Biochemistry, Biophysics, and Structural Biology Commons^™

Effects Of Macromolecular Crowding On Ferredoxin And Ferredoxin-Nadp+ Reductase Kinetics, Danielle Bautista, David W. Seybert Ph.D.

Undergraduate Research and Scholarship Symposium

Macromolecular crowding refers to the intrinsic occurrence of high concentrations of macromolecules found within cells, which may alter biomolecular properties of other macromolecules. Simulating these environments in vitro should enable a more accurate understanding of these modifications. We have employed in vitro studies to specifically examine the effects of macromolecular crowding on biological redox reactions. The iron-sulfur protein ferredoxin (Fdx) and the FAD-containing flavoprotein ferredoxin-NADP+ reductase (FNR), from Spinacea oleracea, were utilized as a model electron transfer system. Utilizing NADPH as a reductant, the Fdx-FNR complex reduces mammalian cytochrome c as a surrogate electron acceptor. Reduction of the heme moiety …

Discovery And Characterisation Of A Novel Toxin From Dendroaspis Angusticeps, Named Tx7335, That Activates The Potassium Channel Kcsa, Ivan O. Rivera-Torres, Tony B. Jin, Martine Cadene, Brian T. Chait, Sébastien F. Poget

Publications and Research

Due to their central role in essential physiological processes, potassium channels are common targets for animal toxins. These toxins in turn are of great value as tools for studying channel function and as lead compounds for drug development. Here, we used a direct toxin pull-down assay with immobilised KcsA potassium channel to isolate a novel KcsA-binding toxin (called Tx7335) from eastern green mamba snake (Dendroaspis angusticeps) venom. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulphide bonds that belongs to the three-finger toxin family, but with a unique modification …

Localisation And Protein-Protein Interactions Of The Helicobacter Pylori Taxis Sensor T1pd And Their Connection To Metabolic Functions, Wiebke Behrens, Tobias Schweinitzer, Jonathan L. Mcmurry, Christine Josenhans

Faculty Articles

The Helicobacter pylori energy sensor TlpD determines tactic behaviour under low energy conditions and is important in vivo. We explored protein-protein interactions of TlpD and their impact on TlpD localisation and function. Pull-down of tagged TlpD identified protein interaction partners of TlpD, which included the chemotaxis histidine kinase CheAY2, the central metabolic enzyme aconitase (AcnB) and the detoxifying enzyme catalase (KatA). We confirmed that KatA and AcnB physically interact with TlpD. While the TlpD-dependent behavioural response appeared not influenced in the interactor mutants katA and acnB in steady-state behavioural assays, acetone carboxylase subunit (acxC) mutant behaviour was altered. TlpD was …

Nonreplicative Dna Helicases Involved In Maintaining Genome Stability, Salahuddin Syed

USF Tampa Graduate Theses and Dissertations

Double-strand breaks and stalled forks arise when the replication machinery encounters damage from exogenous sources like DNA damaging agents or ionizing radiation, and require specific DNA helicases to resolve these structures. Sgs1 of Saccharomyces cerevisiae is a member of the RecQ family of DNA helicases and has a role in DNA repair and recombination. The RecQ family includes human genes BLM, WRN, RECQL4, RECQL1, and RECQL5. Mutations in BLM, WRN, and RECQL4 result in genetic disorders characterized by developmental abnormalities and a predisposition to cancer. All RecQ helicases have common features including a …

Evaluation Of Cytotoxicity Profile And Intracellular Localisation Of Doxorubicin-Loaded Chitosan Nanoparticles, Gabriele Dadalt Souto, Zeineb Farhane, Esen Efeoglu, Alan Casey, Jennifer Mcintyre, Hugh Byrne

