Biochemistry Commons^™

Expression Of Insulin Responsive Genes In Insulin Resistant Conditions, And The Effect Of Selenium On Gene Expression, David L. Ruff

Masters Theses

Chronically high blood glucose levels lead to many problems, such as insulin resistance, the hallmark of Type II diabetes. Increased flux through the hexosamine biosynthesis pathway is one mechanism by which high glucose as well as glucosamine has been shown to induce insulin resistance. This study tests the effects of glucosamine induced insulin resistance on insulin regulation of the metabolic genes glucose-6-phosphate dehydrogenase (G6PDH) and fatty acid synthase (FAS) as well as insulin responsive proteins tribbles homolog (TRIB3) and sterol regulatory element binding protein (SERBP-1c) 1c.

Selenium, a micronutrient has been shown to be an effective insulin mimetic in Type …

Kdac8 Substrate Specificity Quantified By A Biologically Relevant, Label-Free Deacetylation Assay, Tasha B. Toro, Terry J. Watt

Faculty and Staff Publications

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. These modifications regulate a variety of biological processes and are reversed by the lysine deacetylase (KDAC) family of enzymes. Despite the known prevalence and importance of acetylation, the details of KDAC substrate recognition are not well understood. While several methods have been developed to monitor protein deacetylation, none are particularly suited for identifying enzyme-substrate pairs of label-free substrates across the entire family of lysine deacetylases. Here, we present a fluorescamine-based assay which is more biologically relevant than existing methods and amenable …

Investigation Of Pathways For Complex Sphingolipid Biosynthesis In Arabidopsis Thaliana (L.) Heynh, Kyle Luttgeharm

Department of Biochemistry: Dissertations, Theses, and Student Research

Sphingolipids are essential components of eukaryote membranes. The ceramide backbone of complex sphingolipids is composed of an 18 carbon Long Chain Base (LCB) bound to a 16-26 carbon fatty acid (FA) through an amide linkage. Ceramides are synthesized de novo from a free LCB and fatty acyl coA by ceramide synthase (sphingosine N-acyl transferase, EC 2.3.1.24) which can be inhibited by the fungal mycotoxin Fumonisin B₁. Arabidopsis thaliana contains three ceramide synthases denoted LOH1, LOH2, and LOH3 that have previously been hypothesized to have unique substrate preferences that control the final sphingolipid composition, different susceptibilities to …

Structural And Functional Studies Of The Papain-Like Protease 2 From Mouse Hepatitis Virus, Yafang Chen

Open Access Dissertations

Our goal is to establish a system to investigate how the deubiquitinating (DUB) and deISGylating activities of coronavirus (CoV) papain-like protease domains (PLPs) are involved in virus immune evasion. To this end, we chose PLP2 from mouse hepatitis virus (MHV) as our target of study because MHV has historically served as a model system for the study of CoVs, and it has undeniable advantage of ease in culturing in comparison to human coronaviruses.

It is reported here the expression and purification of a region of MHV nsp3 that contains the catalytic core of the PLP2 domain and its neighboring domains. …

Nanoparticle Conversion To Biofilms: In Vitro Demonstration Using Serum-Derived Mineralo-Organic Nanoparticles, Tsui-Yin Wong, Hsin-Hsin Peng, Cheng-Yeu Wu, Jan Martel, David M. Ojcius, Fu-Yung Hsu, John Ding-E. Young

All Dugoni School of Dentistry Faculty Articles

Aims: Mineralo-organic nanoparticles (NPs) detected in biological fluids have been described as precursors of physiological and pathological calcifications in the body. Our main objective was to examine the early stages of mineral NP formation in body fluids. Materials & methods: A nanomaterial approach based on atomic force microscopy, dynamic light scattering, electron microscopy and spectroscopy was used. Results: The mineral particles, which contain the serum proteins albumin and fetuin-A, initially precipitate in the form of round amorphous NPs that gradually grow in size, aggregate and coalesce to form crystalline mineral films similar to the structures observed in calcified human arteries. …

Co-Treatment With Conjugated Linoleic Acid And Nitrite Modulates Mitochondrial Respiration And Electron Transport Chain Activity In Vivo And Attenuates Mitochondrial Dysfunction During Cardiac Injury., Patrick Van Hoose

