Biochemistry Commons^™

Clpxp Degradation System In Escherichia Coli, A Study Of Its Energy Sources And Its Applications In Managing The Expression Levels Of Targeted Membrane And Soluble Proteins, Thilini Abeywansha

Theses and Dissertations--Chemistry

ClpXP is an Escherichia coli protease that carries out energy-dependent intracellular proteolysis. In recent years, this system has been widely studied due to its importance as protein regulatory machinery and a virulence factor. Protein substrates of ClpXP contain degrons with a specific protein sequence. SsrA tag is one of the five degrons known to designate proteins for ClpXP degradation. SsrA is an 11 amino acid peptide added to the C-terminus of nascent polypeptide chains translated from aberrant messenger RNAs lacking stop codons via a process called trans-translation.

ClpXP was known to targets only cytosolic proteins with degrons until recently, …

Investigation Of Multidrug Efflux Transporter Acrb In Escherichia Coli: Assembly, Degradation And Dynamics, Prasangi Irosha Rajapaksha

Theses and Dissertations--Chemistry

The Resistant Nodulation Division (RND) super family member, tripartite AcrA-AcrB-TolC efflux pump, is a major contributor in conferring multidrug-resistance in Escherichia coli. The structure of the pump complex, and drug translocation by functional rotation mechanism have been widely studied. Despite of all these data, the dynamics of the assembly process of the pump and AcrB during functional rotation in the process of drug efflux remains poorly understood. My thesis focuses on understanding the pump assembly process, dynamics of AcrB in functional rotation mechanism, and also investigate the mechanism of degradation of AcrB facilitated by a C-terminal ssrA tag.

In the …

Characterization Of Potential Anti-Infective Agents By Targeting Burkholderia Pseudomallei And Escherichia Coli Ispf, Dakota Lane Grote

Graduate Research Theses & Dissertations

With the likelihood of contracting an antibiotic resistant infection increasing, the need for novel anti-infective agents is critical. The development of novel anti-infective agents benefits from the identification of new target pathways necessary for the survival of the pathogen. One such target is the methylerythritol phosphate (MEP) pathway, responsible for the synthesis of the isoprenoid precursors isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) in bacteria and other pathogenic organisms. The MEP pathway is an attractive target because it is specific to a wide range of pathogenic organisms, while humans have a separate pathway, the mevalonate (MVA) pathway, for the biosynthesis …

Development Of Fluorescence Microscopy Approaches To Study Subcellular Protein Transport And Enzymatic Activity, Anchal Singh

Masters Theses

Understanding the subcellular localization of proteins and their activity is important in understanding their normal function in eukaryotic cells. Fluorescence cellular imaging techniques can selectively and sensitively visualize subcellular biochemistry. Using this approach, two different methods were employed in this thesis. The first focused on studying protein import into peroxisome and the other on monitoring the activity of an endoplasmic reticulum (ER)-localized enzyme, human carboxylesterase 1 (CES1).

Peroxisomes are mainly known as the center for long chain fatty acid b-oxidation as well as the production and detoxification of hydrogen peroxide. Proteins which are needed in the peroxisomes are encoded in …

Xylan-Derived Carbon Nutrient Acquisition By Caulobacter Crescentus, Rhoda Ekuama Inkoom

Masters Theses

Bacterial decomposition of xylan and uptake of xylan-derived carbon compounds require a combination of enzymes, proteins, and complex molecular mechanisms. Bacteria have evolved molecular mechanisms for its utilization depending on its environment and source of xylan. Caulobacter crescentus, a Gram-negative bacterium which lives in an aquatic oligotrophic environment, exhibits asymmetric division which is an adaptive feature for its nutrient poor habitat. Although aromatic hydrocarbon and cellulose-derived mono-, di- and oligosaccharide utilization has been reported in literature, its xylan-derived carbon compound utilization is yet to be characterized. In this study, C. crescentus utilizes xylan-derived monomers arabinose, xylose and acetate as …

Developing Synthetic Strategies For Multifaceted Applications Of Stable Gold-Based Complexes, Randall Tyler Mertens

Theses and Dissertations--Chemistry

Development of stable gold-based complexes has been a rapidly advancing field due to the popularity of gold complexes, particularly for use in biomedical research and catalytic transformations. Given that auranofin, a gold(I) complex with FDA approval for the treatment of rheumatoid arthritis is used in the clinic, the development of stable gold-based molecules of clinical relevance is urgently needed. Herein are reported, synthetic strategies used for the development of new classes of gold(I) and gold(III) complexes for advancement in mitochondrial modulation for use as chemotherapeutics as well as application to gold catalysis due to the unique geometry of complexes presented …

