Biochemistry Commons^™

Phytochrome B Integrates Light And Temperature Signals In Arabidopsis, Martina Legris, Cornelia Klose, E Sethe Burgie, Cecilia Costigliolo Rojas Rojas, Maximiliano Neme, Andreas Hiltbrunner, Philip A. Wigge, Eberhard Schäfer, Richard D. Vierstra, Jorge J. Casal

Biology Faculty Research

Ambient temperature regulates many aspects of plant growth and development, but its sensors are unknown. Here, we demonstrate that the phytochrome B (phyB) photoreceptor participates in temperature perception through its temperature-dependent reversion from the active Pfr state to the inactive Pr state. Increased rates of thermal reversion upon exposing Arabidopsis seedlings to warm environments reduce both the abundance of the biologically active Pfr-Pfr dimer pool of phyB and the size of the associated nuclear bodies, even in daylight. Mathematical analysis of stem growth for seedlings expressing wild-type phyB or thermally stable variants under various combinations of light and temperature revealed …

Quantitative Proteomic Investigation Of Disease Models Of Type 2 Diabetes, Mark Gabriel Athanason

USF Tampa Graduate Theses and Dissertations

PANcreatic DERived factor (PANDER, FAM3B) is a member of a superfamily of FAM3 proteins that are uniquely structured and strongly expressed from the endocrine pancreas and co-secreted with insulin. Unique animal models available to our lab have indicated that PANDER can induce a selective hepatic insulin resistant (SHIR) phenotype whereby insulin signaling is blunted yet lipogenesis is increased. The complexity of the biological networks involved with this process warranted the logical approach of employing quantitative mass spectrometry based proteomic analysis using stable isotope labeling of amino acids in cell culture (SILAC) to identify the global proteome differences between the PANDER …

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

USF Tampa 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 post-translational …

An Iron Detection System Determines Bacterial Swarming Initiation And Biofilm Formation, Chuan-Sheng Lin, Yu-Huan Tsai, Chih-Jung Chang, Shun-Fu Tseng, Tseng-Ru Wu, Chia-Chen Lu, Ting-Shu Wu, Jang-Jih Lu, Jim-Tong Horng, Jan Martel, David M. Ojcius, Hsin-Chih Lai, John Ding-E. Young

All Dugoni School of Dentistry Faculty Articles

Iron availability affects swarming and biofilm formation in various bacterial species. However, how bacteria sense iron and coordinate swarming and biofilm formation remains unclear. Using Serratia marcescens as a model organism, we identify here a stage-specific iron-regulatory machinery comprising a two-component system (TCS) and the TCS-regulated iron chelator 2-isocyano-6,7-dihydroxycoumarin (ICDH-Coumarin) that directly senses and modulates environmental ferric iron (Fe3+) availability to determine swarming initiation and biofilm formation. We demonstrate that the two-component system RssA-RssB (RssAB) directly senses environmental ferric iron (Fe³⁺) and transcriptionally modulates biosynthesis of flagella and the iron chelator ICDH-Coumarin whose production requires the pvc cluster. …

Development Of In Vivo Systems For Detecting And Studying Ribosome Inhibition By Small Molecules, Shijie Huang

Chemistry and Chemical Biology ETDs

The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome controlled green fluorescent protein reporter that produce fluorescent signal when the O-ribosome is inhibited. As a proof …

Regulation Of Palmitoylation Enzymes And Substrates By Intrinsically Disordered Regions, Krishna D. Reddy

USF Tampa Graduate Theses and Dissertations

Protein palmitoylation refers to the process of adding a 16-carbon saturated fatty acid to the cysteine of a substrate protein, and this can in turn affect the substrate’s localization, stability, folding, and several other processes. This process is catalyzed by a family of 23 mammalian protein acyltransferases (PATs), a family of transmembrane enzymes that modify an estimated 10% of the proteome. At this point in time, no structure of a protein in this family has been solved, and therefore there is poor understanding about the regulation of the enzymes and their substrates. Most proteins, including palmitoylation enzymes and substrates, have …

Engineering Small Molecule Based Dimerization To Induce Translation And Provide Optogenetic Control, Catherine Wright

Chemistry and Chemical Biology ETDs

We wanted to develop a system that combines the spatial control of photoactivation and control of translation to build a tool to spatially control translation in neurons. This kind of tool could be used to investigate the role of spatially controlled translation of any protein in neural behavior. In this way the development and growth of neural processes could be studied to elucidate the mechanisms for spatially sensitive events such as pathfinding, repair, or long-term potentiation.