Articles

In the emerging field of nanomedicine, targeted delivery of nanoparticle encapsulated active pharmaceutical ingredients (API) is seen as a potential significant development, promising improved pharmacokinetics and reduced side effects. In this context, understanding the cellular uptake of the nanoparticles and subsequent subcellular distribution of the API is of critical importance. Doxorubicin (DOX) was encapsulated within chitosan nanoparticles to investigate its intracellular delivery in A549 cells in vitro. Unloaded (CS-TPP) and doxorubicin-loaded (DOX-CS-TPP) chitosan nanoparticles were characterised for size (473±41 nm), polydispersity index (0.3±0.2), zeta potential (34±4 mV), drug content (76±7 µM) and encapsulation efficiency (95±1%). The cytotoxic response to …

Genetically Induced Production Of Secondary Metabolites In Bacillus Megaterium, Majie C. Foster, Jay L. Brewster, P. Matthew Joyner

Seaver College Research And Scholarly Achievement Symposium

Historically, the most important source of new antibiotic drug leads has been small organic compounds made by bacteria. Many antibiotics have been developed into pharmaceutical agents from these molecules (often called secondary metabolites) produced by soil bacteria. Bacillus species are soil bacteria known for producing various antimicrobials including gramicidin, bacitracin, surfactin, and others. Bacillus megaterium is a widely used model gram positive bacterium. Although there has been extensive research on this organism, little is known about its secondary metabolites. We hypothesized that the production of secondary metabolites in this organism could be induced by replacing promoters controlling the expression of …

Axonal Transport And Life Cycle Of Mitochondria In Parkinson's Disease Model, Hyun Sung

Open Access Dissertations

In neurons, normal distribution and selective removal of mitochondria are essential for preserving compartmentalized cellular function. Parkin, an E3 ubiquitin ligase associated with familial Parkinson’s disease, has been implicated in mitochondrial dynamics and removal. However, it is not clear how Parkin plays a role in mitochondrial turnover in vivo, and whether the mature neurons possess a compartmentalized Parkin-dependent mitochondrial life cycle. Using the live Drosophila nervous system, here, I investigate the involvement of Parkin in mitochondrial dynamics; organelle distribution, morphology and removal. Parkin deficient animals displayed less number of axonal mitochondria without disturbing organelle motility behaviors, morphology and metabolic state. …

A Unified Analytic Framework For Prioritization Of Non-Coding Variants Of Uncertain Significance In Heritable Breast And Ovarian Cancer, Eliseos J. Mucaki, Natasha G. Caminsky, Ami M. Perri, Ruipeng Lu, Alain Laederach, Matthew Halvorsen, Joan H. M. Knoll, Peter K. Rogan

Biochemistry Publications

Background

Sequencing of both healthy and disease singletons yields many novel and low frequency variants of uncertain significance (VUS). Complete gene and genome sequencing by next generation sequencing (NGS) significantly increases the number of VUS detected. While prior studies have emphasized protein coding variants, non-coding sequence variants have also been proven to significantly contribute to high penetrance disorders, such as hereditary breast and ovarian cancer (HBOC). We present a strategy for analyzing different functional classes of non-coding variants based on information theory (IT) and prioritizing patients with large intragenic deletions.

Methods

We captured and enriched for coding and non-coding variants …

Preliminary Results On Organization On The Court, Physical And Technical Performance Of Brazilian Professional Futsal Players: Comparison Between Friendly Pre-Season And Official Match, Luiz Henrique Palucci Vieira, Sera N. Doğramaci, Ricardo Augusto Barbieri, Fabio Milioni, Felipe Arruda Moura, Vitor Luiz De Andrade, Guilherme Manna Cesar, Paulo Roberto Pereira Santiago

Athletic Performance Research

The main aim of this study was to verify possible differences between a friendly pre-season match (FM) and an official in-season match (OM) regarding physical, technical, and organizational performances of a professional Brazilian futsal team. Ten professional futsal athletes participated in this study. The matches were monitored with video cameras (30 Hz) and athlete trajectories obtained with automatic tracking. The values obtained for distance covered per minute, percentage of distance covered at moderate intensity, team coverage area, spread, passes, possessions, ball touches and successful passes per minute were greater for the OM than FM. On the contrary, percentage of distance …

