Biochemistry Commons^™

Structure Function Studies Of Hiv-1 Protease, Bradley James Keusch

Wayne State University Theses

HIV-1 is the causative agent of the devastating human disease Acquired Immunodeficiency Syndome (AIDS). While much progress has been made over the past two decades, HIV-1 remains a major global health concern. HIV-1 protease is 99-amino acid homodimer aspartyl protease that is essential to the life cycle of HIV. This has rendered it an attractive and very successful drug target. However, due to the high error rate of the HIV -1 reverse transcriptase, drug resistance mutations in the protease can develop very rapidly in some patients, rendering current protease inhibitors (one of the main classes of drug in common antiretroviral …

Beta-Sheet Forming Peptides By Design : Control Of Folding And Applications, Gaius Takor

Legacy Theses & Dissertations (2009 - 2024)

The focus of the present research is the synthesis of polypeptides for the study of protein folding and misfolding and for the development of novel polypeptide-based optical antennas in nanotechnology. It is hypothesized that simple polypeptides can be used as models to mimic in vivo folding of globular proteins. Desired repetitive polypeptides were genetically encoded and expressed in E. coli using conventional methods and characterized using a variety of spectroscopic (including circular dichroism (CD), deep UV resonance Raman (DUVRR), UV-vis and fluorescence) and microscopic (atomic force microscopy (AFM) and transmission electron microscopy (TEM)) techniques. The polypeptides predominantly formed bilayer, fibrillar …

Investigation Of Enzymatically Synthesized Glycogen As A Novel Nanodendrimer For Therapeutic Delivery, Sarah Ann Engelberth

Legacy Theses & Dissertations (2009 - 2024)

The field of medicinal chemistry is ever expanding, designing and discovering new therapeutic strategies. Oftentimes, it is challenging for these therapeutics to undergo clinical translation due to ineffective administration or unwanted toxicity in vivo. As such, drug delivery vehicles are designed to overcome these hurdles, allowing for delivery to the site of action by improving biodistribution, protecting therapeutic cargo, and decreasing toxicity. The work presented here aims to investigate a naturally-derived carbohydrate nanodendrimer, enzymatically synthesized glycogen (ESG) for drug delivery. This nontoxic, highly-branched, glucose-based structure has interior void volumes to allow for cargo encapsulation as well as a large density …

The Chemistry Of New Garlic-Derived Organosulfur Compounds And The Molecular Basis Of Olfaction, Berenice Dethier

Legacy Theses & Dissertations (2009 - 2024)

Garlic is a very popular condiment that has been used around the world for centuries. It is also a source of a remarkably extensive range of organosulfur compounds, whose chemistry is the focus of this thesis. The central reaction in formation of these compounds is the enzymatic cleavage of alk(en)yl cysteine sulfoxides by alliinases, which leads to sulfenic acids. The latter can then undergo condensation and rearrangement into various organosulfur compounds. Three aspects of the chemistry of garlic have been investigated in this thesis.

Study Of Protein-Protein Interaction By Using In-Cell Nmr In Human Cells, Asma Salem M Aldousary

Legacy Theses & Dissertations (2009 - 2024)

We developed a new technology to study protein-protein interaction in mammalian cells. This technology is based high resolution of Nucleic Magnetic Resonance (NMR) spectroscopy. Using this technology we studied interaction between the receptor for advanced glycation endproducts (RAGE). RAGE- is a multiligand receptor of immunoglobulin receptor family that is activated by a multitude of ligands. Activation of RAGE results in signal transduction that leads to the inflammatory response implicated in the complications of Diabetes. RAGE is an emergent drug target that has been explored for the variety of pathologist including cancers, neurological disorders, inflammatory disease, and diabetes. and Intracellular effector …

A Survey Of The Current Drug Screening Techniques To Obtain Rational Design And Study Drug-Target Interactions, Stephen Dansereau

Legacy Theses & Dissertations (2009 - 2024)

Different techniques have been developed over the years for the purpose of studying proteins and understanding their functions. Early techniques typically employed bioluminescence or fluorescence such such as the firefly protein luciferase and the jellyfish green fluorescent protein (GFP), respectively, to localize proteins within the cell. X-ray crystallography has also provided valuable structural details of many different proteins in vitro. Yet, nuclear magnetic resonance (NMR) spectroscopy offers the most realistic insight into proteins' physiologic structures and how proteins function in their native, cellular environments.

