Biochemistry Commons^™

Electropermeabilization Does Not Correlate With Plasma Membrane Lipid Oxidation, Olga Michel, Andrei G. Pakhomov, Maura Casciola, Jolanta Saczko, Julita Kulbacka, Olga N. Pakhomova

Bioelectrics Publications

The permeabilized condition of the cell membrane after electroporation can last minutes but the underlying mechanisms remain elusive. Previous studies suggest that lipid peroxidation could be responsible for the lasting leaky state of the membrane. The present study aims to link oxidation within the plasma membrane of live cells to permeabilization by electric pulses. We have introduced a method for the detection of oxidation by ratiometric fluorescence measurements of BODIPY-C11 dye using total internal reflection fluorescence (TIRF) microscopy, limiting the signal to the cell membrane. CHO-K1 cells were cultured on glass coverslips coated with an electroconductive indium tin oxide (ITO) …

Production And Purification Of Basic Fibroblast Growth Factor Fused To Two Collagen Binding Domains Expressed In E. Coli Bl21 Using Flask And Fed-Batch, Hazim Aljewari

Graduate Theses and Dissertations

Delivering effective and non-toxic doses of bioactive materials that can aid in activating tissue regeneration to wounded tissue has proven to be an enormous challenge. This study was designed to produce a potential therapeutic recombinant protein by fusing two collagen binding domains to basic fibroblast growth factors (bFGF) through a collagenase cleavage site linker, so it can release the bFGF in a wound site by the action of this enzyme. The novel fusion protein was expressed in Escherichia coli BL-21 (E. coli) using traditional flask shaker and fed-batch cultivation. Cell lysate was purified by FPLC using Immobilized metal affinity chromatography …

Structure And Bioactivity Of A Rationally Designed Nicotinic Acetylcholine Receptor Antagonist Targeting The Alpha-3 Beta-2 Subtype, Leanna Marquart

Boise State University Theses and Dissertations

The work presented in this thesis contributes to ongoing development of an efficient workflow for identification of lead compounds, based on the molecular scaffold of α-conotoxin MII, for drug therapies targeting nicotinic acetylcholine receptors. Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated ion channels with distribution throughout the central nervous system and are implicated in a variety of neurological diseases including schizophrenia, nicotine addiction, Alzheimer’s disease, and Parkinson’s disease. However, ligand effect on nAChR function is not well understood. Consequently, drug therapies for neurological diseases either do not exist, or have short-lasting efficacy and/or severe side effects. Barriers to the comprehension …

Asymmetric Synthesis Of The C29-C34 Moiety Of Fragment A Of The Antascomicin B & Thermal Azole Based Claisen Rearrangements, Dharma Theja Nannapaneni

Graduate Theses and Dissertations

The dissertation describes asymmetric synthesis towards C29-C34 moiety of fragment A of the Antascomicin B and Thermal azole based Claisen rearrangements. In chapter 1, we describes asymmetric synthesis towards C29-C34 moiety of fragment A of the Antascomicin B. The non-immunosuppressant Rapamycin, Ascomycin, and Tacrolimus (FK506), strongly binds with FKBP12, the ligand FKBP12 complexes responsible for immunosuppressive activity. Antascomicin B structurally related to Rapamycin, Ascomycin, and Tacrolimus (FK506), binds strongly to FKBP12, yet does not shown immunosuppressive activity. The ligand FKBP12 binding complexes shown to have potent neuroprotective and neurogenerative properties in mouse models of Parkinson’s disease. The linear synthesis of …

Xenobiotic Exposure Requires Mitochondrial Metabolism For Redox Homeostasis And Survival In Astrocytes, Jordan Rose

Department of Biochemistry: Dissertations, Theses, and Student Research

Astrocytes are integral components of glutamatergic neurotransmission, providing essential metabolic processes for neuronal homeostasis and acting as the first line of defense against xenobiotics crossing the blood brain barrier. Arsenic is a xenobiotic with widespread natural and industrial prevalence, and has been linked to impaired neurodevelopment and neuronal death. Given the integrated metabolic nature of astrocytes and neurons, we sought to explore how arsenic impacts astrocyte metabolism in order to better understand the mechanisms of xenobiotic toxicity in the mammalian brain.