Electronic Theses and Dissertations

Cardiovascular disease and subsequent complications, such as myocardial infarction (MI), is the leading cause of death within the United States. Clinical intervention to reduce cardiac damage following myocardial ischemia is limited. Polyunsaturated fatty acids have been linked to an overall beneficial effect on cardiovascular health and function. Conjugated linoleic acid (cLA) is an 18:2 unsaturated fatty acid found within the diet in ruminant meat and dairy products. The cis-9 trans-11 isomer of cLA is the most prominent isomer within the diet and cLA has been linked to favorable outcomes in numerous disease states. The mechanism mediating the effects of cLA …

Normal Glycolytic Enzyme Activity Is Critical For Hypoxia Inducible Factor-1a Activity And Provides Novel Targets For Inhibiting Tumor Growth, Geoffrey Grandjean Phd

Dissertations and Theses (Open Access)

Normal Glycolytic Enzyme Activity is Critical for Hypoxia Inducible Factor-1α Activity and Provides Novel Targets for Inhibiting Tumor Growth

By Geoffrey Grandjean

Advisory Professor: Garth Powis, D. Phil

Unique to proliferating cancer cells is the observation that their increased need for energy is provided by a high rate of glycolysis followed by lactic acid fermentation in a process known as the Warburg Effect, a process many times less efficient than oxidative phosphorylation employed by normal cells to satisfy a similar energy demand ^[1]. This high rate of glycolysis occurs regardless of the concentration of oxygen in the cell and …

Optimizing A Luciferase-Based Tool For Studying The Effects Of Fatty Acid Desaturase 7 On Singlet Oxygen Accumulation In Arabidopsis Thaliana, Abeer Muhammedali Alnasrawi

Graduate Theses and Dissertations

In plants, reactive oxygen species (ROS) are generated as a byproduct of normal metabolism, as well as in response to adverse conditions such as light stress, extreme temperatures, and exposure to pests and pathogens. Singlet oxygen (1O2) is a ROS that is formed during photosynthesis in photosystem II (PSII) of the chloroplasts. Levels of 1O2 and other ROS are tightly controlled in healthy plants, but some studies suggest that levels of fatty acid desaturase (FAD) activity in Arabidopsis thaliana can influence constitutive and/or stress-responsive ROS accumulation. In this study, a luciferase-based reporter gene that is selectively stimulated by 1O2 (AAA-ATPase: …

Production And Harvest Of Microalgae In Wastewater Raceways With Resource Recycling, Alexander Colin Roberts

Master's Theses

Microalgae can be grown on municipal wastewater media to both treat the wastewater and produce feedstock for algae biofuel production. However the reliability of treatment must be demonstrated, as well as high areal algae productivity on recycled wastewater media and efficient sedimentation harvesting. This processes was studied at pilot scale in the present research.

A pilot facility was operated with nine CO₂-supplemented raceway ponds, each with a 33-m² surface area and a 0.3-m depth, continuously from March 6, 2013 through September 24, 2014. The ponds were operated as three sets of triplicates with two sets continuously fed …

Actin Filaments Target The Oligomeric Maturation Of The Dynamin Gtpase Drp1 To Mitochondrial Fission Sites, Wei-Ke Ji, Anna L. Hatch, Ronald A. Merrill, Stefan Strack, Henry N. Higgs

Dartmouth Scholarship

While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, …

Assessment Of Learning Gains In A Flipped Biochemistry Classroom, Deanna Dahlke Ojennus

Chemistry Faculty Scholarship

The flipped classroom has become an increasingly popular pedagogical approach to teaching and learning. In this study, learning gains were assessed in a flipped biochemistry course and compared to gains in a traditional lecture. Although measured learning gains were not significantly different between the two courses, student perception of learning gains did differ and indicates a higher level of satisfaction with the flipped lecture format.

Case 62: Glycolysis, The Pentose Phosphate Pathway, And The Citric Acid Cycle In Tumorigenesis, Dr. Kathleen Cornely

Chemistry & Biochemistry Case Studies

Hypoxia-inducible factors are transcription factors that mediate the cellular response to hypoxia (low cellular oxygen concentration) by enhancing or suppressing the transcription of specific genes involved in metabolic pathways.