Mutant Study Of Sinorhizobium Meliloti Proline Utilization A (Puta), Jacob E. Wilkinson, John J. Tanner, Donald F. Becker

UCARE: Research Products

The purpose of this project is to purify and characterize the reaction kinetics of mutant versions the enzyme Proline Utilization A (PutA) in Sinorhizobium meliloti. The enzyme catalyzes the first step in proline metabolism. It has two active sites. The first is proline dehydrogenase (PRODH) which converts proline to pyrroline-5-carboxylate (P5C). The second is P5C dehydrogenase (P5CDH) which converts P5C to glutamate. Although many bacterial organisms have PutA, there are still significant interspecies variations, resulting in an entire family of PutA enzymes. The main difference is the length of the amino acid sequence. This affects the protein’s structure or …

The Nmr Solution Structure And Function Of Rpa3313: A Hypothetical Protein From R. Palustris, Austin J. Lowe, Jonathan Catazaro, Cheryl Arrowsmith, Robert Powers

UCARE: Research Products

Protein function elucidation often relies heavily on amino acid sequence analysis and other bioinformatics approaches. The reliance is further extended to structure homology modeling for ligand docking and protein-protein interaction mapping. However, sequence analysis of RPA3313 exposes a large, unannotated class of hypothetical proteins mostly from the Rhizobiales order. In the absence of sequence and structure information, further functional elucidation of this class of proteins has been significantly hindered. A high quality NMR structure of RPA3313 reveals that the protein forms a novel split βαβ fold with a conserved ligand binding pocket between the first β-strand and the N-terminus of …

Genetically Incorporated Vinyl Sulfide For Various Bioorthogonal Reactions, Carly Faller, Xin Shang, Jiantao Guo

UCARE: Research Products

No abstract provided.

Analysis Of Histone Deacetylase Involvement In Pseudomonas Syringae-Triggered Chromatin Changes, Jennifer Wies, Victoria Shum, Karin V. Van Dijk

UCARE: Research Products

I had the great privilege of working with Dr. Karin van Dijk, through UCARE, researching the specific pathways through which histone deacetylation occurs in plants during the 2015-2016 academic year. My first year conducting research helped me develop my technical skills and learn about experimental protocols and how to conduct them. I would like to continue my research during the 2016-2017 academic year and now that I have a year of research experience, be able to further develop my skills and learn more about my research project.

Background

Pseudomonas syringae is a bacterial pathogen that is well known for causing …

Hair Analysis In Motor Vehicles, Jaclyn E. Humrich, Larry Barksdale

UCARE: Research Products

The purpose of this research was to examine the frequency of human and animal hair in motor vehicles in the Lincoln, NE area. We were looking specifically at animal hair to see if there is a significant difference in the distribution of animal hair in pet-owner and non pet-owner vehicles. Finding a significantly high distribution of animal hair in pet-owner vehicles would lead us to believe that an association could be made between a suspect owning an animal and their presence at a crime scene.

Our null hypothesis is that there is no significance in the distribution of cat/dog hairs …

Binding Of Oxaliplatin And Its Analogs With Dna Nucleotides At Variable Ph And Concentration Levels, Rippa Sehgal

Masters Theses & Specialist Projects

Oxaliplatin is one of the three FDA-approved platinum anticancer drugs and considered a third generation drug, discovered after the first generation drug cisplatin and second generation drug carboplatin. It is known to react with proteins and DNA nucleotides in the body. Reaction with DNA occurs primarily at guanosine residues and secondarily at adenine residues for oxaliplatin and other platinum drugs. We have previously studied oxaliplatin and an analog with additional steric hindrance in the amine ligand and found that the analog had different reactivity with methionine. Now, we have prepared oxaliplatin and its three analogs Pt(Me2dach)(ox), Pt(en)(ox) and Pt(Me4en)(ox) and …

Section Abstracts: Structural Biology, Biochemistry, And Biophysics

Virginia Journal of Science

Abstracts of the Structural Biology, Biochemistry, and Biophysics Section for the 94th Annual Virginia Academy of Science Meeting, May 18-20, 2016, at University of Mary Washington, Fredericksburg, VA.

