Biochemistry Commons^™

Mutational Analysis And Domain Characterization Of The Apolipoprotein L-1 Ion Channel, Charles M. Schaub

Dissertations, Theses, and Capstone Projects

The human innate immunity factor Apolipoprotein L-1 (APOL1) protects against Trypanosoma brucei brucei infection. Recent studies have shown recombinant APOL1 (rAPOL1) inserts into planar lipid bilayers at an acidic pH 5.6 and forms a cation-selective channel, which opens upon subsequent neutralization, pH 7.2. This corresponds with the pH changes APOL1 would encounter during endosome recycling, suggesting that APOL1 forms a pH-gated ion channel in the plasma membrane of the parasite, leading to uncontrolled ion flux and osmotic imbalance. However, structural and domain characteristics of the APOL1 channel are poorly understood, despite potential similarities to diphtheria and colicin toxins. Utilizing E. …

Through The Back Door: Proteins Escape Cells Without Conventional Permission, Michael J. Cohen

Dissertations, Theses, and Capstone Projects

Proteins secreted to the extracellular environment play a fundamental role as signals, in metabolism, and a variety of other processes. The process of secretion through the endoplasmic reticulum and Golgi to the plasma membrane is well documented, and all cargo in this pathway contains a signal peptide. However, a variety of proteins secreted from eukaryotes lack a signal peptide and are called unconventionally secreted proteins. Here we discuss known mechanisms of unconventional protein secretion, as well as model proteins which follow characterized pathways. Additionally, we summarize the roles various unconventionally secreted proteins play outside of cells and suggest criteria for …

Hydrolysis Of Palm Oil Mill Effluent Using Enzymes From Spent Mushroom Substrate Of Pleurotus Pulmonarius (Fr.) Quel, Mat Yunan Nurul Anisa

Student Works (2020-2029)

Mushroom cultivation and palm oil industry in Malaysia have contributed to the large volumes of lignocellulosic residues. Lignocellulolytic enzymes profiles (lignin peroxidase, laccase, xylanase, endoglucanase, and β-glucosidase) of Pleurotus pulmonarius (parents strains UMP001 and UMP 002, and the hybrid UMH004) during growth in sawdust substrate bags were investigated. The enzymes extracted from the spent mushroom substrate (SMS) of the strain with the highest enzymes productivity was used to hydrolyse palm oil mill effluent (POME). Optimization of parameters for POME hydrolysis to release reducing sugar was conducted using central composite design (CCD) of response surface methodology (RSM). The potential of hydrolysed …

Molecular Composition And Photochemical Lifetimes Of Brown Carbon Chromophores In Biomass Burning Organic Aerosol, Lauren T. Fleming, Peng Lin, James M. Roberts, Vanessa Selimovic, Robert Yokelson, Julia Laskin, Alexander Laskin, Sergey A. Nizkorodov

Chemistry and Biochemistry Faculty Publications

To better understand the effects of wildfires on air quality and climate, it is important to assess the occurrence of chromophoric compounds in smoke and characterize their optical properties. This study explores the molecular composition of light-absorbing organic aerosol, or brown carbon (BrC), sampled at the Missoula Fire Sciences laboratory as a part of the FIREX Fall 2016 lab intensive. A total of 12 biomass fuels from different plant types were tested, including gymnosperm (coniferous) and angiosperm (flowering) plants and different ecosystem components such as duff, litter, and canopy. Emitted biomass burning organic aerosol (BBOA) particles were collected onto Teflon …

Synthesis And Preliminary Ex Vivo Testing Of Sirna Targeting Tcrb: A Proposed Therapy For The Treatment Of Autoimmunity, Nicholas J. Magazine

LSU Doctoral Dissertations

Abstract

Background:

As of 2018, the United States National Institutes of Health estimate that over half a billion people worldwide are affected by autoimmune disorders. Though these conditions are prevalent, treatment options remain relatively poor, relying primarily on various forms of immunosuppression which carry potentially severe side effects and often lose effectiveness overtime. Given this, new forms of therapy are needed. We propose small-interfering RNA (siRNA) for hypervariable regions of the T-cell receptor β-chain gene (TCRb) as a highly targeted, novel means of therapy for the treatment of autoimmune disorders.

Objectives:

To develop methods to produce siRNA targeting …

Dominant Negative Effects By Inactive Spa47 Mutants Inhibit T3ss Function And Shigella Virulence, Jamie L. Burgess, Heather B. Case, R. Alan Burgess, Nicholas E. Dickenson

Chemistry and Biochemistry Faculty Publications

Type three secretion systems (T3SS) are complex nano-machines that evolved to inject bacterial effector proteins directly into the cytoplasm of eukaryotic cells. Many high-priority human pathogens rely on one or more T3SSs to cause disease and evade host immune responses, underscoring the need to better understand the mechanisms through which T3SSs function and their role(s) in supporting pathogen virulence. We recently identified the Shigella protein Spa47 as an oligomerization-activated T3SS ATPase that fuels the T3SS and supports overall Shigella virulence. Here, we provide both in vitro and in vivo characterization of Spa47 oligomerization and activation in the presence and absence …

Case 34: Kinetics Of Alkaline Phosphatase, Dr. Kathleen Cornely

Chemistry & Biochemistry Case Studies

Kinetic studies are carried out using the alkaline phosphatase, an enzyme that has an important role in skeletal mineralization.