Flavin Modification And Redox Tuning In The Bifurcating Electron Transfer Flavoprotein From Rhodopseudomonas Palustris: Two Arginines With Different Roles, Nishya Mohamed-Raseek

Theses and Dissertations--Chemistry

Electron bifurcation is considered as a third fundamental mode of energy conservation mechanism in addition to two well-known mechanisms, substrate level phosphorylation and Oxidative phosphorylation, in electron bifurcation endergonic and exergonic redox reactions are coupled. The newly discovered flavin based electron bifurcation in electron transfer flavoproteins (ETFs) helps to reduce low potential ferredoxin, which provides electrons to drive biologically demanding reactions such as atmospheric dinitrogen fixation in diazotroph and methane production in methanogens.

Current research demonstrates the capacity for electron bifurcation in the Rhodopseudomonas palustris ETF (RpalETF) system. RpalETF contains two chemically identical but functionally different FADs: …

Tet1-Mediated Dna Hydroxymethylation Regulates Adult Remyelination In Mice, Sarah Moyon, Rebecca Frawley, Damien Marechal, Dennis Huang, Katy L.H. Marshall-Phelps, Linde Kegel, Boguslawa Sadowski, Yong-Hui Jiang, David A. Lyons, Wiebke Möbius, Patrizia Casaccia

Advanced Science Research Center

The mechanisms regulating myelin repair in the adult central nervous system (CNS) are unclear. Here, we identify DNA hydroxymethylation, catalyzed by the Ten-Eleven- Translocation (TET) enzyme TET1, as necessary for myelin repair in young adults and defective in old mice. Constitutive and inducible oligodendrocyte lineage-specific ablation of Tet1 (but not of Tet2), recapitulate this age-related decline in repair of demyelinated lesions. DNA hydroxymethylation and transcriptomic analyses identify TET1-target in adult oligodendrocytes, as genes regulating neuro-glial communication, including the solute carrier (Slc) gene family. Among them, we show that the expression levels of the Na+/K+/Cl− transporter, SLC12A2, are …

Elucidation Of The Structure Of Supramolecular Polymorphs In Peptide Nanofibres Using Raman Spectroscopy, Sian Sloan-Dennison, Ayala Lampel, Eileen Raßlenberg, Rein V. Ulijn, Ewen Smith, Karen Faulds, Duncan Graham

Advanced Science Research Center

Peptide fibre formation via molecular self-assembly is a key step in a range of cellular processes and increasingly considered as an approach to produce supramolecular biomaterials. We previously demonstrated the self-assembly of the tripeptide lysine-dityrosine (KYY) as a substrate for the formation of proton-conducting melanin-like materials. Point based Raman scattering is one of several techniques which were used to characterise the secondary structure of the KYY nanofibre but as is often the case with this type of fibre, the spectra are rather complex and in addition there were variations in intensity between samples making interpretation difficult. Using Raman mapping we …

Development Of Linked-Domain Protein Inhibitors Of The E2-Conjugating Enzyme Ube2d, Anneroos E. Nederstigt

University of the Pacific Theses and Dissertations

In most eukaryotic organisms, the ubiquitination pathway is one of the most important and versatile signaling systems in use. It is integral to processes such as protein degradation and homeostasis, DNA repair cell cycle regulation, signaling and regulation, epigenetics, and many more. Ubiquitin (Ub) is a short polypeptide of 8.6 kDa, 76 residues that functions as a reversible post-translation modification (PTM). It furthermore contains 7 different lysine residues (K6, K11, K27, K29, K33, K48, K63), all of which can form isopeptide linkages with one another to link individual Ub moieties to form unique polyUb chains onto substrates. The type of …

Optimizing Dna Extraction From Microorganisms Living In Wind Cave, Allison Warming

Honors Program Theses

Wind Cave National Park in South Dakota is home to many creatures, from the giant buffalo to microscopic single-celled organism. This study focused on the single-celled organisms living in Wind Cave. A total of 81 samples were collected and grown on culture media. The goal of this project was to determine which commercially available kit would yield the highest amount of purified DNA from the samples cultured from the cave. Following the protocols provided by the manufacturer, a control sample as well as samples from three plates had their DNA isolated and purified using seven commercially available DNA purification kits. …

In Silico And In Vitro Tudies For Identification Of Novel Egfr Tyrosine Kinase Inhibitors, Duangjai Todsaporn