Fipronil Promotes Adipogenesis Via Ampkα-Mediated Pathway In 3t3-L1 Adipocytes, Quancai Sun, Jeremy Yang, Kyong-Sup Yoon, John M. Clark, Yeonhwa Park

SIUE Faculty Research, Scholarship, and Creative Activity

Emerging evidence suggests that organochlorine, organophosphorus and neonicotinoid insecticide exposure may be linked to the development of obesity and type 2 diabetes. However, there is no knowledge of the potential influence of fipronil, which belongs to the phenylpyrazole chemical family, on obesity. Thus, the goal of this study was to determine the role of fipronil in adipogenesis using 3T3-L1 adipocytes. Fipronil treatment, at 10 mM, increased fat accumulation in 3T3-L1 adipocytes as well as promoted key regulators of adipocyte differentiation (CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor gamma-γ), and key regulators of lipogenesis (acetyl-CoA carboxylase and fatty acid synthase). The …

Defining The Roles Of Serine Palmitoyltransferase-Interacting Proteins In The Regulation Of Sphingolipid Homeostasis, Athen N. Kimberlin

Department of Biochemistry: Dissertations, Theses, and Student Research

Sphingolipids are major structural components of the plasma membrane and endomembrane system. Research suggests that sphingolipids are involved with the formation of lipid microdomains, also known as lipid rafts, which may help to organize proteins within the membrane and may be important for membrane trafficking. Aside from their structural roles in membranes, sphingolipids and their metabolic products have been implicated in several cellular signaling responses like programmed cell death (PCD). Because of this, maintenance of sphingolipid homeostasis is critical for eukaryotic cell growth and development. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is the primary regulatory …

Synthesis And Mechanistic Investigations Of Transition Metal Complexes And Ligands For Chemotherapeutic Applications, Jimmie Lee Bullock Jr.

Chemistry & Biochemistry Theses & Dissertations

Transition metal complexes have played a critical role in antitumor therapy with many complexes incorporating platinum, ruthenium, and lanthanum having been investigated in preclinical and clinical trials. The best known transition metal therapeutic is cisplatin, which is utilized in nearly 50% of all cancer therapies, despite its significant toxic side effects. The toxic side effects of current FDA approved platinum-based chemotherapeutics are often overlooked due to the “special status” granted to these drugs due to their ability to fight, what is often considered an incurable disease with life expectancies often measured in months. Oncology drug development has therefore now focused …

Biophysical Studies Of The Allosteric Regulatory Mechanism Of Syk Tandem Sh2 Domains Interacting With Immunoreceptor Tyrosine-Based Activation Motifs, Chao Feng

Open Access Dissertations

The non-receptor spleen tyrosine kinase (Syk) is an important player in signal transduction from immunoreceptors to various downstream targets. It is widely expressed in both haematopoietic and epithelial cells. Syk disorder is closely related with many inflammatory and autoimmune diseases, as well as cancers.

Syk associates with immunoreceptors through its tandem SH2 domains (tSH2), which contain two SH2 domains connected by interdomain A. The association of Syk with immunoreceptors is regulated by Y130 phosphorylation in interdomain A. The unphosphorylated tSH2 can bind with the doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the cytoplasmic domains of immunoreceptors with very high …

Learning The Abc's Of Ribose Transport Using Biophysical Methods, Satchal K. Erramilli

Open Access Dissertations

ATP-binding cassette transporters comprise a large superfamily of proteins that are involved in a variety of biological phenomenon, from bacterial metabolism to cellular homeostasis, antigen-presentation, and drug resistance. These proteins are implicated in a variety of clinically relevant phenomenon, including the human diseases cystic fibrosis, macular degeneration, and cancer. Understanding their structure-function can guide therapeutics and contribute to our overall understanding of these biological phenomena.

