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2013

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Full-Text Articles in Life Sciences

Spectroscopic Studies Of Peptide-Membrane Interactions, Kathryn Blakey Dupont Jan 2013

Spectroscopic Studies Of Peptide-Membrane Interactions, Kathryn Blakey Dupont

Publicly Accessible Penn Dissertations

Understanding the structure-dynamics-function relationship is a fundamental motivation for studying how proteins fold. Over the past several decades, significant progress has been made in elucidating the folding energy landscapes and dynamics of soluble, globular proteins. In contrast, the folding kinetics and mechanisms of membrane proteins are much less studied and understood, due in part to the fact that they reside in the heterogeneous and complex membrane environment. To provide new mechanistic insights into membrane protein folding, herein we studied the folding kinetics of the influenza hemagglutinin fusion peptide (HAfp), which folds into a representative helix-turn-helix structure in model membranes. Our ...


Magnetic Field Effects On Molecular Emissivity In Solutions, Tomoyasu Mani Jan 2013

Magnetic Field Effects On Molecular Emissivity In Solutions, Tomoyasu Mani

Publicly Accessible Penn Dissertations

Control over dynamics of excited states of molecules is fundamental to utilization of these states in all areas of technologies, including optical microscopy and tomography. We explored the possibility of magnetically controlling molecular emissivity by influencing spin dynamics in radical pairs (RPs) and triplet-triplet pair. We envisioned that by including RPs into a pathway leading to the formation (or decay) of an emissive triplet state, magnetic influence on phosphorescence could be realized via modulation of the RP's spin dynamics. RPs can initially be produced in their singlet or triplet spin state. These two cases were explored in the studies ...


Investigations Of Bioactivity, Disposition, And Metabolism Of Lipids Through Liquid Chromatography-Mass Spectrometry, Nathaniel W. Snyder Jan 2013

Investigations Of Bioactivity, Disposition, And Metabolism Of Lipids Through Liquid Chromatography-Mass Spectrometry, Nathaniel W. Snyder

Publicly Accessible Penn Dissertations

Inflammatory diseases and multiple human cancers are associated with increased cyclooxygenase-2 (COX-2) expression together with decreased expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH). This has been hypothesized to lead to a milieu of bioactive lipids modulating angiogenesis and cellular proliferation. Further insight into these processes were gained by traditional cell and molecular biology approaches, which were combined with cutting edge liquid chromatography mass spectrometry (LC-MS) methods to elucidate the function, disposition, and metabolism of a major COX-2/15-PGDH derived metabolite, 11-oxo-eicosatetraenoic acid (11-oxo-ETE). 11-oxo-ETE was found to possess significant anti-proliferative activity (IC50 2 microM) in human umbilical endothelial cells (HUVECS), which was ...


Large Protein Folding And Dynamics Studied By Advanced Hydrogen Exchange Methods, Benjamin Thomas Walters Jan 2013

Large Protein Folding And Dynamics Studied By Advanced Hydrogen Exchange Methods, Benjamin Thomas Walters

Publicly Accessible Penn Dissertations

Protein folding studies over the past 50 years have been largely focused on small proteins (< 200 residues) leading to a dearth of information about large protein folding. Regardless of protein size, research has generally lacked the structural tools with necessary temporal resolution to provide mechanistic insight into the process. This goal requires incisive information on transient kinetic intermediate conformations that describe the folding pathway. In this work special challenges that hinder large protein folding studies are addressed, and advancements to both HX NMR and HX MS experiments are described that provide unparalleled temporal resolution of structure formation than has been previously possible. These various advanced hydrogen exchange methods are used to study folding behaviors of the large, 370-residue, two-domain maltose binding protein from E. coli and provide a description of its folding pathway in structural detail. This work sheds light on two basic unresolved problems regarding the mechanisms of protein folding, the first being the enigmatic nature of the initial folding collapse event seen in many proteins, and the second concerning the nature of the folding pathway. We find that from an initially heterogeneous hydrophobic collapse, an obligatory intermediate emerges with a 7-second time constant followed by an apparent sequential pathway to the native state. These results add the largest protein studied at structural resolution to-date to the list of proteins known to fold through obligatory, native-like intermediates in distinct pathways and this work highlights strategies that may be employed to interrogate other large systems in future work.


Association Of Protein Helices And Assembly Of Foldamers: Stories In Membrane And Aqueous Environments, Shaoqing Zhang Jan 2013

Association Of Protein Helices And Assembly Of Foldamers: Stories In Membrane And Aqueous Environments, Shaoqing Zhang

Publicly Accessible Penn Dissertations

Solvents play an important role in association and assembly of molecules. Here we studied solvent effects on proteins and organic chemicals in different contexts. First, X-ray crystal structures show that helix dimers in membrane- and water-soluble proteins have distinct behaviors in packing and sequence selection. Transmembrane dimers are stabilized by compact packing and hydrogen bonding between small residues. Meanwhile, water-soluble dimers utilize hydrophobic residues for packing irrespective of the size of the interface and tight dimers are rare. Secondly, we apply the results learned above to a complex system in which a designed protein binds to single-walled carbon-nanotube in aqueous ...