The Use Of Ion Mobility Spectrometry-Mass Spectrometry For The Structural Elucidation Of Progressively Larger Nucleic Acids, Jennifer Lynn Lippens

Legacy Theses & Dissertations (2009 - 2024)

The biomedical community and pharmaceutical industry have come to rely in ever

The Gammaturc Nanomachine Mechanism And Future Applications, Timothy Riehlman

Legacy Theses & Dissertations (2009 - 2024)

The complexity and precision of the eukaryotic cell’s cytoskeletal network is unrivaled by any man-made systems, perfected by billions of years of evolution, mastering elegant processes of self-assembly, error correction, and self-repair. Understanding the capabilities of these networks will have important and far reaching applications in human medicine by aiding our understanding of developmental processes, cellular division, and disease mechanisms, and through biomimicry will provide insights for biosynthetic manufacturing at the nanoscale and across scales. My research utilizes cross species techniques from Human to the model organism of Fission Yeast to investigate the structure and mechanisms of the g-tubulin ring …

Effects Of High Voltage Nanosecond Electric Pulses On Eukaryotic Cells (In Vitro): A Systematic Review, Tina Batista Napotink, Matej Reberšek, P. Thomas Vernier, Babara Mali, Damijan Miklavčič

Bioelectrics Publications

For this systematic review, 203 published reports on effects of electroporation using nanosecond high-voltage electric pulses (nsEP) on eukaryotic cells (human, animal, plant) in vitro were analyzed. A field synopsis summarizes current published data in the field with respect to publication year, cell types, exposure configuration, and pulse duration. Published data were analyzed for effects observed in eight main target areas (plasma membrane, intracellular, apoptosis, calcium level and distribution, survival, nucleus, mitochondria, stress) and an additional 107 detailed outcomes. We statistically analyzed effects of nsEP with respect to three pulse duration groups: A: 1–10 ns, B: 11–100 ns and C: …

Contribution Of Cx3cr1/Fractalkine (Cx3cl1) Axis To The Progression Of Non-Small Cell Lung Cancer (Nsclc), Rhonda Prince

Wayne State University Theses

A thesis is presented on the characterization of the contribution of the chemokine axis, CX_3CR1/Fractalkine, to the progression of Non-Small Cell Lung Cancer (NSCLC). Cancer is the second leading cause of death in the United States, and Lung cancer is one of the four leading causes of the disease. Carcinoma cells, unlike their normal counterparts, gain the ability to express chemokine receptors. Understanding if/how this gain of expression contributes to the progression of cancer could provide a lucrative therapeutic target. Goals of the study include investing if the NSCLC cell line A549, an adenocarcinoma cell line, express the chemokine receptor …

Characterization Of The Yeast Cysteine Desulfurase Complex Within The Mitochondrial Fe-S Cluster Biogenesis, Dulmini Pabasara Barupala

Wayne State University Dissertations

Disrupted iron homeostasis within the human body materializes as various disorders. Pathophysiology of many of them relates to iron induced oxidative damage to key cellular components caused by iron accumulation within the tissues. Pertaining to the growing occurrence, cost of patient care and devastating burden associated with these diseases, the call for understanding the role of iron homeostasis within these disorders becomes inevitable. Being an abundant iron containing cofactor, the role of Fe-S clusters in cellular iron homeostasis is indisputable in the case of Friedreich’s ataxia, a disease caused by a deficiency in the protein frataxin that is indispensable during …

A Disease Spectrum For Itpa Variation: Advances In Biochemical And Clinical Research, Nicholas E. Burgis

Chemistry and Biochemistry Faculty Publications

Human ITPase (encoded by the ITPA gene) is a protective enzyme which acts to exclude noncanonical (deoxy) nucleoside triphosphates ((d)NTPs) such as (deoxy)inosine 5′-triphosphate ((d)ITP), from (d)NTP pools. Until the last few years, the importance of ITPase in human health and disease has been enigmatic. In 2009, an article was published demonstrating that ITPase deficiency in mice is lethal. All homozygous null offspring died before weaning as a result of cardiomyopathy due to a defect in the maintenance of quality ATP pools. More recently, a whole exome sequencing project revealed that very rare, severe human ITPA mutation results in early …