This study focuses on understanding the motor protein of the bacterial ribose ABC transporter in the context of transport. This complex is required for the uptake of the nucleotide precursor, ribose. Using biophysical methods, we …

Characterization Of Cu-Rich Aggregates In Neurogenic Niches Of The Rodent Brain By X-Ray Fluorescence Microscopy, Brendan T. Sullivan

Open Access Dissertations

Copper is an essential element in the brain playing several critical roles ranging from neurotransmitter synthesis to ATP production. As Cu is typically present in micromolar concentrations and has a spatially capricious distribution in the brain, determining concentrations has historically been challenging. X-ray fluorescence microscopy (XRF) offers excellent spatial resolution (down to 30~nm) and detection limits (sub parts per million), making it an excellent tool for analyzing metal distributions in the brain. Using XRF, it is demonstrated that Cu-rich aggregates with concentrations in the hundreds of millimolar are present in the subventricular zone of rats and mice. As the subventricular …

Mechanistic Characterization Of Acetic Acid Resistance Enzymes Of Acetobacer Aceti, Jesse R. Murphy

Open Access Dissertations

Acetobacter aceti (A. aceti) is a Gram-negative, acidophilic bacterium that is used for the industrial production of acetic acid from ethanol. Oxidation of ethanol by membrane-bound oxidoreductases provides energy for A. aceti and the production of high concentrations of acetic acid is an effective defense mechanism. Acetic acid diffuses through cell membranes at low pH and effectively kills many bacteria, including E. coli, at low millimolar concentrations. The ability of A. aceti to thrive in molar concentrations of acetic acid is partially due to the twin subjects of this thesis, the acetic acid resistance factors AarA (citrate synthase, …

Molecular Analysis Confirming The Introduction Of Nile Crocodiles, Crocodylus Niloticus Laurenti 1768 (Crocodylidae), In Southern Florida, With An Assessment Of Potential For Establishment, Spread, And Impacts., Michael R. Rochford, Kenneth L. Krysko, Frank J. Mazzotti, Matthew W. Shirley, Mark W. Parry, Joseph A. Wasilewski, Jeffrey S. Beauchamp, Christpher R. Gillette, Edward F. Metzger Iii, Michiko A. Squires, Louis A. Somma

Papers in Herpetology

The state of Florida, USA, has more introduced herpetofauna than any other governmental region on Earth. Four species of nonnative crocodilians have been introduced to Florida (all since 1960), one of which is established. Between 2000–2014 we field-collected three nonnative crocodilians in Miami-Dade County, Florida, and one in Hendry County, Florida. We used DNA barcoding and molecular phylogenetics to determine species identification and native range origin. Also, we described diet, movement, and growth for one crocodile. Our molecular analyses illustrated that two of the crocodiles we collected are most closely related to Nile Crocodiles (Crocodylus niloticus) from South Africa, suggesting …

Comparison Of Interactive Voice Response (Ivr) With Paper Administration Of Instruments To Assess Functional Status, Sexual Function, And Quality Of Life In Elderly Men, Rc Rosen, Aj Stephens-Shields, Gr Cunningham, D Cifelli, D Cella, Jt Farrar, E Barrett-Connor, Ce Lewis, M Pahor, X Hou, Pj Snyder

Faculty, Staff and Students Publications

PURPOSE: Patient-reported outcome (PRO) measures are essential for assessing subjective patient experiences. Interactive voice response (IVR) data collection provides advantages for clinical trial design by standardizing and centralizing the assessment. Prior to adoption of IVR as a mode of PRO administration in the Testosterone Trials (TTrials), we compared IVR to paper versions of the instruments to be used. METHODS: IVR versions of the FACIT-Fatigue scale and Psychosexual Daily Questionnaire, Question 4, were developed. In one pilot study, IVR versions of these scales were compared to paper versions in 25 men = 65 years at each of two clinical sites. In …

Water Soluble Cationic Porphyrin Sensor For Detection Of Hg2+, Pb2+, Cd2+, And Cu2+, Christopher Orr, Miranda Uherek, Matibur Zamadar