We demonstrate that astrocyte viability depends upon de novoglutathione (GSH) synthesis during arsenic exposure, and sub-lethal arsenic exposure …

Impact Of Metazooplankton Filter Feeding On Escherichia Coli Under Variable Environmental Conditions, Niveen S. Ismail, Brittney M. Blokker, Tyler R. Feeney, Ruby H. Kohn, Jingyi Liu, Vivian E. Nelson, Mariah C. Ollive, Sarah B. L. Price, Emma J. Underdaha

Engineering: Faculty Publications

The fecal indicator bacterial species Escherichia coli is an important measure of water quality and a leading cause of impaired surface waters. We investigated the impact of the filter-feeding metazooplankton Daphnia magna on the inactivation of E. coli. The E. coli clearance rates of these daphnids were calculated from a series of batch experiments conducted under variable environmental conditions. Batch system experiments of 24 to 48 h in duration were completed to test the impacts of bacterial concentration, organism density, temperature, and water type. The maximum clearance rate for adult D. magna organisms was 2 ml h1 organism1. Less than …

A Novel Switch-Like Function Of Delta-Catenin In Dendrite Development, Ryan Baumert

Dissertations and Theses (Open Access)

The formation of neuronal networks in the brain is tightly regulated, and dependent on the morphology of dendrites, the branch-like signal-receiving structures extending from neurons. Disruptions in dendrite development, or dendritogenesis, can lead to the atypical neuronal connectivity associated with multiple neurodevelopmental diseases. My research addresses molecular processes that underlie dendritogenesis via analysis of a pair of novel interactions involving the protein delta-catenin.

In neurons, delta-catenin localizes to dendrites and synapses, where it functions in their development and maintenance. Structurally, delta-catenin possesses a central Armadillo domain and a C-terminal PDZ-binding motif. This motif associates with PDZ domain-containing proteins, and is …

Investigation Of Phosphoserine Aminotransferase 1(Psat1) In Breast Cancer Progression., Stephanie Metcalf

Electronic Theses and Dissertations

This dissertation describes my research into the involvement of phosphoserine aminotransferase 1 (PSAT1) in breast cancer progression; specifically, in triple negative breast cancer (TNBC) metastasis and endocrine resistance in estrogen receptor positive breast cancer (ER+BC). Breast cancer is the most common tumor diagnosis among women. While the overall 5-year survival for breast cancer is reaching 90%, the 5-year survival for metastatic disease is only 22%. Metastasis and endocrine resistance combined can affect over 50% of patients. One of the proteins and pathways implicated in both metastasis and endocrine resistance in breast cancer is phosphoserine aminotransferase 1 (PSAT1) and the serine …

Probing Of Carbohydrate-Protein Interactions Using Galactonoamidine Inhibitors, Jessica B. Pickens

Graduate Theses and Dissertations

Glycoside hydrolases are ubiquitous and one of the most catalytically proficient enzymes known, and thus understanding their mechanisms are crucial. Most research has focused on the interaction of the glycon of substrates and their inhibitors within the active site of glycoside hydrolases. The inhibitors employed to probe these interactions generally had small aglycons (i.e. a hydrogen atom, amidines, small aliphatic groups, or benzyl groups). Here, the interactions of the aglycon with glycoside hydrolases are examined by probing the active sites with a library of 25 galactonoamidines. The studies described in this dissertation aim to increase the understanding of stabilization of …

Toward Understanding The Mechanism Of Protein Targeting In The Chloroplast Signal Recognition Particle Pathway, Mercede Furr

Graduate Theses and Dissertations

Protein targeting is a vital cellular function. The signal recognition particle (SRP) pathway is a universally conserved targeting system present in the cytosol and used to co-translationally target many proteins to the inner membrane of prokaryotes and the endoplasmic reticulum of eukaryotes. The chloroplast has a homologous SRP system which post-translationally targets light harvesting chlorophyll binding proteins (LHCPs) to the thylakoid membrane for integration. The chloroplast SRP (cpSRP) is a heterodimer with a 54 kDa subunit equivalent to SRP54 in the canonical pathway. In addition, cpSRP contains a novel 43 kDa subunit which is a unique and irreplaceable component. cpSRP43 …