Chemically induced dimerization was used to install a switch into the activation of translation for specific genes. An abscisic acid (ABA) dependent dimerization of …

Design Of Novel Ion Channel Modulators, Vladimir Yarov-Yarovoy

Science Seminar Series

Function and modulation of neuronal sodium channels are critical for the neuromodulation of electrical excitability and synaptic transmission in neurons - the basis for many aspects of signal transduction, learning, memory and physiological regulation. Mutations in neuronal voltage-gated sodium channel genes are responsible for various human neurological disorders. Furthermore, human neuronal voltage-gated sodium channels are primary targets of therapeutic drugs used as local anesthetics and for treatment of neurological and cardiac disorders. Yarov-Yarovoy's lab is working on rational design of novel therapeutically useful blockers of voltage-gated sodium channels for treatment of pain and epilepsy. Serious, chronic pain affects at least …

A Pipette-Based Calibration System For Fast-Scan Cyclic Voltammetry With Fast Response Times, Eric S. Ramsson

Peer Reviewed Articles

Fast-scan cyclic voltammetry (FSCV) is an electrochemical technique that utilizes the oxidation and/or reduction of an analyte of interest to infer rapid changes in concentrations. In order to calibrate the resulting oxidative or reductive current, known concentrations of an analyte must be introduced under controlled settings. Here, I describe a simple and cost-effective method, using a Petri dish and pipettes, for the calibration of carbon fiber microelectrodes (CFMs) using FSCV.

Polymorphisms In Mthfr, Ms And Cbs Genes And Premature Acute Myocardial Infarction In A Pakistani Population, Mohammad Perwaiz Iqbal, Khalida Iqbal, Asal Khan Tareen, Siddiqa Parveen, Naseema Mehboobali, Ghulam Haider, Saleem Perwaiz Iqbal

Department of Biological & Biomedical Sciences

High prevalence of premature coronary heart disease in Pakistanis compared to other populations points towards the genetic predisposition of this population to develop this disease. Since no investigations have been carried out in Pakistan to study the relationship of polymorphisms in genes involved in homocysteine cycle, the objective of the present study was to find out if there is any association of methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C; methionine synthase (MS) A2756G; cystathionine-beta-synthase (CBS) 844ins68, G919A polymorphisms with premature acute myocardial infarction (AMI) in a population of Pakistani patients with this disease. In a cross-sectional study, DNA samples of 143 AMI …

A Polyaniline-Based Sensor Of Nucleic Acids, Partha Sengupta, Jared Gloria, Marcus K. Parker, Alex S. Flynt

Faculty Publications

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive …

The Biological Function Of Hammerhead Rana Ribozyme In Human Genome, Ibtehal M. A. Awad

Theses

Hammerhead RNA [HHR] sequence originally found in viroids is also detected in many species. HHR are often found in introns of different genes ranging from prokaryotes to

eukaryotes. HHR can form specific tertiary structure that make specific region susceptible to cleavage. However, the role of the HHR is not clear in other life forms. This study aims to

Investigate the role of HHR in the intron 6 of (RECK) gene. Our study shows that when blocking the HHR sequence in the intron6, the production of RECK protein declines which may be due to the effect of HHR in splicing and …

Expression, Purification, And Crystallization Of An Endoxylanase From Bacteroides Vulgatus, Jesslyn Park, Jason Hurlbert

The Winthrop McNair Research Bulletin

Sustainable sources of energy are growing in demand as fossil fuels are rapidly expended. One such energy source is fuel ethanol generated from the fermentation of plant biomass by engineered bacterial biocatalysts. The creation of a biocatalyst capable of converting nearly any plant matter to fuel ethanol requires the identification of novel enzymes capable of degrading specific carbohydrate polymers and cloning these enzymes into a bacterial host. This study seeks to structurally characterize a novel xylanase of glycosyl hydrolase family 30 (GH30) from Bacteroides vulgatus, a bacterium found in the human gut microbiome, via x-ray crystallography. The gene for …