Undergraduate Research Conference

Here we report the sensing properties of the aqueous solution of mesotetra(N-methyl-4-pyridyl)porphine tetrachloride (1) for simultaneous detection of toxic metal ions by using UV-vis spectroscopy. Cationic porphyrin 1 displayed different electronic absorptions in UV-vis region upon interacting with Hg2+, Pb2+, Cd2+, and Cu2+ ions in neutral water solution at room temperature. Quite interestingly, the porphyrin 1 showed that it can function as a single optical chemical sensor and/ or metal ion receptor capable of detecting two or more toxic metal ions, particularly, Hg2+, Pb2+, and Cd2+ ions coexisting in a water sample. Porphyrin 1 in an aqueous solution provides a …

Antimicrobial Properties Of Isomers Of Benzofuranylethanol, Nick Cheatwood, Jessica Bronstad, Michele Harris

Undergraduate Research Conference

A biotransformaDon reacDon is a chemical conversion of a substance into a desired product with the aid of whole, living cells containing the necessary enzymes. In addiDons to living cells, other substances, such as vegetable strips, will also catalyze biotransformaDon reacDons. Advantages of biotransformaDon reacDons include the following: the ability to recycle material, the use of less hazardous chemicals, the ability to compost vegetable strips, the use of inexpensive materials that are locally available, and the ability to produce a single isomer. The biotransformaDon reacDon for the conversion of benzofuranyl methyl ketone (BMK) to (-)-benzofuranylethanol (BMA) using carrot strips as …

Biophysical Investigation Of Amyloid Formation And Their Prion-Like Self-Replication, Mentor Mulaj

USF Tampa Graduate Theses and Dissertations

Growth and deposition of amyloid fibrils, polymers of proteins with a cross beta-sheet structure, are associated with a significant number of human pathologies including Alzheimer’s disease, Parkinson’s disease, prion diseases, type II diabetes, and senile systematic or dialysis-related amyloidoses. The broader objective of my research is to identify the basic mechanisms regulating nucleation and growth of amyloid fibrils. There is increasing evidence that amyloid formation may proceed along at least two distinct assembly pathways for the formation of rigid fibrils. One pathway involves the nucleated polymerization of the characteristic rigid fibrils from partially denatured monomers and the other proceeds via …

Cost-Effectiveness Of Using A Gene Expression Profiling Test To Aid In Identifying The Primary Tumour In Patients With Cancer Of Unknown Primary., M B Hannouf, E Winquist, S M Mahmud, M Brackstone, S Sarma, G Rodrigues, P Rogan, J S Hoch, G S Zaric

Biochemistry Publications

We aimed to investigate the cost-effectiveness of a 2000-gene-expression profiling (GEP) test to help identify the primary tumor site when clinicopathological diagnostic evaluation was inconclusive in patients with cancer of unknown primary (CUP). We built a decision-analytic-model to project the lifetime clinical and economic consequences of different clinical management strategies for CUP. The model was parameterized using follow-up data from the Manitoba Cancer Registry, cost data from Manitoba Health administrative databases and secondary sources. The 2000-GEP-based strategy compared to current clinical practice resulted in an incremental cost-effectiveness ratio (ICER) of $44,151 per quality-adjusted life years (QALY) gained. The total annual-budget …

Novel Techniques In Chemical Ecology To Examine Life Histories In Fishes, Orian Tzadik

USF Tampa Graduate Theses and Dissertations

Diet and movements in fishes are often logistically challenging to study. Trace element and stable isotope analyses have advanced these fields considerably, but are still constrained by methodological impediments, such as the tendency towards lethal sampling. Studying endangered fishes is particularly challenging as representative samples are difficult to obtain. However, the information gained from such studies is often critical to the recovery of endangered fishes as knowledge of life history attributes has the potential to greatly influence the success of management strategies.

I tested the viability of using fin rays in fishes as a non-lethal approach to study diet and …