion and metastasis ...


Mechanism Elucidation And Inhibitor Discovery Against Serine And Metallo-Beta-Lactamases Involved In Bacterial Antibiotic Resistance, Orville A. Pemberton Nov 2017

Mechanism Elucidation And Inhibitor Discovery Against Serine And Metallo-Beta-Lactamases Involved In Bacterial Antibiotic Resistance, Orville A. Pemberton

Graduate Theses and Dissertations

The emergence and proliferation of Gram-negative bacteria expressing β-lactamases is a significant threat to human health. β-Lactamases are enzymes that degrade the β-lactam antibiotics (e.g., penicillins, cephalosporins, monobactams, and carbapenems) that we use to treat a diverse range of bacterial infections. Specifically, β-lactamases catalyze a hydrolysis reaction where the β-lactam ring common to all β-lactam antibiotics and responsible for their antibacterial activity, is opened, leaving an inactive drug. There are two groups of β-lactamases: serine enzymes that use an active site serine residue for β-lactam hydrolysis and metalloenzymes that use either one or two zinc ions for catalysis. Serine ...


A Structural Biology Approach To The Problem Of Antibiotic Resistance In Bacteria, Jared Armand Delmar Jan 2017

A Structural Biology Approach To The Problem Of Antibiotic Resistance In Bacteria, Jared Armand Delmar

Graduate Theses and Dissertations

X-ray crystallography remains the most robust method to determine protein structure at the atomic level. We demonstrate how these structural studies can directly contribute to unsolved problems in biology, with a focus on the growing problem of antibiotic resistance in bacterial infections. Multi-drug efflux transporters are common and powerful resistance mechanisms that are capable of extruding a number of structurally unrelated antimicrobials, including antibiotics and toxic heavy metal ions, from the bacterial cell. We begin by presenting the crystal structures of the individual pump components of the Escherichia coli Cus system, a paradigm for efflux machinery, and speculate on how ...


An Enzymology And Structural Biology Approach Towards Elucidation Of E. Coli Fatty Acid Biosynthesis And Its Application To The Production Of Bio-Renewables, Aaron Michael Marcella Jan 2017

An Enzymology And Structural Biology Approach Towards Elucidation Of E. Coli Fatty Acid Biosynthesis And Its Application To The Production Of Bio-Renewables, Aaron Michael Marcella

Graduate Theses and Dissertations

Prokaryotic fatty acid biosynthesis is an important process to study due to its necessity for cell survival and production of highly reduced carbon chains. A characterization of several proteins involved in E. coli fatty acid biosynthesis is presented. This work provides a method for generating large amounts of pure malonyl acyl-carrier-protein using both the wild type malonyl-CoA:holo-ACP transacylase (FabD) and a variant studied further in our lab. This preparation overcomes the phenomena of a 50:50 mixture of malonyl and acetyl-ACP in the reaction and is crucial for the efficacy of future assays involving malonyl-ACP. A new assay has ...


The Effects Of Phytohormones And Isoprenoids In Dihydroartemisinin-Induced Dormancy In The Erythrocytic Stages Of Plasmodium Falciparum, Marvin Duvalsaint Duvalsaint Nov 2016

The Effects Of Phytohormones And Isoprenoids In Dihydroartemisinin-Induced Dormancy In The Erythrocytic Stages Of Plasmodium Falciparum, Marvin Duvalsaint Duvalsaint

Graduate Theses and Dissertations

Our ability to control malaria has been challenged by increasing antimalarial resistance. Plasmodium falciparum undergoes dormancy in the blood stages which is hypothesized to be a means by which they are able to survive under drug pressure. This helps select for resistant parasites which grow following removal of drug. The mechanisms behind dormancy and the subsequent recrudescence are not fully understood but translating knowledge from related organisms which undergo a similar phenomenon might shed some light. Higher plants utilize dormancy during the early development stages to survive under unfavorable conditions, increasing fitness of the seedling and ensuring viability when this ...