N-Terminal Protein Modification By Aminoacyl Transferase: Enzymology And Applications., Anne Wagner Jan 2013

N-Terminal Protein Modification By Aminoacyl Transferase: Enzymology And Applications., Anne Wagner

Publicly Accessible Penn Dissertations

Protein modification is an important tool for understanding how a protein moves, folds, and operates in its cellular environment. We aim for a minimalist approach to protein modification in order to learn more about the protein while not perturbing its native state. Our method for ligation of small molecules to proteins has high N-terminal specificity and creates natural amide bond linkages under mild conditions. We utilized E. coli aminoacyl L/F transferase (AaT) in our ligation reaction to recognize a small synthetic substrate analog, which mimics aminoacylated tRNA, and can be chemically synthesized in only three steps. We have studied ...


Fatty Acid And Polyketide Synthesis Enzymes: Sequences, Structures, And Mechanisms, David Cantu Jan 2013

Fatty Acid And Polyketide Synthesis Enzymes: Sequences, Structures, And Mechanisms, David Cantu

Graduate Theses and Dissertations

The fatty acid and polyketide synthesis enzymes were studied, and findings compose this dissertation. The thioester-active enzyme database (ThYme) was constructed, which provides almost all known sequences of fatty acid and polyketide synthesis enzymes classified and organized into families based on amino acid sequence similarity. ThYme aids in predicting active sites, catalytic residues, and mechanisms of individual sequences, as well as providing a standardized nomenclature. Detailed and timely reviews on thioesterases, ketoacyl reductases, hydroxyacyl dehydratases, enoyl reductases, and acyl carrier proteins, as well as their products and pathways, were also done.

Normal vibrational mode analysis showed that the dynamic behavior ...


Computational Approaches To Anti-Toxin Therapies And Biomarker Identification, Rebecca Jane Swett Jan 2013

Computational Approaches To Anti-Toxin Therapies And Biomarker Identification, Rebecca Jane Swett

Wayne State University Dissertations

This work describes the fundamental study of two bacterial toxins with computational methods, the rational design of a potent inhibitor using molecular dynamics, as well as the development of two bioinformatic methods for mining genomic data.

Clostridium difficile is an opportunistic bacillus which produces two large glucosylating toxins. These toxins, TcdA and TcdB cause severe intestinal damage. As Clostridium difficile harbors considerable antibiotic resistance, one treatment strategy is to prevent the tissue damage that the toxins cause. The catalytic glucosyltransferase domain of TcdA and TcdB was studied using molecular dynamics in the presence of both a protein-protein binding partner and ...


Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T, Sandamali Amarasingha Ekanayaka Jan 2013

Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T, Sandamali Amarasingha Ekanayaka

Wayne State University Dissertations

DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO FRET ASSAY TO CHARACTERIZE THE SACCHAROMYCES CEREVISIAE GPI TRANSAMIDASE

By

SANDAMALI AMARASINGHA EKANAYAKA

December 2013

Advisor: Dr. Tamara L. Hendrickson

Major: Biochemistry

Degree: Doctor of Philosophy

The enzyme glycosylphosphatidylinositol transamidase (GPI-T) mediates the attachment of a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of specific proteins to produce GPI anchored proteins. This post-translational modification is essential for viability of eukaryotic organisms. However, very little is known about GPI-T and its catalytic activity. Thus, the research described in this abstract was conducted to develop an in vitro assay to monitor GPI-T. A high-throughput assay for ...


Development Of Peptide Inhibitors Targeting Clostridium Difficile Toxins A/B And Characterizing The Regulatory Role Of A Putative Negative Regulator Tcdc In Clostridium Difficile Toxin Gene Expression, Sanofar Jainul Abdeen Jan 2013

Development Of Peptide Inhibitors Targeting Clostridium Difficile Toxins A/B And Characterizing The Regulatory Role Of A Putative Negative Regulator Tcdc In Clostridium Difficile Toxin Gene Expression, Sanofar Jainul Abdeen

Wayne State University Dissertations

Clostridium difficile infections cause one of the most common and vital hospitalacquired

diseases often associated with broad-spectrum antibiotic usage. TcdA and TcdB

are the key virulence factors involved in major patho-physiology. While standard

antibiotics provide some respite, due to the high relapse rates and the emergence of more

severe disease presentations, antibiotics alone have often proven to be suboptimal.