Chulalongkorn University Theses and Dissertations (Chula ETD)

Epidermal growth factor receptor (EGFR) has been identified as a promising target in the treatment of non-small cell lung cancer (NSCLC). Mutations, including L858R/T790M double and L858R/T790M/C797S triple mutations, in the TK domain of EGFR have been linked to the severity of several cancers, especially NSCLC. A combination of comprehensive molecular modeling and in vitro kinase inhibition assay was used to unravel the mutational effects of EGFR on the susceptibility of five commercial drugs (erlotinib, gefitinib, afatinib, dacomitinib, and osimertinib) from each generation of EGFR in comparison with the wild-type EGFR. The binding affinity of five-FDA-approved drugs towards the L858R/T790M …

Neuroprotective Effect Of Passiflora Edulissims Against Amyloid Beta-Induced Neurotoxicity, Shuqin Cao

Chulalongkorn University Theses and Dissertations (Chula ETD)

Alzheimer’s disease (AD) is a common and devastating disease characterized by pathological aggregations of beta-amyloid (Aβ) plaques extracellularly, and Tau tangles intracellularly. While our understandings of the aetiologies of AD have greatly expanded over the decades, there is no drug available to stop disease progression. This study involves two parts. First, we demonstrate the potential of P. edulis pericarp (PP) extract in protecting against Aβ-mediated neurotoxicity in mammalian cells and Caenorhabditis elegans models of AD. We show PP protects against memory deficit, neuronal loss, and promotes longevity in the Aβ model of AD via stimulation of mitophagy, the selective cellular …

Substrate Binding Mechanism Of Glycerophosphodiesterase Towards Pesticides And Improvement Of Stability By Mutational Analysis, Nayana Bhat

Chulalongkorn University Theses and Dissertations (Chula ETD)

Molecular dynamics studies have been providing a great number of atomistic details of biological systems to understand enzymatic activity and underlying reaction mechanisms. Herein, molecular dynamic (MD) simulations were employed to study the binding mechanisms of organophosphate pesticides towards two metallohydrolases, i.e., glycerophosphodiesterase (GpdQ) and methyl parathion hydrolase (MPH). OPH are the class of enzymes which breaks down harmful organophosphates, including the pesticides and warfare agents into less harmful byproducts. Hence, these enzymes are excellent candidates for bioremediation. In the present study, various nonspecific organophosphate pesticides were docked into GpdQ and MPH enzymes to construct the pesticide/protein complex for performing …

Functional Characterization Of Rice Malate Synthase In Arabidopsis Responding To Salt Stress, Supisara Thanabut

Chulalongkorn University Theses and Dissertations (Chula ETD)

Salt stress is one of the major abiotic stresses that seriously limit plant growth and development and can lead to a significant reduction in crop yield, especially rice. Salt stress affects plants via ionic toxicity and osmotic stress. Osmotic stress results in a reduction of water absorption ability. Ionic toxicity causes Na+ toxicity, which disrupts photosynthesis, protein synthesis, and enzyme activity. The previous study of KDML105 rice transcriptome profile and qRT-PCR revealed that rice malate synthase (OsMS) encoding malate synthase, which is one of the key enzymes in glyoxylate cycle was up-regulated under salt stress, so OsMS may involve in …

In Silico And In Vitro Screening Of Newly Designed Compounds Against Coxsackivirus A16 And Enterovirus A71, Amita Sripattaraphan

Chulalongkorn University Theses and Dissertations (Chula ETD)

Outbreaks of hand, foot, and mouth disease (HFMD) occur around the world. It is caused by the Coxsackievirus-A16 (CV-A16) and Enterovirus-A71 (EV-A71) that belong to the Enterovirus genus. Unfortunately, neither an anti-HFMD drug nor a vaccine is currently available. Rupintrivir, one of the drug candidates for HFMD treatment, has been attractive for the development of its analogs with broad biological activities. This drug is an inhibitor for 3C protease of CV-A16 and EV-A71, an enzyme that plays a crucial role in the viral replication process. ). In the present study, we focused on designing 50 novel rupintrivir analogs against CV-A16 …

In Silico And In Vitro Studies On Inclusion Complexation Of Anthraquinone Derivatives With Beta-Cyclodextrin Derivatives, Amy Oo

Chulalongkorn University Theses and Dissertations (Chula ETD)