Significance Of Pten Phosphorylation And Its Nuclear Function In Lung Cancer, Prerna Malaney Nov 2016

Significance Of Pten Phosphorylation And Its Nuclear Function In Lung Cancer, Prerna Malaney

Graduate Theses and Dissertations

Phosphorylation mediated inactivation of PTEN leads to multiple malignancies with increased severity. However, the consequence of such inactivation on downstream functions of PTEN are poorly understood. Therefore, the objective of my thesis is to ascertain the molecular mechanisms by which PTEN phosphorylation drives lung cancer. PTEN phosphorylation at the C-terminal serine/threonine cluster abrogates its tumor suppressor function. Despite the critical role of the PTEN C-tail in regulating its function, the crystal structure of the C-tail remains unknown. Using bioinformatics and structural analysis, I determined that the PTEN C-tail is an intrinsically disordered region and is a hot spot for ...


Investigation Of Respiratory Syncytial Virus Structural Determinants And Exploitation Of The Host Ubiquitin System, Jillian Nicole Whelan Apr 2016

Investigation Of Respiratory Syncytial Virus Structural Determinants And Exploitation Of The Host Ubiquitin System, Jillian Nicole Whelan

Graduate Theses and Dissertations

Respiratory syncytial virus (RSV) is a globally circulating, non-segmented, negative sense (NNS) RNA virus that causes severe lower respiratory infections. This study explored several avenues to ultimately expand upon our understanding of RSV pathogenesis at the protein level. Evaluation of RSV intrinsic protein disorder increased the relatively limited description of the RSV structure-function relationship. Global proteomics analysis provided direction for further hypothesis-driven investigation of host pathways altered by RSV infection, specifically the interaction between the RSV NS2 protein and the host ubiquitin system. NS2 primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. The goal ...


Strep-Tag Ii Fusion Technology For The Immobilization Of Lipase B From Candida Antarctica, Sumreet Singh Johar Jan 2016

Strep-Tag Ii Fusion Technology For The Immobilization Of Lipase B From Candida Antarctica, Sumreet Singh Johar

Graduate Theses and Dissertations

Fusion tags are genetically coded protein or peptides that can be expressed as attached moieties to the desired enzyme to improve downstream purification of the target protein. Fusion tags have the potential to provide specific immobilization with repeatable attachment of the enzyme, eliminate the need for purification and post-modification and enable dissociation of spent enzyme and regeneration of the support. Lipase is an industry-relevant enzyme utilized for the production of natural flavors, biodegradable polyesters for food packaging, structured lipids, antioxidant esters, sugar esters and several other important compounds used in food and agriculture industries. In this research work, we are ...


Introducing Multiple Sites Of Acetylation To Histone H3 Via Nonsense Suppression, Isaac Young Jan 2016

Introducing Multiple Sites Of Acetylation To Histone H3 Via Nonsense Suppression, Isaac Young

Graduate Theses and Dissertations

A common post-translational modification (PTM) of proteins is lysine acetylation. This is an especially ubiquitous PTM in the histones of chromatin, and is important for helping to regulate both structural and mechanistic aspects of chromatin. The fundamental unit of chromatin is called the nucleosome and is made up of DNA that wraps around a histone protein octamer. Protruding from the nucleosome are 10 unstructured “tails” which protrude into the aqueous environment. A number of strategies exist for generating acetylated nucleosomes for the in-vitro study of chromatin including: Purification from eukaryotic organisms, chemical acetylation, amino acid analog incorporation, enzyme mediated acetylation ...