Eosinophils From Murine Lamina Propria Induce Differentiation Of Naïve T Cells Into Regulatory T Cells Via Tgf-Β1 And Retinoic Acid, Hong-Hu Chen, Ai-Hua Sun, David M. Ojcius, Wei-Lin Hu, Yu-Mei Ge, Xu'ai Lin, Lan-Juan Li, Jian-Ping Pan, Jie Yan

All Dugoni School of Dentistry Faculty Articles

Treg cells play a crucial role in immune tolerance, but mechanisms that induce Treg cells are poorly understood. We here have described eosinophils in lamina propria (LP) that displayed high aldehyde dehydrogenase (ALDH) activity, a rate-limiting step during all-trans retinoic acid (ATRA) synthesis, and expressed TGF-β1 mRNA and high levels of ATRA. Co-incubation assay confirmed that LP eosinophils induced the differentiation of naïve T cells into Treg cells. Differentiation promoted by LP eosinophils were inhibited by blocked either TGF-β1 or ATRA. Peripheral blood (PB) eosinophils did not produce ATRA and could not induce Treg differentiation. These data identifies LP eosinophils …

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

USF Tampa Graduate Theses and Dissertations

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

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

USF Tampa Graduate Theses and Dissertations

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

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

In Situ Measurements And Modeling Of Reactive Trace Gases In A Small Biomass Burning Plume, M. Muller, B. Anderson, A. Beyersdorf, J. H. Crawford, G. Diskin, P. Eichler, A. Fried, F. N. Keutsch, T. Mikoviny, K. L. Thornhill, J. G. Walega, A. J. Weinheimer, M. Yang, Robert Yokelson, A. Wisthaler

Chemistry and Biochemistry Faculty Publications

An instrumented NASA P-3B aircraft was used for airborne sampling of trace gases in a plume that had emanated from a small forest understory fire in Georgia, USA. The plume was sampled at its origin for deriving emission factors and followed ~ 13.6 km downwind for observing chemical changes during the first hour of atmospheric aging. The P-3B payload included a proton-transfer-reaction time-of-flight mass spectrometer (PTR-ToF-MS), which measured non-methane organic gases (NMOGs) at unprecidented spatio-temporal resolution (10m/0.1 s). Quantitative emission data are reported for CO₂, CO, NO, NO₂, HONO, NH₃ and 16 NMOGs (formaldehyde, methanol, …

Inquiry-Based Experiments For Large-Scale Introduction To Pcr And Eestriction Enzyme Digests, Kelly E. Johanson, Terry J. Watt

Faculty and Staff Publications

Polymerase chain reaction and restriction endonuclease digest are important techniques that should be included in all Biochemistry and Molecular Biology laboratory curriculums. These techniques are frequently taught at an advanced level, requiring many hours of student and faculty time. Here we present two inquiry-based experiments that are designed for introductory laboratory courses and combine both techniques. In both approaches, students must determine the identity of an unknown DNA sequence, either a gene sequence or a primer sequence, based on a combination of PCR product size and restriction digest pattern. The experimental design is flexible, and can be adapted based on …

Chemical Inhibition Of Fatty Acid Absorption And Cellular Uptake Limits Lipotoxic Cell Death, Constance Ahowesso, Paul N. Black, Nipun Saini, David Montefusco, Jessica Chekal, Chrysa Malosh, Craig W. Lindsley, Shaun R. Stauffer, Concetta Dirusso

Department of Biochemistry: Faculty Publications

Chronic elevation of plasma free fatty acid (FFA) levels is commonly associated with obesity, type 2 diabetes, cardiovascular disease and some cancers. Experimental evidence indicates FFA and their metabolites contribute to disease development through lipotoxicity. Previously, we identified a specific fatty acid transport inhibitor CB16.2, a.k.a. Lipofermata, using high throughput screening methods. In this study, efficacy of transport inhibition was measured in four cell lines that are models for myocytes (mmC2C12), pancreatic ß-cells (rnINS-1E), intestinal epithelial cells (hsCaco-2), and hepatocytes (hsHepG2), as well as primary human adipocytes. The compound was effective in inhibiting uptake with IC₅₀s between 3 …

Detection And Characterization Of Mineralo-Organic Nanoparticles In Human Kidneys, Tsui-Yin Wong, Cheng-Yeu Wu, Jan Martel, Cheng-Wei Lin, Fu-Yung Hsu, David M. Ojcius, Paul Y. Lin, John Ding-E. Young