Rotational Tuning Of Transmembrane Helix Properties Based On The Precise Placements Of Aromatic And Charged Residues, Matthew J. Mckay

Graduate Theses and Dissertations

Designed model transmembrane peptides and oriented 2H and 15N solid-state nuclear magnetic resonance (NMR) spectroscopy were used to analyze how simple sequence modifications can influence peptide structure, behavior and dynamics as well as for determining the pKa of glutamic acid at the membrane interface. The GW5,19ALP23 (acetyl-GGALW(LA)6LWLAGA-amide) peptide framework adopts a well-defined tilted orientation in lipid bilayers (DLPC, DMPC and DOPC) and undergoes low amounts of dynamic motion. The sequence was initially modified by moving the Trp residues outwards to positions 4 and 20. This new sequence GW4,20ALP23 (acetyl-GGAW(AL)7AWAGA-amide) displays high amounts of signal averaging of NMR observables caused by …

Single Molecule Fluorescence Studies Of Protein Structure And Dynamics Underlying The Chloroplast Signal Recognition Particle Targeting Pathway, Dustin R. Baucom

Graduate Theses and Dissertations

The work presented in this dissertation explores the structural dynamics in the chloroplast signal recognition particle pathway. Findings include cpSRP shows scanning functionality similar to that in the cytosolic SRP with the ribosome. The intrinsically disordered C-terminal tail of the Albino3 protein has some transient secondary structure. Upon binding to cpSRP43 in solution, separate secondary structure formation was identified in the C-terminal tail of Albino3. Finally, to increase efficiency of analyzing fluorescence time traces for this work, a modular software was produced.

Influence Of Single And Multiple Histidine Residues And Their Ionization Properties On Transmembrane Helix Dynamics, Orientations And Fraying, Fahmida Afrose

Graduate Theses and Dissertations

Since aromatic and charged residues are often present in various locations of transmembrane helices of integral membrane proteins, their impacts on the molecular properties of transmembrane proteins and their interactions with lipids are of particular interest in many studies. In this work, I used solid-state deuterium NMR spectroscopy in designed model peptide GWALP23 [GGALW(LA)6LWLAGA] with selective deuterium labels to addresses the pH dependence and influence of single and multiple “guest” histidine residues in the orientation and dynamic behaviors of transmembrane proteins. The mutations include Gly to His (G2/22 to H2/22), Trp to His (W5/19 to H5/19) and Leu to His …

Exploration Of Ataxia Telangiectasia And Rad3-Related’S (Atr’S) Role In Cell Death Regulation: Implications In Development, Cancer, And Stroke, Brian Cartwright

Electronic Theses and Dissertations

From gametogenesis until death an organism’s genome is under constant bombardment from endogenous and exogenous sources of DNA damage. To maintain genomic integrity amid this damage, cells have evolved responses which allow them to either preserve viability for recovery or initiate self-destructive pathways depending on the severity of DNA damage. One protein involved in initiating and carrying out these responses is the protein kinase ataxia telangiectasia and Rad3-related (ATR). ATR is known primarily for its regulatory role in initiating the checkpoint-signaling cascade following DNA damage and replicative stress. These signaling events lead to cell cycle arrest, DNA repair, or apoptosis …

Deepep: A Deep Learning Framework For Identifying Essential Proteins, Min Zeng, Min Li, Fang-Xiang Wu, Yaohang Li, Yi Pan

Computer Science Faculty Publications

Background: Essential proteins are crucial for cellular life and thus, identification of essential proteins is an important topic and a challenging problem for researchers. Recently lots of computational approaches have been proposed to handle this problem. However, traditional centrality methods cannot fully represent the topological features of biological networks. In addition, identifying essential proteins is an imbalanced learning problem; but few current shallow machine learning-based methods are designed to handle the imbalanced characteristics. Results: We develop DeepEP based on a deep learning framework that uses the node2vec technique, multi-scale convolutional neural networks and a sampling technique to identify essential proteins. …