Investigation Of Snare Mediated Membrane Fusion And Its Regulation By Optimized Single Molecule Method, Yicheng Zhu Jan 2016

Investigation Of Snare Mediated Membrane Fusion And Its Regulation By Optimized Single Molecule Method, Yicheng Zhu

Graduate Theses and Dissertations

Neurotransmitter release going through synaptic vesicle cycle is one key step how signal is transported in our brains. The mechanism on molecular level has been under development and debated for decades. Many milestones have been made including, the identification of SNARE as core assembly machinery, the clarification of synaptotagmin as calcium sensor, the recognition of NSF and SNAP as disassembly apparatus, the determination of complexin and SM protein as regulatory protein. However, the sequence of their involvement in synaptic vesicle cycle, the relationship between the structure and psychological function, microscale fusion mechanism and are under further investigation. This puzzle is ...


Role Of Non-Catalytic Ligands In Macromolecule Function, Muneaki Watanabe Jan 2016

Role Of Non-Catalytic Ligands In Macromolecule Function, Muneaki Watanabe

Graduate Theses and Dissertations

Human hexokinase Type-I (HKI) binds to the outer mitochondrial membrane, and in so doing protects the mitochondrion, controls adenine nucleotide flux through the membrane and blocks mitochondrion-linked apoptosis. Adenosine 5’-triphosphate (ATP) releases HKI from the outer mitochondrial membrane, but the mechanism of ATP release is unclear. ATP-release is not due to the generation of glucose 6-phosphate (G6P) from ATP and glucose or by the binding of ATP to HKI at its catalytic or regulatory domains. Instead, the voltage dependent anion channel (VDAC) has a high affinity binding site for ATP as revealed by titrations using ATP and fluorescent analogs ...


Exploring The Genetic Redundancy Of Zea Mays Fatty Acid Elongase (Fae), Kayla S. Flyckt Jan 2016

Exploring The Genetic Redundancy Of Zea Mays Fatty Acid Elongase (Fae), Kayla S. Flyckt

Graduate Theses and Dissertations

Very-long-chain fatty acids (VLCFAs) consist of greater than 18 carbon atoms and have diverse biological functions including energy storage, protection from biotic and abiotic stresses, signaling, and are components of the plasma membrane. The biosynthesis of VLCFAs occurs by the iterative extension of fatty acyl-CoAs through a series of reactions catalyzed by four enzyme components known as fatty acid elongase (FAE). The FAE enzyme 3-ketoacyl CoA synthase (KCS) is hypothesized to be responsible for the substrate specificity of FAE, controlling the amount and identity of VLCFAs produced in specific cell types and tissues. The KCS gene family consists of multiple ...


Targeting Autopalmitoylation To Modulate Protein S-Palmitoylation, Laura Dawn Hamel Nov 2015

Targeting Autopalmitoylation To Modulate Protein S-Palmitoylation, Laura Dawn Hamel

Graduate Theses and Dissertations

Palmitoylation refers to the covalent attachment of fatty acids, such as palmitate, onto the cysteine residues of proteins. This process may subsequently alter their localization and function. Nearly all of the enzymes that catalyze palmitoylation, zDHHC protein acyl transferases (PATs), are implicated in neurological disorders, infectious diseases, and cancer in humans. Of particular interest to those who study palmitoylation are Ras family GTPas and zDHHC9-GCP16, the zDHHC PAT that palmitoylates Ras proteins. Erf2-Erf4 is the zDHHC PAT that palmitoylates Ras proteins in Saccharomyces cerevisiae. Currently, there are no methods to therapeutically target palmitoylation for the treatment of disease. One of ...


Novel Enzyme Perspectives: Arylalkylamine N-Acyltransferases From Bombyx Mori & 1-Deoxy- D-Xylulose-5-Phosphate Synthase From Plasmodium Falciparum And Plasmodium Vivax, Matthew R. Battistini Nov 2015

Novel Enzyme Perspectives: Arylalkylamine N-Acyltransferases From Bombyx Mori & 1-Deoxy- D-Xylulose-5-Phosphate Synthase From Plasmodium Falciparum And Plasmodium Vivax, Matthew R. Battistini

Graduate Theses and Dissertations

This dissertation is dedicated to the research and investigation of novel enzymes and the methods used to study them, with physiological roles ranging from isoprenoid biosynthesis to neurotransmitter production. Using a combination of bioinformatics, recombinant cloning, enzymology, and proteomics, we have contributed to the understanding and exploration of several human illnesses, including malaria, cancer, and endocrine dysfunction.