All Dugoni School of Dentistry Faculty Articles

Ectopic calcification is associated with various human diseases, including atherosclerosis, cancer, chronic kidney disease, and diabetes mellitus. Although mineral nanoparticles have been detected in calcified blood vessels, the nature and role of these particles in the human body remain unclear. Here we show for the first time that human kidney tissues obtained from end-stage chronic kidney disease or renal cancer patients contain round, multilamellar mineral particles of 50 to 1,500 nm, whereas no particles are observed in healthy controls. The mineral particles are found mainly in the extracellular matrix surrounding the convoluted tubules, collecting ducts and loops of Henle as …

Hirsutella Sinensis Mycelium Attenuates Bleomycin-Induced Pulmonary Inflammation And Fibrosis In Vivo, Tsung-Teng Huang, Hsin-Chih Lai, Yun-Fei Ko, David M. Ojcius, Ying-Wei Lan, Jan Martel, John Ding-E. Young, Kowit-Yu Chong

All Dugoni School of Dentistry Faculty Articles

Hirsutella sinensis mycelium (HSM), the anamorph of Cordyceps sinensis, is a traditional Chinese medicine that has been shown to possess various pharmacological properties. We previously reported that this fungus suppresses interleukin-1β and IL-18 secretion by inhibiting both canonical and non-canonical inflammasomes in human macrophages. However, whether HSM may be used to prevent lung fibrosis and the mechanism underlying this activity remain unclear. Our results show that pretreatment with HSM inhibits TGF-β1–induced expression of fibronectin and α-SMA in lung fibroblasts. HSM also restores superoxide dismutase expression in TGF-β1–treated lung fibroblasts and inhibits reactive oxygen species production in lung epithelial cells. Furthermore, …

Two-Ply Channels For Faster Wicking In Paper-Based Microfluidic Devices, Conor K. Camplisson, Kevin M. Schilling, William L. Pedrotti, Howard A. Stone, Andres W. Martinez

Chemistry and Biochemistry

This article describes the development of porous two-ply channels for paper-based microfluidic devices that wick fluids significantly faster than conventional, porous, single-ply channels. The two-ply channels were made by stacking two single-ply channels on top of each other and were fabricated entirely out of paper, wax and toner using two commercially available printers, a convection oven and a thermal laminator. The wicking in paper-based channels was studied and modeled using a modified Lucas–Washburn equation to account for the effect of evaporation, and a paper-based titration device incorporating two-ply channels was demonstrated.

The Generation, Exploitation And Future Of Induced Pluripotent Stem Cells, Jacob Steenwyk

Scholarly Undergraduate Research Journal at Clark (SURJ)

The foundational advancements of John Gurdon and Shinya Yamanaka have improved understanding of dedifferen- tiation of cells to a pluripotent state. The seminal discovery established a novel system to study disease pathogenesis, drug screening, and toxicity, as well as sprouted the new field of regenerative medicine. In this article, the method- ology to obtain dedifferentiated cells, known as induced pluripotent stem (iPS) cells, subsequent validation, and application of which are reviewed. The experiments investigated here aim to demonstrate the capacity of iPS cells to replace the ethically-gray human embryonic cells by developing human livers and viable, healthy animals. It is …

Biochemical Effects Of Meditation: A Literature Review, William C. Daube, Charles E. Jakobsche

Scholarly Undergraduate Research Journal at Clark (SURJ)

Meditation is an activity that can help reduce stress and anxiety from daily life as well as help cultivate overall feel- ings of peacefulness, relaxation, and contentment. This review describes several studies that have been used to assess how meditation can influence the body at the molecular level. The presented results focus on small-molecule metab- olites, which are broadly defined as naturally-produced molecules that weigh less than approximately 1000 Da. The results show that meditation can significantly affect hormones and neurotransmitters such as cortisol, dehydroepi- androstrone, serotonin, melatonin, and epinephrine. Some common and modern experimental techniques that are relevant to …

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

USF Tampa Graduate Theses and Dissertations

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

Cobalt-Centred Boron Molecular Drums With The Highest Coordination Number In The Cob16− Cluster, Ivan A. Popov, Tian Jian, Gary V. Lopez, Alexander I. Boldyrev, Lai-Sheng Wang

Chemistry and Biochemistry Faculty Publications

The electron deficiency and strong bonding capacity of boron have led to a vast variety of molecular structures in chemistry and materials science. Here we report the observation of highly symmetric cobalt-centered boron drum-like structures of CoB16⁻, characterized by photoelectron spectroscopy and ab initio calculations. The photoelectron spectra display a relatively simple spectral pattern, suggesting a high symmetry structure. Two nearly degenerate isomers with D8d(I) and C4v (II) symmetries are found computationally to compete for the global minimum. These drum-like structures consist of two B8 rings sandwiching a cobalt atom, which has the highest …