Pyk2 Activates The Nlrp3 Inflammasome By Directly Phosphorylating Asc And Contributes To Inflammasome-Dependent Peritonitis, I-Che Chung, Chun-Nan Ouyang, Sheng-Ning Yuan, Hsin-Pai P. Li, Jeng-Ting Chen, Hui-Ru Shieh, Yu-Jen Chen, David M. Ojcius, Ching-Liang Chu, Jau-Song Yu, Yu-Sun Chang, Lih-Chyang Chen

All Dugoni School of Dentistry Faculty Articles

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, …

Infrared Spectroscopy Of The Nitrogenase Mofe Protein Under Electrochemical Control: Potential-Triggered Co Binding, P. Paengnakorn, Philip A. Ash, Sudipta K. Shaw, Karamatullah Danyal, T. Chen, Dennis R. Dean, Lance C. Seefeldt, Kylie A. Vincent

Chemistry and Biochemistry Faculty Publications

We demonstrate electrochemical control of the nitrogenase MoFe protein, in the absence of Fe protein or ATP, using europium(III/II) polyaminocarboxylate complexes as electron transfer mediators. This allows the potential dependence of proton reduction and inhibitor (CO) binding to the active site FeMo-cofactor to be established. Reduction of protons to H2 is catalyzed by the wild type MoFe protein and Β-98^Tyr→His and Β-^99Phe→His variants of the MoFe protein at potentials more negative than -800 mV (vs. SHE), with greater electrocatalytic proton reduction rates observed for the variants compared to the wild type protein. Electrocatalytic proton reduction is strongly …

Purification Of 26s Proteasomes And Their Subcomplexes From Plants, Richard S. Marshall, David C. Gemperline, Richard D. Vierstra

Biology Faculty Research

The 26S proteasome is a highly dynamic, multisubunit, ATP-dependent protease that plays a central role in cellular housekeeping and many aspects of plant growth and development by degrading aberrant polypeptides and key cellular regulators that are first modified by ubiquitin. Although the 26S proteasome was originally enriched from plants over 30 years ago, only recently have significant advances been made in our ability to isolate and study the plant particle. Here, we describe two robust methods for purifying the 26S proteasome and its subcomplexes from Arabidopsis thaliana; one that involves conventional chromatography techniques to isolate the complex from wild-type …

Atomic Tiles: Manipulative Resources For Exploring Bonding And Molecular Structure, Alan L. Kiste, Rebecca G. Hooper, Gregory E. Scott, Seth Bush

Chemistry and Biochemistry

A simple manipulative resource, Atomic Tiles, is described for scaffolding the learning of Lewis structures without using algorithmic, rule-based methods of drawing. Students use Atomic Tiles to (1) create models of bonding that lead to drawing Lewis structures, (2) use the structures they create to infer patterns required for rational structures and common organic functional groups, (3) translate between Lewis structures and molecular models, and (4) use molecular models to identify isomers.

A Study Of Defensive Mechanisms Employed By Two Species Of Nudibranchs Using Toxicity And Unpalatability Analyses, Sean Youn

Independent Study Project (ISP) Collection

Nudibranchs are marine invertebrates that have developed an intriguing defense mechanism, including warning coloration and the use of chemicals accumulated through their sponge diet. The goal of this study was to determine whether the strength of chemical defenses differs between dietary and accumulated secondary metabolites for two species: Glossodoris vespa and Ceratosoma brevicaudatum. First, NMR spectroscopy was used to not only identify specific compounds in the mantle (outer covering) and the viscera (gut) but also to analyze the possibility of nudibranch species transporting more toxic compounds for defensive purposes. Next, toxicity (brine shrimp) and palatability (Palaemon shrimp) assays were used …

Effects Of Herbicides On Industrial Hemp (Cannabis Sativa) Phytotoxicity, Biomass, And Seed Yield, Brett A. Maxwell

Masters Theses & Specialist Projects

Field studies were established in 2015 at Bowling Green and Lexington, KY to evaluate industrial hemp (Cannabis sativa) tolerance to various herbicides. Hemp was planted into conventionally tilled soils in mid to late June at a seeding rate of 39 kg/ha in Bowling Green and 22 kg/ha in Lexington. Five herbicide active ingredients were applied preemergence (PRE) the day of planting and six postemergence (POST) treatments were applied to 30 cm hemp with a CO2-backpack sprayer delivering 140 L/ha. Plots were 3.1 m wide by 6.1 m long and were sprayed with a 2.1 m boom sprayer leaving a 0.46 …