Isotopic Characterization Of Nitrogen Oxides (NoX), Nitrous Acid (Hono), And Nitrate (PNo3-) From Laboratory Biomass Burning During Firex, Jiajue Chai, David J. Miller, Eric Scheuer, Jack Dibb, Vanessa Selimovic, Robert Yokelson, Kyle K. Zarzana, Steven Brown, Abigail R. Koss, Carsten Warneke, Meredith Hastings

Chemistry and Biochemistry Faculty Publications

New techniques have recently been developed and applied to capture reactive nitrogen species, including nitrogen oxides (NOx D NOCNO2), nitrous acid (HONO), nitric acid (HNO3), and particulate nitrate (pNO3 ), for accurate measurement of their isotopic composition. Here, we report – for the first time – the isotopic composition of HONO from biomass burning (BB) emissions collected during the Fire Influence on Regional to Global Environments Experiment (FIREX, later evolved into FIREX-AQ) at the Missoula Fire Science Laboratory in the fall of 2016. We used our newly developed annular denuder system (ADS), which was verified to completely capture HONO associated …

Case 67: Phenylalanine Hydroxylase: An Investigation Of Its Structure Leads To The Development Of Pku New Therapies, Dr. Kathleen Cornely

Chemistry & Biochemistry Case Studies

The characteristics of phenylalanine hydroxylase, the enzyme missing in persons afflicted with the genetic disorder phenylketonuria (PKU), are examined. Understanding how the enzyme works will allow the development of drugs to treat this condition.

Constituent Partitioning Consensus Docking Models And Application In Drug Discovery, Rainer Metcalf

USF Tampa Graduate Theses and Dissertations

This work expounds on some of the current computational tools and programs available and the best practices associated with their use. A high-level introduction, intended for both novices and the semi-experienced, focusing on the more common programs used in scientific literature is the scope of this topic. Both classical and quantum techniques are described. Classical methodologies include Molecular Dynamics, Monte Carlo, energy minimization methods, molecular docking, low-mode, and homology modeling. Quantum chemistry techniques are also discussed encompassing Hartree-Fock, Post-Hartree-Fock theories, and Density Functional Theory along with associated basis sets.

Along with established methodologies, novel theoretical methods are introduced for furthering …

The Role Of Atypical Protein Kinase C In Colorectal Cancer Cells Carcinogenesis, S M Anisul Islam

USF Tampa Graduate Theses and Dissertations

Colorectal cancer (CRC) is the third most common malignancy and the fourth most common cause of cancer-related death worldwide. CRC is a life-threatening disease due to therapy-resistant cancerous cells. The exact mechanisms of cell growth, survival, metastasis and inter & intracellular signaling pathways involved in CRC are still a significant challenge. Moreover, the treatment of metastatic CRC considered palliative for many years aimed for an improved life, with little hope of a cure, highlighting the need for developing novel targeted therapy for CRC. Hence, investigating new molecular mechanism(s) that lead to colorectal carcinogenesis may give insight into the therapeutic target. …

Comparison Of Montage With Conventional Stereoscopic Seven‑Field Photographs For Assessment Of Etdrs Diabetic Retinopathy Severity, Nam V. Nguyen, Erin M. Vigil, Muhammad Hassan, Muhammad S. Halim, Sean C. Baluyot, Hugo A. Guzman, Rubbia Afridi, Diana V. Do, Yasir J. Sepah

Department of Biochemistry: Faculty Publications

Background: The ETDRS stereoscopic seven-field (7F) has been a standard imaging and grading protocol for assessment of diabetic retinopathy (DR) severity score in many clinical trials. To the best of our knowledge, the comparison between montage and stereoscopic 7F has not been reported in the literature. Therefore, the main purpose of this study is to compare agreement between montage and stereoscopic seven-field (7F) photographs in the assessment of DR severity.