Therefore there is a desperate need to develop an effective non-antimicrobial

therapeutics. Part of this work focuses on identification and further characterization of

peptide therapeutic that target the major virulence factor TcdA/TcdB. Towards

development of mechanistic-based anti-toxin agent, phage display ...


Synthesis And Application Of Atp Analogs For Phosphorylation-Dependent Kinase-Substrate Crosslinking, Satish Kumar Garre Venkata Raghavendra Jan 2013

Synthesis And Application Of Atp Analogs For Phosphorylation-Dependent Kinase-Substrate Crosslinking, Satish Kumar Garre Venkata Raghavendra

Wayne State University Dissertations

Phosphorylation is an important post-translational modification that plays a key role in a variety of signaling cascades and cellular functions. Kinases phosphorylate protein substrates in a highly regulated manner and are promiscuous. Understanding kinase-substrate specificity has been challenging and there is a need for new chemical tools. To this end we developed -phosphate modified ATP photocrosslinking analogs ATP-ArN3 and ATP-BP, that crosslink substrate and kinase in a phosphorylation dependent manner. We have successfully demonstrated that ATP-ArN3 and ATP-BP can be used with natural kinase and substrates using cell lysates in vitro. We used our approach to identify novel kinases of ...


Single-Stranded Dna Scanning And Deamination With Single-Molecule Resolution, Padhuk Gamarala Senavirathne Jan 2013

Single-Stranded Dna Scanning And Deamination With Single-Molecule Resolution, Padhuk Gamarala Senavirathne

Wayne State University Dissertations

SINGLE-STRANDED DNA SCANNING AND DEAMINATION WITH SINGLE-MOLECULE RESOLUTION

by

GAYAN SENAVIRATHNE

December 2013

Advisor: Dr. David Rueda & Dr.Ashok Bhagwat

Major: Chemistry (Analytical)

Degree: Doctor of Philosophy

APOBEC3G (Apo3G) and Activation-Induced cytidine Deaminase (AID) are the most notable members of APOBEC enzymes, a family of cytidine (C) deaminases with crucial functions in the immune system. In T cells, Apo3G deaminates C in viral cDNA to halt the replication of HIV 1 strains lacking viral infectivity factor (vif). In B cells, AID is required for the diversification of antibodies through initiation of somatic hypermutation (SHM), and class switch recombination (CSR), by ...


Oligodeoxynucleotide Synthesis Using Protecting Groups And A Linker Cleavable Under Non-Nucleophilic Conditions, Xi Lin Jan 2013

Oligodeoxynucleotide Synthesis Using Protecting Groups And A Linker Cleavable Under Non-Nucleophilic Conditions, Xi Lin

Dissertations, Master's Theses and Master's Reports - Open

Oligodeoxynucleotides (ODNs) containing latent electrophilic groups can be highly useful in antisense drug development and many other applications such as chemical biology and medicine, where covalent cross-linking of ODNs with mRNA, protein and ODN is required. However, such ODN analogues cannot be synthesized using traditional technologies due to the strongly nucleophilic conditions used in traditional deprotection/cleavage process.

To solve this long lasting and highly challenging problem in nucleic acid chemistry, I used the 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) function to protect the exo-amino groups on the nucleobases dA, dC and dG, and to design the linker between the nascent ODN and ...


X-Band Rapid-Scan Epr, Deborah Gale Mitchell Jan 2013

X-Band Rapid-Scan Epr, Deborah Gale Mitchell

Electronic Theses and Dissertations

The advantages of rapid-scan EPR relative to CW and pulse techniques for samples with long longitudinal relaxation time T1 (Ns0 defects in diamond, N@C60, and amorphous hydrogenated silicon), heterogeneous samples (crystalline 1:1 α,γ-bisdiphenylene-β-phenylallyl (BDPA):benzene), lossy samples (aqueous nitroxyl radicals), and transient radicals (5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO)-superoxide adduct) were studied.

For samples with long relaxation times, CW (continuous wave) EPR is challenging due to power saturation and distortions from passage effects. In rapid-scan EPR, the field is swept through resonance in a time that is short relative to T2. In rapid-scan EPR, the ...


Seeded Propagation Of Tau Fibrils, Paul David Dinkel Jan 2013

Seeded Propagation Of Tau Fibrils, Paul David Dinkel

Electronic Theses and Dissertations

In various neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, Pick's disease, and corticobasal degeneration, the deposition of fibrils composed of misfolded tau protein is observed. Recent evidence suggests that tau fibrils transfer between cells and spread throughout the brain, underscoring the significance of fibril propagation.

Six tau isoforms exist in the adult human brain that can be grouped into 4-repeat (4R) tau and 3-repeat (3R) tau based on the presence or absence of the second of four microtubule binding repeats. We demonstrate in vitro that seeded fibril growth, a prerequisite for the spreading of the tau pathology ...