Our first project involved studying the enzymes responsible for N-acylarylalkylamide biosynthesis in Bombyx mori. Very little is known how these potent signaling molecules are produced in vivo, however, one possible pathway is the direct conjugation of an acyl-CoA to a corresponding arylalkylamide ...


Structure Based Drug Design Targeting Bacterial Antibiotic Resistance And Alzheimer's Disease, Eric Michael Lewandowski Oct 2015

Structure Based Drug Design Targeting Bacterial Antibiotic Resistance And Alzheimer's Disease, Eric Michael Lewandowski

Graduate Theses and Dissertations

Structure based drug design is a rapidly advancing discipline that examines how protein targets structurally interact with small molecules, or known inhibitors, and then uses this information to lead inhibitor optimization efforts. In the case of novel inhibitors, protein structural information is first obtained via X-ray crystallography, NMR studies, or a combination of both approaches. Then, computational molecular docking is often used to screen, in silico, millions of small molecules and calculate the potential interactions they may have with the target protein’s binding pocket, in hopes of identifying novel low affinity inhibitors. By examining the interactions these small, low ...


Regulation And Targeting Of The Fancd2 Activation In Dna Repair, Valentina Celeste Caceres Jan 2015

Regulation And Targeting Of The Fancd2 Activation In Dna Repair, Valentina Celeste Caceres

Graduate Theses and Dissertations

Fanconi anemia (FA) is a genome instability syndrome that is clinically manifested by bone marrow failure, congenital defects, and elevated cancer susceptibility. The FA pathway is known to regulate the repair of DNA interstrand crosslinks in part through DNA homologous recombination (HR) repair. Up to today 16 FA proteins have been discovered that may participate in the common pathway. Cells that have mutations in the FA genes are hypersensitive to DNA damaging agents and display chromosome instability. A key regulatory event in the FA pathway is monoubiquitination of FANCD2-FANCI heterodimer that is mediated by a multi-component E3 ubiquitin ligase complex ...


Understanding The Bases For Catalysis In Class Ii Diterpene Cyclases Results In Novel Product Chemistries, Kevin Charles Potter Jan 2015

Understanding The Bases For Catalysis In Class Ii Diterpene Cyclases Results In Novel Product Chemistries, Kevin Charles Potter

Graduate Theses and Dissertations

Labdane-related diterpenoids are a large group of natural products (over 7,000 known) with applications in many industries. The cyclization of the general diterpene precursor, (E,E,E)-geranylgeranyl diphosphate, into various bicyclic diterpene precursors catalyzed by class II diterpene cyclases characterizes labdane-related diterpenoid biosynthesis. The catalytic acid in the protonation-initiated cyclization reaction has been previously identified, however the catalytic base was not known. Using ent-copalyl diphosphate synthase from Arabidopsis thaliana, involved in gibberellin phytohormone biosynthesis, site-directed mutagenesis studies have uncovered the catalytic base responsible for deprotonation, which consists of a water molecule ligated, in part, by the side-chains from ...


Eukaryotic Gene Expression Patterns Of Microorganisms In The Amazon River Plume Parallel The Biogeochemistry Of Plume Waters, Brian L. Zielinski Sep 2014

Eukaryotic Gene Expression Patterns Of Microorganisms In The Amazon River Plume Parallel The Biogeochemistry Of Plume Waters, Brian L. Zielinski

Graduate Theses and Dissertations

Unraveling the microbiological processes that occur as water travels from a river's mouth into the ocean is critical to understanding the role of river plumes in global biogeochemical cycles. Metranscriptomics, the gene expression of a whole community of organisms, was utilized to examine six stations along the Amazon River Plume (ARP) in 2010 to test thehypothesis that there were measurable differences in gene expression for key biogeochemical genes along the ARP. This body of work focuses on methods developed to identify which genes are biogeochemically important for a particular environment along extreme salinity, nutrient and community gradients in the ...