Characterization Of Hemerythrin-Like Protein Rv2633c, Michelle D. Cherne

Honors Undergraduate Theses

Hemerythrin-like protein Rv2633c is a small 18 kDa protein that is expressed in Mycobacterium tuberculosis (Mtb). Sequence analysis of Rv2633c predicts the presence of a hemerythrin-like domain, which binds dioxygen using a µ-oxo-bridge (Fe-O-Fe), rather than a heme group. Though it is noticeably upregulated during macrophage infection and during in vitro acidification, the role of Rv2633c in Mtb survival has yet to be elucidated. This project aims to characterize the function of Rv2633c by studying the in vitro response of the recombinant protein to conditions present in the macrophage lysosome, such as reduced oxygen levels or the …

Aptameric Sensors: In Vitro Selection Of Dna That Binds Bromocresol Purple, Derek B. Miller

Honors Undergraduate Theses

Aptamers being used as sensors is an emerging field that has capabilities of being tomorrow’s diagnostic tools. As aptameric sensors have become more popular, their visualization systems have been limited. The majority of today’s aptameric sensors require expensive machinery such as a fluorometer in order to visualize results. We propose a system that will cut the need for instrumentation and be detected via the naked eye. With the selection of an aptamer to bind the pH indicating dye bromocresol purple (BCP) this may be achieved. When rendered active, the binding towards BCP will facilitate a color change from yellow to …

Variance Of The Amyloid Beta Peptide As A Metric For The Diagnosis Of Alzheimer's Disease, Christina Beckett

Theses and Dissertations--Medical Sciences

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder associated with aging. AD is by far the best understood and most studied neurodegenerative disease. Substantial advances have been made over the last decade, however it is debatable how much closer we are to a clinically useful therapy. A long standing goal in the AD field has been to improve the accuracy of early detection, with the assumption that the ability to intervene earlier in the disease process will lead to a better clinical outcome. Major facets of this effort have been the continued development and improvement of AD biomarkers, with …

Elucidating Proteasome Catalytic Subunit Composition And Its Role In Proteasome Inhibitor Resistance, Kimberly C. Carmony

Theses and Dissertations--Pharmacy

Proteasome inhibitors bortezomib and carfilzomib are FDA-approved anticancer agents that have contributed to significant improvements in treatment outcomes. However, the eventual onset of acquired resistance continues to limit their clinical utility, yet a clear consensus regarding the underlying mechanisms has not been reached.

Bortezomib and carfilzomib are known to target both the constitutive proteasome and the immunoproteasome, two conventional proteasome subtypes comprising distinctive sets of catalytic subunits. While it has become increasingly evident that additional, ‘intermediate’ proteasome subtypes, which harbor non-standard mixtures of constitutive proteasome and immunoproteasome catalytic subunits, represent a considerable proportion of the proteasome population in many cell …

Towards Elucidation Of The Mechanism Of Biological Nanomotors, Zhengyi Zhao

Theses and Dissertations--Pharmacy

Biological functions such as cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging, and cell entry all involve biomotor-driven DNA translocation. In the past, the ubiquitous biological nanomotors were classified into two categories: linear and rotation motors. In 2013, we discovered a third type of biomotor, revolving motor without rotation. The revolving motion is further found to be widespread among many biological systems. In addition, the detailed sequential action mechanism of the ATPase ring in the phi29 dsDNA packaging motor has been elucidated: ATP binding induces a conformational entropy alternation of ATPase …

Molecular And Biochemical Signaling Underlying Arabidopsis-Bacterial/Virus/Fungal Interactions, Mohamed H. El-Shetehy

Theses and Dissertations--Plant Pathology

Systemic acquired resistance (SAR) is a form of inducible defense response triggered upon localized infection that confers broad-spectrum disease resistance against secondary infections. Several factors are known to regulate SAR and these include phenolic phytohormone salicylic acid (SA), phosphorylated sugar glycerol-3-phosphate (G3P), and dicarboxylic acid azelaic acid (AzA). This study evaluated a role for free radicals nitric oxide (NO) and reactive oxygen species (ROS) in SAR. Normal accumulation of both NO and ROS was required for normal SAR and mutations preventing NO/ROS accumulation and/or biosynthesis compromised SAR. A role for NO and ROS was further established using pharmacological approaches. Notably, …

Repositioning Of Drugs Using Open-Access Data Portal Dtome: A Test Case With Probenecid (Review), Mohammad U. Ahmed, Dylan J. Bennett, Tze-Chen Hsieh, Barbara B. Doonan, Saba Ahmed, Joseph M. Wu