Development Of Dihydrochalcone Functionalized Gold Nanoparticles For Augmented Antineoplastic Activity, Jason N. Payne

Masters Theses & Specialist Projects

Phloridzin, an antidiabetic and antineoplastic agent usually found in fruit trees, is a dihydrochalcone constituent that has a clinical/pharmaceutical significance as a sodiumglucose linked transport 2 (SGLT2) inhibitor. Phloridzin never experienced widespread clinical usage in the pharmaceutical market due to its side effects and poor bioavailability when compared to other antidiabetic therapeutics. The poor bioavailability is primarily attributed to the degradation of the glycosidic bond of the phloridzin, resulting in the formation of phloretin, the aglycone of phloridzin and glucose. While phloretin displays a reduced capacity of SGLT2 inhibition, this nutraceutical shows enhanced antineoplastic activity in comparison to phloridzin. Gold …

Visible-Light Generation Of High-Valent Metal-Oxo Intermediates And A Biomimetic Oxidation Catalyzed By Manganese Porphyrins With Iodobenzene Diacetate, Ka Wai Kwong

Masters Theses & Specialist Projects

High-valent iron-oxo intermediates play central roles as active oxidants in enzymatic and synthetic catalytic oxidations. Many transition metal catalysts are designed for biomimetic studies of the predominant oxidation catalysts in Nature, the cytochrome P450 enzymes.

In this work, a new photochemical method to generate high-valent iron-oxo porphyrin models was discovered. As controlled by the electronic nature of porphyrin ligands, iron(IV)-oxo porphyrin radical cations (Compound I model) and iron(IV)-oxo porphyrin derivatives (Compound II model) were produced. These observations indicate that the photochemical reactions involve a heterolytic cleavage of O-Br in precursors to give a putative iron(V)-oxo intermediate, which might relax to …

Characterization Of The Rcf1 Q-X3-R-X-R-X3-Q Motif And Its Role In Supporting Oxidative Phosphorylation Enzyme Complexes, Joshua Garlich

Dissertations (1934 -)

Mitochondrial respiratory complexes are critical components of cellular energy production that require tight regulation to ensure optimal function. Rcf1 and Rcf2 are mitochondrial proteins that can physically associate with the yeast respiratory complexes III and IV, and the higher-ordered III-IV respiratory supercomplex that also contains the ADP/ATP translocase, AAC. Rcf1 can physically associate with both complex III and IV independently, and can be chemically crosslinked to AAC, indicating a close physical proximity to a predominant regulator of energy flux within the cell. It was therefore hypothesized that Rcf1, through its physical association with complexes III and IV, and its close …

Design Of Drug Nano-Carriers For Study Of Multidrug Resistance In Single Live Cells, Pavan Kumar Cherukuri

Chemistry & Biochemistry Theses & Dissertations

Multidrug resistance (MDR) exists in both prokaryotic and eukaryotic cells. MDR is responsible for ineffective treatment of a wide range of diseases, such as infections and cancer. The ATP-binding cassette (ABC) membrane transporters (efflux pumps) are one of the largest and most diverse super-families of membrane proteins found in all living organisms, ranging from bacteria to humans. All ABC transporters share a common structure of four core domains; two transmembrane domains (TMD) with variable sequence and topology and two nucleotide-binding domains (NBD) with conserved sequences. Conventional methods for the study of the efflux functions are radioactively labeled substrates and fluorescent …

Bioenergetics: Experimental Demonstration Of Excess Protons And Related Features, Haitham A. Saeed

Chemistry & Biochemistry Theses & Dissertations

Over the last 50 years, ever since the Nobel-prize work of Peter Mitchell’s Chemiosmotic theory, the question whether bioenergetics energy transduction occurs through localized or delocalized protons has been a controversial issue among scientists. Recently, a proton-electrostatics localization hypothesis was formulated which may provide a new and clear understanding of localized and delocalized proton-coupling energy transduction in many biological systems. The aim of this dissertation was to test this new hypothesis.