Method Development For A Chemical And Biological Analysis Of Glucocorticoids In Wastewater, Maria Carmen Lozano Jan 2013

Method Development For A Chemical And Biological Analysis Of Glucocorticoids In Wastewater, Maria Carmen Lozano

Open Access Theses & Dissertations

Glucocorticoids are a class of steroid hormones that can either be produced synthetically or naturally by the adrenal glands. The synthetic glucocorticoids are highly prescribed in the United States for their anti-inflammatory and immunosuppressive properties to treat a variety of ailments and diseases; however, these have been implicated in a number of adverse human conditions such as obesity, metabolic syndrome, immune-suppression, delayed puberty, adrenal insufficiency and Cushing Syndrome, among others. Ongoing research has shown that synthetic and natural hormones are transported to aquatic environments via mammalian excretion or wastewater effluent, and as a result, the release of glucocorticoids into the ...


Novel Copper (Ii) Mediated Transfection Nano-Systems And Biological Properties Of Pyridinium Surfactants, Sarit Pal Jan 2013

Novel Copper (Ii) Mediated Transfection Nano-Systems And Biological Properties Of Pyridinium Surfactants, Sarit Pal

Open Access Theses & Dissertations

Efficacy of gene therapy is relied upon the improvement of gene delivery vectors into mammalian cells. Although viral-based polynucleotide carriers are still considered as the most common method for gene delivery but the cost effectiveness, nonspecific lethal immune response has evoked the renaissance of non-viral synthetic delivery systems. An initial discovery by Felgnar et al showed Cationic Liposomes (CLs) (having an overall positive charge) when mixed with DNA to form CL-DNA complexes can enhance the transfer of polynucleotides into cells followed by its expression. Presently, a flurry of experimental works is taking place for the better understanding of structural characterization ...


Transformations, Bioavailability And Toxicity Of Manufactured Zno Nanomaterials In Wastewter, Sewwandi Rathnayake Jan 2013

Transformations, Bioavailability And Toxicity Of Manufactured Zno Nanomaterials In Wastewter, Sewwandi Rathnayake

Theses and Dissertations--Plant and Soil Sciences

In order to properly evaluate the ecological and human health risks of ZnO Manufactured nanomaterials (MNMs) released to the environment, it is critical to understand the likely transformation products in the wastewater treatment process and in soils receiving biosolids. To address this critical knowledge gap, we examined the transformation reactions of 30 nm ZnO MNMs in single component and multi-component systems, with phosphate and natural organic matter (NOM). We also assessed the influence of nano ZnO transformation on the bioavailability, and toxicity of ZnO transformation products to Triticum aestivum. The data revealed that ZnO MNMs react with phosphate at concentrations ...


Fate Of 17Α-Estradiol, 17Β-Estradiol, And Estrone In Agricultural Soils And Sediments, Michael L. Mashtare Jr Jan 2013

Fate Of 17Α-Estradiol, 17Β-Estradiol, And Estrone In Agricultural Soils And Sediments, Michael L. Mashtare Jr

Open Access Dissertations

The shift to concentrated animal production facilities and increasing rural-urban migration has increased the localized land application of nearly 1 billion tons of manure and biosolids annually. Although these applications provide nutrients and contribute to soil tilth, they also serve as a source for an estimated 49 tons of the natural manure-borne estrogens, 17α-estradiol (17α-E2), 17β-estradiol (17β-E2), and estrone (E1). While these estrogens are critical to endocrine systems, the low concentrations observed in the environment can disrupt endocrine function in non-target organisms, e.g., altering secondary sex characteristics which can lead to changes in wildlife communities.

Research presented here focuses ...


Amalgamation Of Nucleosides And Amino Acids In Antibiotic Biosynthesis, Sandra H. Barnard Jan 2013

Amalgamation Of Nucleosides And Amino Acids In Antibiotic Biosynthesis, Sandra H. Barnard

Theses and Dissertations--Pharmacy

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading ...


Computational Approaches For Structure Based Drug Design And Protein Structure-Function Prediction, Sai Lakshmana Kumar Vankayala Jan 2013

Computational Approaches For Structure Based Drug Design And Protein Structure-Function Prediction, Sai Lakshmana Kumar Vankayala

Graduate Theses and Dissertations

This dissertation thesis consists of a series of chapters that are interwoven by solving interesting biological problems, employing various computational methodologies. These techniques provide meaningful physical insights to promote the scientific fields of interest. Focus of chapter 1 concerns, the importance of computational tools like docking studies in advancing structure based drug design processes. This chapter also addresses the prime concerns like scoring functions, sampling algorithms and flexible docking studies that hamper the docking successes. Information about the different kinds of flexible dockings in terms of accuracy, time limitations and success studies are presented. Later the importance of Induced fit ...