Exploration Of Mutations In Erythroid 5-Aminolevulinate Synthase That Lead To Increased Porphyrin Synthesis, Erica Jean Fratz May 2014

Exploration Of Mutations In Erythroid 5-Aminolevulinate Synthase That Lead To Increased Porphyrin Synthesis, Erica Jean Fratz

Graduate Theses and Dissertations

5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the first committed step of heme biosynthesis in animals, the condensation of glycine and succinyl-CoA yielding 5-aminolevuliante (ALA), CoA, and CO2. Murine erythroid-specific ALAS (mALAS2) variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. Transfection of HeLa cells with expression plasmids for mALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatment of HeLa ...


Structure-Based Design Of Novel Inhibitors And Ultra High Resolution Analysis Of Ctx-M Beta-Lactamase, Derek Allen Nichols Jan 2014

Structure-Based Design Of Novel Inhibitors And Ultra High Resolution Analysis Of Ctx-M Beta-Lactamase, Derek Allen Nichols

Graduate Theses and Dissertations

The emergence of CTX-M class-A extended-spectrum β-lactamases, which confer resistance to second and third-generation cephalosporins, poses a serious health threat to the public. CTX-M β-lactamases use a catalytic serine to hydrolyze the β-lactam ring. Specifically, the hydrolysis reaction catalyzed by CTX-M β-lactamase proceeds through a pre-covalent complex, a high-energy tetrahedral acylation intermediate, a low-energy acyl-enzyme complex, a high-energy tetrahedral deacylation intermediate after attack via a catalytic water, and lastly, the hydrolyzed β-lactam ring product which is released from the enzyme complex. The crystallographic structure of CTX-M at sub-angstrom resolution has enabled us to study enzyme catalysis as well as perform ...


Structural Basis For Ternary Complex Formation Between Tau, Hsp90, And Fkbp51, Alexander Steven Barrett Jan 2013

Structural Basis For Ternary Complex Formation Between Tau, Hsp90, And Fkbp51, Alexander Steven Barrett

Graduate Theses and Dissertations

The accumulation of the microtubule associated protein tau has been implicated in several neurological disorders; however, its interaction with chaperones along its normal degradation pathway remains largely uncharacterized at single residue resolution. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to probe the interaction between tau, the molecular chaperone Hsp90, and the immunophilin FKBP51. Resonance intensity changes were observed for specific residues in the heteronuclear single quantum coherence (HSQC) spectra of 15N-labeled tau in the presence of Hsp90 and/or FKBP51. Analysis of the HSQC spectra identified the two hydrophobic hexapeptide motifs located at residues V275 - K280 and ...


Specificity Of The Cocaine Aptamer And Its Application In Measurement Of Cocaine And Metabolites In Homogeneous Samples For Forensic Analysis, Ashish Sachan Jan 2013

Specificity Of The Cocaine Aptamer And Its Application In Measurement Of Cocaine And Metabolites In Homogeneous Samples For Forensic Analysis, Ashish Sachan

Graduate Theses and Dissertations

Detection and quantification of molecules has an enormous importance and potential in the field of scientific development and basic discovery research. Advances in colorimetric techniques, fluorescent labeled probes and biosensor based technologies have resulted in the specific detection and precise quantification of analytes. The effective development of a particular detection method needs optimization in terms of overall speed, efficiency, and accuracy of the sensing system. Current biosensor approaches often rely on cumbersome procedural steps and reagent requirements. Along with the complexity of the assay, the size of the analytical instrument is a deterrent factor for on-site applications. To overcome such ...