In this study, we investigated the cytotoxicity of 6 anthraquinones (ventilanone K, emodin, chrysophanol, aurantio-obtusin, 1-O-methyl-2-methyoxychrysophanol, and questin) toward A549 lung cancer cells and emodin (ED) exerted the most potent cytotoxicity with IC50 value of 34.27 ± 1.27 µM. The anti-cancer activity of ED has been well reported but its usage in practical applications has been restricted due to its poor solubility. To address this issue, we performed inclusion complexation of ED with βCD, hydroxypropyl-β-cyclodextrin (HPβCD), 2,6-di-O-methyl-β-cyclodextrin (DMβCD), and sulfobutylether-β-cyclodextrin (SBEβCD) theoretically using molecular dynamics simulations and experimentally using phase solubility study and cytotoxicity screening toward cholangiocarcinoma cell lines (KKU-213A …

Mechanistic Insights Of Mitochondrial Reactive Oxygen Species In Alzheimer's Disease Models, Jakob C. Green

Theses and Dissertations

Alzheimer’s disease (AD) is a neurodegenerative disease that results in impaired cognition, disorientation, confusion, poor judgement, and behavioral changes. The mitochondrial cascade hypothesis (MCH), which postulates that accumulating oxidative stress and mitochondrial dysfunction plays an integral role in the development of the disease, potentially upstream, in conjunction with or independent of Aβ and tau. The literature supports the critical role of mitochondrial dysfunction in the progression and development of AD, but is inconclusive about what precise role, as initiator or delegate, it performs. Regardless, the impact that oxidative stress and mitochondrial dysfunction have on AD deserves further study and investigation. …

Cryogenic Electron-Microscopy (Cryo-Em) Analysis Of Mscs, Ynai, Connexin 26 And Bioinformatic Analysis Of Cysteine And Serine In Experimental Protein Structures, Claudio Catalano

Theses and Dissertations

Functional insights into macromolecules lie in the arrangement of their molecular structures. Investigating the atomic framework of a biomolecule could unwind its biochemical mechanism, providing aid to many relevant biological questions, thus facilitating drug discovery processes. Two approaches have been used to gain knowledge at the atomic level of a protein function: experimental and computational methodologies.

From an experimental perspective, membrane proteins (MPs) were investigated. Such proteins represent ~20% of the human proteome, and ~60% of them are drug targets since they are exposed to the surface of cells and are responsible for a wide range of fundamental functions like …

Characterization Of Cytochrome P450 3a4 Structure And Ligand Binding Mechanism In Anionic Nanodiscs, David T. Sweeney

Theses and Dissertations

The Cytochromes P450 superfamily are ubiquitous throughout life and are present in some viruses. Humans encode for 57 of these enzymes that are involved in the metabolism of ~90% of all chemicals. These membrane-bound monooxygenase enzymes utilize a heme protoporphyrin at their active site to activate molecular oxygen and oxidize their substrates through an intermediate deemed nature's most powerful oxidizer. They are a powerful system at our disposal yet understanding of their structure and ligand binding mechanisms in the presence of a lipid bilayer remains poorly understood. The focus of this work is on Cytochrome P450 3A4 (CYP3A4). We set …

Development Of Unnatural Amino Acid Mutagenesis Technologies To Control Enzyme Activity And Synthesis Of Chemically Reactive Ubiquitin Probes, Jenny Cheung

Theses and Dissertations

Most of the work vital for the survival of the cell are performed by proteins. The primary sequence of proteins is composed of 20 canonical amino acids. However, these 20 amino acids do not offer the full range of functionalities needed for proteins to do their job. Often times, post-translational modifications (PTM’s) occur to add the required functional group. This interaction between all the different proteins in the proteome in addition to PTM’s create a complex system that scientists are still trying to unravel to this day. Many different tools had been developed for this purpose, and one such tool …

Detrimental Role Of Irf1 In Myeloid Cells During Influenza Infection, Bianca L. Baum

Theses and Dissertations

The influenza virus is constantly evolving new ways to overcome both innate and vaccine-induced host defenses, making the innate immune response a critical therapeutic target for minimizing morbidity and mortality caused by infection. Here we examined the role that interferon regulatory factor 1 (IRF1) plays in regulating host innate immune responses in myeloid cells. We found that knocking out IRF1 from myeloid cells in mice (IRF1^DMy) led to a partial protection during IAV infection. Interestingly, recruitment of bone marrow derived myeloid cells as well as NLRP3 inflammasome expression were decreased in the lungs of IRF1^DMy mice, whereas …