NYMC Faculty Publications

The one gene-one enzyme hypothesis, first introduced by Beadle and Tatum in the 1940s and based on their genetic analysis and observation of phenotype changes in Neurospora crassa challenged by various experimental conditions, has witnessed significant advances in recent decades. Much of our understanding of the association between genes and their phenotype expression has benefited from the completion of the human genome project, and has shown continual transformation guided by the effort directed at the annotation and characterization of human genes. Similarly, the idea of one drug‑one primary disease indication that traditionally has been the benchmark for the labeling and …

Synthesis And Biochemical Evaluation Of 3-Phenoxy-1,4-Diarylazetidin-2-Ones As Tubulin-Targeting Antitumor Agents, Thomas F. Greene, Shu Wang, Lisa M. Greene, Seema M. Nathwani, Jade K. Pollock, Azizah M. Malebari, Thomas Mccabe, Brendan Twamley, Niamh O'Boyle, Daniela M. Zisterer, Mary J. Meegan

Articles

Structure-activity relationships for a series of 3-phenoxy-1,4-diarylazetidin-2-ones were investigated leading to the discovery of a number of potent antiproliferative compounds, including trans-4-(3-hydroxy-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (78b) and trans-4-(3-amino-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (90b). X-ray crystallography studies indicate the potential importance of the torsional angle between the 1-phenyl ‘A’ ring and 4-phenyl ‘B’ ring for potent antiproliferative activity, and that a trans configuration between the 3-phenoxy and 4-phenyl rings is generally optimal. These compounds displayed IC₅₀ values of 38 nM and 19 nM respectively in MCF-7 breast cancer cells, inhibited the polymerization of isolated tubulin in vitro, disrupted the microtubular …

Mitochondrial Function Assessed By 31p Mrs And Bold Mri In Non-Obese Type 2 Diabetic Rats, Yuchi Liu, Xunbai Mei, Jielei Li, Nicola Lai, Xin Yu

Electrical & Computer Engineering Faculty Publications

The study aims to characterize age-associated changes in skeletal muscle bioenergetics by evaluating the response to ischemia-reperfusion in the skeletal muscle of the Goto-Kakizaki (GK) rats, a rat model of non-obese type 2 diabetes (T2D). ³¹P magnetic resonance spectroscopy (MRS) and blood oxygen level-dependent (BOLD) MRI was performed on the hindlimb of young (12 weeks) and adult (20 weeks) GK and Wistar (control) rats. ³¹P-MRS and BOLD-MRI data were acquired continuously during an ischemia and reperfusion protocol to quantify changes in phosphate metabolites and muscle oxygenation. The time constant of phosphocreatine recovery, an index of mitochondrial oxidative capacity, …

Computational Studies Of Paradifluorobenzene Cations And Hydrogen Cyanide Molecule, John C. Rowe Iv

Honors Theses

Polycyclic aromatic hydrocarbons (PAHs) are hypothesized to comprise a significant portion of interstellar carbon identified from the Diffuse Interstellar Bands (DIBs). Gas phase cation-molecule reactions between compounds that previously existed as weakly bound species provides the best explanation for the emergence of PAHs in the interstellar medium (ISM). In this work, we use computational methods to characterize one of these weakly-bound systems. We discovered that intramolecular charge distribution (calculated using the Natural Population Analysis) dictate the complexes formed between a paradifluorobenzene cation and either one or two HCN molecules. Additionally, the relative stabilities of the complexes and the binding energies …

Characterization Of Catecholamine Receptors And Transporters In Murine Macrophages, Elizabeth Gonye

Honors Theses

Macrophages are a critical part of the immune response. When circulating monocytes move into tissues they differentiate int macrophages to mount the first line of defense against pathogens.

Calcium (Ii) Catalyzed Nitrone Additions, Elizabeth A. Congdon

Honors Theses

Calcium(II) complexes have be shown to be successful catalysts for nitrone reactions. The addition of n-­‐methyl and n-­‐phenyl nitrones to donor-­‐acceptor cyclopropanes was achieved with calcium triflate (Ca(OTf). Differentially substituted tetrahydro-­‐1,2-­‐oxazines were synthesized in good to excellent yields. Calcium triflate was also found to catalyze the addition of silyl enol ethers to n-­‐phenyl nitrones along the Mukaiyama-­‐Mannich addition pathway. B-­‐amino carbonyls were synthesized from a variety of substituted nitrones. Bulky and cyclic silyl enol ethers were also found to be reactive, the products of which were isolated in good to excellent yields.