The Panicum Mosaic Virus-Like 3' Cap-Independent Translation Element: A Translation Enhancer That Functions In Mammalian Systems, Mariko Sada Peterson Jan 2013

The Panicum Mosaic Virus-Like 3' Cap-Independent Translation Element: A Translation Enhancer That Functions In Mammalian Systems, Mariko Sada Peterson

Graduate Theses and Dissertations

In canonical eukaryotic translation initiation, the recognition of mRNA by the translation machinery is facilitated through the binding of the 5' m7G cap structure to eukaryotic initiation factor 4E (eIF4E). This recognition is necessary for efficient translation of the message to occur. In contrast, some viruses lack the 5' cap structure but have alternative mechanisms to initiate translation and express the viral proteins necessary for replication. The positive-strand RNA plant viruses of the panico-, carmo-, umbra-, (1-3) and aureusviruses (4) contain an RNA structure in their 3' UTRs that binds eIF4E (2) and through communication with the 5' UTR, are ...


Molecular Regulation Of Il-2 Inducible T-Cell Kinase (Itk) And The Tec Kinases: A Combined Experimental And Computational Study, With Emphasis On The N-Terminal Pleckstrin Homology Domain, Scott Edward Boyken Jan 2013

Molecular Regulation Of Il-2 Inducible T-Cell Kinase (Itk) And The Tec Kinases: A Combined Experimental And Computational Study, With Emphasis On The N-Terminal Pleckstrin Homology Domain, Scott Edward Boyken

Graduate Theses and Dissertations

This dissertation investigates the complex regulation of IL-2 inducible T-cell kinase (Itk) and its related Tec family kinase members, using a combination of experimental and computational approaches. Tec kinases are expressed primarily in hematopoietic cells and regulate key signaling events in lymphocyte activation, differentiation, and development. Itk functions in immune response, fine-tuning signaling downstream of the T-cell Receptor (TCR), and is a putative drug target for allergies and autoimmune disease. Btk, a Tec kinase found in B-cells, carries out a similar role to that of Itk, only downstream of B-cell Receptor (BCR) signaling. The chapters of this dissertation focus on ...


Investigating Higher-Order Chromatin Structure And Saga Cooperativity Using Existing And Modified In Vitro Chromatin Systems, Divya Sinha Jan 2013

Investigating Higher-Order Chromatin Structure And Saga Cooperativity Using Existing And Modified In Vitro Chromatin Systems, Divya Sinha

Graduate Theses and Dissertations

The organization of genomic DNA with histones and other proteins ensures the proper storage, utilization and segregation of DNA's encoded information in a healthy eukaryotic cell. These components comprise chromatin, and knowledge of its structure, mechanisms of its formation, and the dynamic changes it undergoes is essential to gain insights into chromatin's role in controlling gene regulation and the defects associated with misregulation. Here we describe our efforts to understand the different aspects of chromatin by using existing and modified in vitro chromatin model systems.

To better understand histone-histone interactions involved in the formation of higher-order chromatin structure ...


Biochemical And Pharmacological Characterization Of Cytochrome B5 Reductase As A Potential Novel Therapeutic Target In Candida Albicans, Mary Jolene Patricia Holloway Jan 2011

Biochemical And Pharmacological Characterization Of Cytochrome B5 Reductase As A Potential Novel Therapeutic Target In Candida Albicans, Mary Jolene Patricia Holloway

Graduate Theses and Dissertations

The opportunistic fungus Candida albicans is a commensal member of the human microflora and is the most common causative agent of fungal-related disease with particular significance in immunocompromised individuals. Emerging drug resistance is a major problem in Candida, contributed by enzymes involved in the detoxification of xenobiotics and pharmacological agents. One such enzyme, cytochrome b5 reductase (cb5r), has a high pharmacological significance owing to its role in fatty acid elongation, ergosterol (or cholesterol in mammals) biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics.

We have compared the kinetic, biochemical, and pharmacological characteristics of C. albicans cb5r isoforms, Cbr1 and Mcr1, as ...