Methods: Stereoscopic 7F photographs were captured from subjects with DR. Montages of monoscopic 7F images were created using Adobe Photoshop CS6 Extended©. The best quality image of each stereo pair …

Targeting Bacterial Resistance And Cancer Metastasis: A Structure Based Approach, Kyle Galen Kroeck

USF Tampa Graduate Theses and Dissertations

Current research in pharmaceutical development commonly utilizes a profusion of methods in molecular modeling in order to probe intricate biological problems. Many original and promising compounds have been identified and developed by integrating experimental and computational methods. Structural biology utilizes many different research techniques including x-ray crystallography, NMR, and electron microscopy in order to develop molecular models of macromolecules that are of biological interest. Such techniques can be used in conjunction with molecular docking, which utilizes those molecular models in order to target macromolecules of therapeutic interest by computationally analyzing the conformations adopted by ligands upon interaction with a desired …

Altered Proteasome Expression And Nuclear Factor (Erythroid-Derived 2)-Like Signaling In Experimental Autoimmune Encephalomyelitis, Kara Leasure Shanley

Biomedical Sciences ETDs

Multiple sclerosis (MS) is a complex neurological disorder characterized by the interactions between heightened inflammation, oxidative stress and neurodegeneration. We and others have previously demonstrated that proteasome dysfunction and its consequences are also important factors in the pathology of both MS and its rodent model, experimental autoimmune encephalomyelitis (EAE). While proteasome subunit alterations in EAE have been observed, the underlying mechanisms are poorly understood. The first goal of this dissertation was to characterize the mechanisms that regulate proteasome expression and composition in neural cells in EAE and in vitro.

Immunohistochemical analysis of the EAE spinal cord shows changes in …

An Investigation Into The Heterogeneity Of Insect Arylalkylamine N-Acyltransferases, Brian G. O'Flynn

USF Tampa Graduate Theses and Dissertations

Arylalkylamine N-acyltransferases (AANATs) have in recent years been suggested as potential new insecticide targets. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. Mammalian AANAT is predominantly associated with circadian rhythm regulation, as it catalyzes the formation of N-acetylserotonin, the precursor of melatonin, from serotonin. In insects, this process is a key step in melanism, as well as hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each insect AANAT (iAANAT) isoform characterized indicates that while catalyzing similar reactions, each insect accommodates …

The Democratization And Development Of Cell-Free Protein Synthesis, Max Z. Levine

Master's Theses

Cell-free protein synthesis (CFPS) using crude lysates has developed into a robust platform technology over the last 60 years to express numerous types of recombinant proteins. The open-nature, elimination of reliance on cell viability, and focus of all energy towards production of the protein of interest represent substantial advantages of CFPS over in vivo protein expression methods. CFPS has provided new opportunities across a series of research fields that include metabolic engineering, therapeutic and vaccine development, education, biosensors, and many more. In recent years, optimizations of CFPS have even allowed the platform to reach the industrial level of protein production. …

Multiple Cytosolic Dna Sensors Bind Plasmid Dna After Transfection, Nina Semenova, Masa Bosnjak, Katarina Znidar, Maja Cemazar, Loree Heller

Bioelectrics Publications

Mammalian cells express a variety of nucleic acid sensors as one of the first lines of defense against infection. Despite extensive progress in the study of sensor signaling pathways during the last decade, the detailed mechanisms remain unclear. In our previous studies, we reported increased type I interferon expression and the upregulation of several proposed cytosolic DNA sensors after transfection of several tumor cell types with plasmid DNA (pDNA). In the present study, we sought to reveal the early events in the cytosolic sensing of this nucleic acid in a myoblast cell line. We demonstrated that DNA-dependent activator of interferon …

Transcriptomic Analyses And Computational Modeling Reveal That Fatty Acid Transport Protein 2 (Fatp2) Impacts The Transcriptional Activity Of Pparα Thus Altering The Lipid Metabolic Landscape, Vincent M. Perez