Impact Of Vector Range Expansion On Pathogen Transmission Dynamics Of Lyme Disease In Southwestern Virginia, Bishan Bhattarai

Honors Theses

Blacklegged ticks, Ixodes scapularis is the primary vector of Lyme disease in eastern United States. Borrelia Burgdorfei, the etiological agent of Lyme disease is transferred by ticks of Ixodes species. In recent years, its congener, Ixodes affinis has been expanding its range northwards from its southern population. We were interested in studying how the introduction of this new vector affected the interaction between the pathogen genotype and the host. We hypothesized that differential host use by I. affinis and I. scapularis would partly explain observed differences in B. burgdorferi infection prevalence and genotypic structure in southeastern Virginia. The result …

Pluralistic And Stochastic Gene Regulation: Examples, Models And Consistent Theory, Elisa N. Salas, Jiang Shu, Matyas F. Cserhati, Donald P. Weeks, Istvan Ladunga

Department of Biochemistry: Faculty Publications

We present a theory of pluralistic and stochastic gene regulation. To bridge the gap between empirical studies and mathematical models, we integrate pre-existing observations with our meta-analyses of the ENCODE ChIP-Seq experiments. Earlier evidence includes fluctuations in levels, location, activity, and binding of transcription factors, variable DNA motifs, and bursts in gene expression. Stochastic regulation is also indicated by frequently subdued effects of knockout mutants of regulators, their evolutionary losses/gains and massive rewiring of regulatory sites. We report wide-spread pluralistic regulation in ≈800 000 tightly co-expressed pairs of diverse human genes. Typically, half of ≈50 observed regulators bind to both …

Structure And Biological Activity Of A D3g Mutation In The Human Mitochondrial Chaperonin Hsp60, Jihui Li

Open Access Theses & Dissertations

Proteins are essential components in all cellular activities, including signal transduction, gene expression regulation, immune response, structural support and many others. Following their synThesis on the ribosome, proteins need to fold into their precise three-dimensional structure to obtain functionality. If they fold incorrectly they are prone to aggregation and cause a wide range of neurodegenerative diseases. Some proteins are capable of folding without assistance but others need help from protein complexes known as chaperonins. Chaperonins are a family of proteins that assemble into barrel-shaped cages the create a protected chamber for misfolded proteins to refold into their biologically active state. …

Investigation Of Dna Interaction With Bis-Cationic Imidazolium Salts, Kyle Daily 6440928

Williams Honors College, Honors Research Projects

Imidazolium salt compounds have shown promise in vitro against non-small cell lung cancer (NSCLC) lines, but the exact mechanism of activity these compounds is still unknown. Due to their planar aromatic structure, we hypothesized that our bis-cation imidazolium salt series could act through DNA intercalation similar to known intercalators ethidium bromide, acridine orange, and echinomycin. Here, we tested bis-cation imidazolium salts with linkers of 2, 4, 10, and 12 carbons for DNA intercalation via viscosity and fluorescent intercalator displacement (FID) assays. Despite some solubility issues that may have decreased results, we found that our butyl and dodecyl linked compounds had …

Lipid-Protein Interactions Via Fluorescence Quenching In Small Unilamellar Vesicles, Talia Kordahi

Williams Honors College, Honors Research Projects

Studying peptide interactions with model membranes have aided in understanding protein-lipid interactions more clearly. In this report, fluorescent small unilamellar vesicles (SUVs) of a known composition were made. Two peptides, RND1 and MARCKS, were also fluorescently labeled and added to a solution of SUVs. The interactions between these were observed via fluorescence quenching, read out with a spectrofluorimeter. In addition, two environments of buffer were tested: 1X PBS and 0.1X PBS. After obtaining data, it was concluded that MARCKS quenched the SUVs more than RND1. This could be due to the presence of more lysine amino acids in MARCKS, which …

The Role Of Tgfβ1 And Ep Receptors In The Differentiation Of Normal And Diseased Lung Fibroblasts, Madison A. Hexter

Williams Honors College, Honors Research Projects

Fibroblasts, and their differentiation into myofibroblasts, are important in the pathology of several lung diseases including asthma and idiopathic pulmonary fibrosis (IPF). The differentiation process is dependent on several factors, such as soluble factors like transforming growth factor beta 1 (TGFβ1) and mechanical signals such as matrix stiffness. Additionally, the soluble arachidonic acid metabolite Prostaglandin E₂ (PGE₂) prevents TGFβ1 induced differentiation of fibroblasts to myofibroblasts, but the molecular mechanism is not completely understood¹⁵. Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive ion channel, has previously been implicated in the differentiation of cardiac fibroblasts^12,13. …