Virtual Reality Tool For Visualizing The Oral Microbiome, Alexus M. Harris

Theses and Dissertations

There are limited teaching tools for helping both students and patients understand the oral microbiome. It is important to understand the oral microbiome as it heavily influences the health of your oral cavity and the dysbiosis of disease. The oral microbiome influences other aspects of health and serves as an indicator for health. With a greater understanding of the oral microbiome, patients can make healthier lifestyle changes and dentists can better educate their patients. For that reason, the goal of this project was to create a virtual reality tool for visualizing the oral microbiome. The assets for the VR tool …

Conformational Modulation Of Α-Helical N-Acetylated Α-Synuclein, Ricardo Fernandez

Theses and Dissertations

Human N-acetylated α-synuclein (^NAcαSyn) has long been accepted as an intrinsically disordered protein (IDP) predominantly found in terminal nerve regions in healthy subjects. ^NAcαSyn structural fluidity enables the formation of transient or dynamic assemblies. Some of these assemblies may serve functional, yet undefined purposes. ^NAcαSyn is highly susceptible to the surrounding environment, leading to misfolding under high cytosolic oxidative stress conditions, which in turn assemble in the form of toxic oligomers or amyloid fibrils. These ^NAcαSyn inclusions along with their accretion and migration within neurons, are pathological hallmarks of Parkinson’s disease (PD), dementia, and multiple …

In Vitro Screening Of Tp5801 Against Kinases For Nature Communications Publication, Joshua Andersen

ScholarsArchive Data

Data from screen of TP-5801 against in vitro kinases

Improvement In 14-3-3 Binding Site Prediction, Katherine K. Mccormack

ScholarsArchive Data

The 14-3-3 family of phospho-binding proteins regulate a variety of major cellular processes through interaction with a network of dynamic proteins. Deregulation of the 14-3-3 interaction network contributes to a variety of degenerative disorders and cancers. Our lab focuses on identifying novel 14-3-3 interactions and understanding how 14-3-3 binding regulates protein function. A major gap in this process is that identifying the phospho-site where 14-3-3 docks on a given protein is time- and resource-consuming. Prediction algorithms have been developed to predict canonical 14-3-3 binding sites, however, there are many non-canonical sites that existing software is unable to predict. To fill …

Kinase Screen Data For Nature Communications Publication, Joshua Andersen

ScholarsArchive Data

A TNK1 peptide encompassing S502 was screened against 245 Ser/Thr kinases. This file contains kinase activity values (against S502) from an n=1 experiment. These data support a manuscript to be published in Nature Communications (Chan and Egbert et al.)

Stomatal Conductance And Reed Canary Grass Suppression Of Native Michigan Trees, David E. Martinez Vasquez, Martin L. Vanderschoot, David Warners

Faculty and Professional Research

Reed canary grass (Phalaris arundinacea), a prominent invasive understory species, threatens native Michigan biodiversity statewide. A notable affected area includes a stretch of agricultural land along Schooley Drain in Caledonia, MI, located in the headwaters of the Plaster Creek watershed. Our study pursues the following questions: “Do native trees provide enough shade to reduce the growth of Reed canary grass?” and “Which native trees best transpire water from a floodplain?”: To answer these questions, we initiated a long-term evaluation of seven different species of native Michigan trees to investigate which species best inhibits the growth of reed canary grass and …

Plaster Creek Floodplain And Adjacent Ravines Near Stanaback Park: Botanical Inventory Highlights And Management Considerations, David Warners, Garrett Crow, Haley Weesies

Faculty and Professional Research

In the summer of 2021, the research team visited this area of ravines and floodplain a total of twenty-one times to conduct our field research. During the visits, they carefully walked throughout different sections of the study site, identifying each vascular plant species they saw growing there and observing how the flora developed throughout the season. In addition to keeping detailed field notes, they documented permanent records of the findings of our inventory by collecting voucher specimens that have been deposited in the Calvin University Herbarium. Additionally, they sent duplicate specimens to the Michigan State University Herbarium and/or University of …

Botanical Inventory Highlights And Management Considerations For Covenant Park, David Warners, Garrett Crow, Haley Weesies

Faculty and Professional Research

In the spring and summer of 2021, Calvin University students and faculty conducted a botanical inventory and assessment of the natural areas in Covenant Park, located at 3724 Shaffer Avenue SE, Kentwood, MI 49512. Given that the property was recently acquired by the City of Kentwood Parks & Recreation, the purpose of this inventory is to inform Kentwood Parks and Recreation staff about the site’s floristic quality, diversity, and ecological significance. The specific objectives of this study are to 1) provide a comprehensive inventory of the species of plants occurring on the property, 2) identify particular areas that would benefit …