Department of Biochemistry: Dissertations, Theses, and Student Research

Fatty acid transport protein 2 (FATP2) is highly expressed in liver, small intestine, and kidney where it functions in both the uptake of exogenous long chain fatty acids (LCFAs) and in the activation of very long chain fatty acids (VLCFAs). Here we address the phenotypic impacts of deleting FATP2 with the following three separate approaches: [1] Utilizing an unbiased next-generation sequencing analysis of FATP2-null (fatp2-/-) mice fed a standard chow diet; [2] Utilizing an unbiased next-generation sequencing analysis of fatp2-null (fatp2-/-) mice fed a high-fat ketogenic diet (KD) and fasted for 24-hours [3] Building dynamic computer models built with data …

How Oxygen Availability Affects The Antimicrobial Efficacy Of Host Defense Peptides: Lessons Learned From Studying The Copper-Binding Peptides Piscidins 1 And 3, Adenrele Oludiran, David S. Courson, Malia D. Stuart, Anwar R. Radwan, John C. Putsma, Myriam L. Cotten, Erin B. Purcell

Chemistry & Biochemistry Faculty Publications

The development of new therapeutic options against Clostridioides difficile (C. difficile) infection is a critical public health concern, as the causative bacterium is highly resistant to multiple classes of antibiotics. Antimicrobial host-defense peptides (HDPs) are highly effective at simultaneously modulating the immune system function and directly killing bacteria through membrane disruption and oxidative damage. The copper-binding HDPs piscidin 1 and piscidin 3 have previously shown potent antimicrobial activity against a number of Gram-negative and Gram-positive bacterial species but have never been investigated in an anaerobic environment. Synergy between piscidins and metal ions increases bacterial killing aerobically. Here, we …

Remodeling Of Chlamydomonas Metabolism Using Synthetic Inducers Results In Lipid Storage During Growth, Nishikant Wase, Boqiang Tu, Girish Kumar Rasineni, Ronald Cerny, Ryan Grove, Jiri Adamec, Paul N. Black, Concetta Dirusso

Department of Biochemistry: Faculty Publications

Microalgae accumulate lipids during stress such as that of nutrient deprivation, concomitant with cessation of growth and depletion of chloroplasts. By contrast, certain small chemical compounds selected by high-throughput screening in Chlamydomonas reinhardtii can induce lipid accumulation during growth, maintaining biomass. Comprehensive pathway analyses using proteomics, transcriptomics, and metabolomics data were acquired from Chlamydomonas cells grown in the presence of one of two structurally distinct lipid activators. WD10784 stimulates both starch and lipid accumulation, whereas WD30030-treated cells accumulate only lipids. The differences in starch accumulation are largely due to differential effects of the two compounds on substrate levels that feed …

P-20 Characterization Of Cgx Tautomerase And Mutants With Acetylenecarboxylic Acid, Daniel Chi, Karen Draths, Amaya Sirinimal

Celebration of Research and Creative Scholarship

In the production of commodity chemicals through microbial synthesis, the use of renewable sugars as a raw material is increasingly common. However, this generates competition between its use in the chemical industry and the food industry. Therefore, developing alternative biosynthetic pathways involving other renewable feedstocks to replace starch-derived feedstocks is crucial. CgX, a tautomerase native to Corynebacterium glutamicum, catalyzes the hydration and subsequent decarboxylation of acetylenecarboxylic acid (ACA), a compound that can be produced from methane gas and carbon dioxide, both abundant greenhouse gases. The products formed from the CgX (wild-type)-catalyzed reaction of ACA results in a mixture of …

Intrinsic Cleavage Inhibition By Thermophilic Type Ii-S Restriction Endonucleases, Dino Jake Ciccone

Master of Science in Chemical Sciences Theses

Restriction endonucleases (REs) are the cornerstone research tools used in molecular biology and biotechnology. These enzymes are essential to several ground-breaking achievements, including DNA cloning, gene editing, and genome mapping. A subset of these enzymes, Type II-S restriction endonucleases (IISREs), cleave double-stranded DNA (dsDNA) not governed by sequence specificity, but rather at a fixed distance from their recognition sequence. These enzymes maintain complete cleavage promiscuity, allowing utilization in techniques such as Golden Gate assembly, Serial Analysis of Gene Expression (SAGE), and Restriction Endonuclease Protection, Selection, and Amplification (REPSA). However, laboratories generated evidence that intrinsically cleavage refractory sequences exist. Understanding the …