To demonstrate the fundamental behavior of localized protons in a pure water-membrane-water system in relation to the newly derived pmf equation, excess protons and excess hydroxyl anions were generated …

Identification And Characterization Of The Anticancer Potential Of Indigenous Medicinal Plants Of The Arabian Peninsula, Sameera Omar Mohammed Saeed Balhamar

Theses

Indigenous plant species historically used for their medicinal properties are a tremendous source for bringing newer and safer drugs to the market. A concerted effort is needed to characterize their medicinal potential and identify new molecules that could be exploited in modern medicine. The current study was undertaken to study the anticancer properties of several indigenous plants that are used by the local population of the Arabian Peninsula and beyond for various medicinal purposes. Towards this end, we acquired different plant extracts from five plants, namely Boswellia sacra (BS), Cleome droserifolia (CD), Teucrium muscatensis (TM), Orchadenus arabicus (OA), and Acredocarpus …

Therapeutic Targets For Alzheimer's Disease: Insights From In Vitro And In Vivo Models Of Inflammation, Magdalena J. Kiprowska

Dissertations, Theses, and Capstone Projects

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive neuronal loss that over the years spreads from the hippocampus to the neural cortex and impairs memory and cognitive functions. At the cellular level AD is linked to the presence of β-amyloid plaques and neurofibrillary tangles but despite decades of research little is known about their contribution to neurodegeneration and whether they are a cause or rather a result of the disease. It is well established that proteasome activity is impaired in AD brains and some studies suggest that this could be one of the initial factors leading to development …

Role Of Hnrnp A1 In The Regulation Of Hdm2 Gene Expression, Heriberto Moran

Dissertations, Theses, and Capstone Projects

hnRNP A1 is one of the most abundant and ubiquitously expressed member of hnRNPs (Heterogenous Nuclear Ribonucleoproteins) family of proteins that play multiple roles in gene expression by participating in major steps in the processing of nascent RNA transcripts 1. It is involved in mRNA biogenesis mechanisms such as the transcription, splicing, stability, export and translation of cellular and viral transcripts. The functions of hnRNP A1 extend to the processing of microRNAs, telomere maintenance and DNA repair 1. Our previous studies have shown that hnRNPA1 had reduced protein level and increased cytoplasmic accumulation in senescent human diploid fibroblasts 2,10. Our …

Specific Binding Affinity Of The Non-Catalytic Domain Of Eukaryotic Like Type Ib Topoisomerase Of Vaccinia Virus, Benjamin R. Reed

Dissertations, Theses, and Capstone Projects

Topoisomerases are ubiquitous proteins that alter supercoiling in double stranded DNA (dsDNA) during transcription and replication and. vaccinia and the closely related poxvirus variola virus, at 314 amino acids in length, encode the smallest of the type I topoisomerases(TopIB). TopIB is a two domain protein that recognizes the sequence 5’-T/CCCTT, cleaves at the 3’-end and relaxes supercoiling through rotation. The C-terminal domain (CTD) alone contains the catalytic activity and specificity. Deletion of the N-terminal domain results in a greatly reduced rate of relaxation and rapid dissociation. Biochemical data suggests that the N-terminal domain (NTD) is important for pre-cleavage binding and …

Molecular Analysis Of Ftsz-Ring Assembly In E. Coli Cytokinesis, Kuo-Hsiang Huang

Dissertations, Theses, and Capstone Projects

An essential first step in bacterial division is the assembly of a cytokinetic ring (Z-ring) formed by the tubulin-like FtsZ at midcell. The highly conserved core domain of FtsZ has been reported to mediate assembly of FtsZ polymers in vivo and in vitro. Species-specific differences in the FtsZ C-terminal domain such as the FtsZ CTV region and interactions with several modulatory proteins such as ZapC and ZapD, restricted to certain bacterial classes, also serve as key determinants of FtsZ protofilament bundling. Here, we characterize (i) the roles of the FtsZ CTV region in mediating both longitudinal and lateral interactions …

Tuning Into Toxins And Channels: The Characterizations Of Tv1 And A Human Cardiac Sodium Channel Voltage-Sensor Domain, Mohammed H. Bhuiyan

Dissertations, Theses, and Capstone Projects

In nature, peptide toxins are an abundant resource, produced both by marine and terrestrial organisms. A major target of these peptide toxins is the group of the highly important voltage-gated ion channels. Due to their high specificity and affinity, peptide toxins have been used for over a decade in discovery and characterization of voltage-gated ion channels. Although peptide toxins have been extensively characterized structurally, the structural characterization of eukaryotic voltage-gated sodium channels has seen much less progress, due to their large size and high hydrophobicity. Voltage-gated sodium channels play crucial roles in many physiological processes, and when these processes are …