Temporal Variation In Optical Properties Of Chromophoric Dissolved Organic Matter (Cdom) In Southern California Coastal Waters With Nearshore Kelp And Seagrass, Catherine D. Clark, Warren J. De Bruyn, Paige Aiona

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Optical properties of chromophoric dissolved organic matter (CDOM) were measured in surf zone waters in diurnal field studies at a Southern California beach with nearshore kelp and seagrass beds and intertidal plant wrack. Absorption coefficients (aCDOM(300 nm)) ranged from 0.35 m21 to 3.7 m21 with short-term variability<1 h, increases at ebb and flood tides and higher values (6 m21) during an offshore storm event. Spectral slopes (S) ranged from 0.0028 nm21 to 0.017 nm21, with higher values after the storm; S was generally inversely correlated with aCDOM(300 nm). 3-D excitation–emission matrix spectra (EEMs) for samples with lower S values had humic-type peaks associated with terrestrial material (A, C), marine microbial material (M) and protein peaks, characteristic of freshly produced organic material. Samples with high S values had no or reduced protein peaks, consistent with aged material. Fluorescent indexes (f450/f500 >2.5, BIX>1.1) were consistent with microbial aquatic sources. Leachates of senescent kelp and seagrass had protein and humic-type EEM peaks. After solar simulator irradiation (4 h), protein peaks rapidly photochemically degraded, humic-type peak C increased in intensity and peak M disappeared. Optical characteristics of kelp leachates were most similar to field samples, …

Multi-Dimensional Glycan Microarrays With Glyco-Macroligands, Satya Nandana Narla, Huan Nie, Yu Li, Xue-Long Sun

Chemistry Faculty Publications

Glycan microarray has become a powerful high-throughput tool for examining binding interactions of carbohydrates with the carbohydrate binding biomolecules like proteins, enzymes, antibodies etc. It has shown great potential for biomedical research and applications, such as antibody detection and profiling, vaccine development, biomarker discovery, and drug screening. Most glycan microarrays were made with monovalent glycans immobilized directly onto the array surface via either covalent or non-covalent bond, which afford a multivalent glycans in two dimensional (2D) displaying. A variety of glyco-macroligands have been developed to mimic multivalent carbohydrate-protein interactions for studying carbohydrate-protein interactions and biomedical research and applications. Recently, a …

Characterization Of Inhibitors Of Fatty Acid Transport Protein-2 In Cell And Animal Models, Nipun Saini

Department of Biochemistry: Dissertations, Theses, and Student Research

Obesity is correlated with insulin resistance and elevated levels of glucose, triglycerides and free fatty acids in blood. This affects overall metabolism and leads to disease. In the obese state, fat also accumulates in non-adipose tissue including liver, muscle and pancreas, where it can lead to cellular dysfunction and death. Currently, only a limited number of drugs are available to combat obesity and it is clear that new drugs, which more narrowly target the metabolic pathways involved, are required. Fatty Acid Transport Proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme …

Ligand Frameworks For Transition-Metal Complexes That Model Metalloenzyme Active, Denan Wang

Dissertations (1934 -)

Advances in the field of biomimetic inorganic chemistry require the design of sophisticated ligand frameworks that reflect the amazing complexity of metalloenzyme active sites. For instance, most active sites feature extensive hydrogen-bonding interactions between ligands bound to the metal center (the “first” coordination sphere) and nearby units in the outer (or “second”) sphere. Since these interactions modify the structural and electronic properties of the active sites, a number of inorganic chemists have sought to design ligands that permit outer-sphere functional groups to interact with first-sphere donors. This dissertation describes our contribution to these broader efforts to model the second coordination …

Image-Charge Detection €“ Novel Instrumentation And Applications, Brandon Lee Barney

Theses and Dissertations

Image-charge detection is an analytical technique in which a highly-charged particle is detected by the magnitude of the image current that it generates in a detecting electrode. This current is represented as a voltage between the charged particle and the sensing electrode. It is a single particle detection method, ideal for the analysis of large, variable mass particles such as biological cells. Some of the physical properties of Bacillus subtilis spores were explored using different applications of image-charge detection. B. subtilis is a gram-negative spore-forming bacteria that has been shown to exhibit extremophile behavior. The particular extremophile behavior that was …