An Evolutionarily Conserved Iron-Sulfur Cluster Underlies Redox Sensory Function Of The Chloroplast Sensor Kinase, Iskander M. Ibrahim, Huan Wu, Roman Ezhov, Gilbert E. Kayanja, Stanislav D. Zakharov, Yanyan Du, Weiguo Andy Tao, Yulia Pushkar, William A. Cramer, Sujith Puthiyaveetil

Purdue University Libraries Open Access Publishing Fund

Photosynthetic efficiency depends on equal light energy conversion by two spectrally distinct, serially-connected photosystems. The redox state of the plastoquinone pool, located between the two photosystems, is a key regulatory signal that initiates acclimatory changes in the relative abundance of photosystems. The Chloroplast Sensor Kinase (CSK) links the plastoquinone redox signal with photosystem gene expression but the mechanism by which it monitors the plastoquinone redox state is unclear. Here we show that the purified Arabidopsis and Phaeodactylum CSK and the cyanobacterial CSK homologue, Histidine kinase 2 (Hik2), are iron-sulfur proteins. The Fe-S cluster of CSK is further revealed to be …

A Proposed Mechanism To Induce Multi-Layer Polydiacetylene-Coated Filter Color Response To Bacteria , Timothy Hanks

Chemistry Publications

The purpose of this study was to develop a multi-layer diacetylene (PDA)-coated filter and to verify the proposed color response mechanism of PDA-coated filters to bacteria. Unlike other reports which immobilize PDA lipo- somes on solid support or create monolayer Langmuir films, multi-layer PDA-coated filters were generated by directly evaporating organic solvents. Different from incorporating functional headgroups or inserting phospho- lipids into PDA, we reported that bacterial growth can trigger the color change of PDA sensors without any mod- ification or phospholipid insertion. The mechanism that pH change from bacteria metabolites lead to color change of PDA filter was proposed …

Quantification Of Mitochondrial Zinc Homeostasis And Analysis Of Zinc And Polyamine-Mediated Axonal Trafficking, Dylan Hunt Fudge

Electronic Theses and Dissertations

The zinc ion is an important emerging signaling molecule for biological processes. In this work we engineered improved zinc sensors based on our previously developed fluorescent sensor GZnP1 to provide sensors with a higher fluorescent readout, faster kinetics, and a superior sensitivity to zinc. We utilized these zinc sensors and further developed the sensors to answer questions pertaining to biological zinc. We showed the labile zinc concentration in the mitochondrial matrix was less than 1 pM while the labile zinc concentration in the cytosol and mitochondrial IMS were comparable (~100 pM) across four different cell lines. Using these zinc sensors …

Characterization Of The Dimerization And Salt Dependent Aggregation Of The Als-Linked Protein Tdp-43, Elizabeth Ehrhardt

Electronic Theses and Dissertations

Trans-active response (TAR) DNA-binding protein 43 (TDP-43) is essential for RNA processing but can also form toxic cytoplasmic inclusions in neurons of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA-binding has been shown to have the potential to decrease or inhibit the aggregation of TDP-43, indicating that supplying RNA therapeutics may be a solution to treat these neurodegenerative disorders. However, the mechanism of aggregation, transitioning from TDP-43’s native dimeric state to small oligomers to toxic aggregates, is still relatively unknown. This needs to be established before determining how to target and disrupt this aggregation. Using GFP-TDP-43, …

A Look At Gene Control: Tracking The Ccnd1 Gene, Bryan Anders

Mahurin Honors College Capstone Experience/Thesis Projects

Cancer occurs when the cell does not properly control its own cell cycle. It then replicates in an out of control fashion leading to the death of various organs and then the demise of the organism as a whole. As it seems to have always been a problem for cell-based life, certain safeguards against cancer have been evolved over time. One such method comes in the form of prevention via cyclin proteins, which are encoded from cyclin genes. The gene that is the focus of this research is the CCND1, or cyclin D1, gene that controls the progression through various …

Development And Application Of Chemical Tools To Identify Kinase-Substrate Interactions, Aparni Kithulgoda Gamage

Wayne State University Dissertations

Post translational modifications regulate a variety of biological processes inside the cell.Protein phosphorylation is one such PTM modification catalyzed by protein kinases, which aid to transfer a signal from one place to another inside the cell. However, irregularities in kinase-mediated signaling are often implicated in many diseases, making kinases effective drug targets. To understand kinase-related disease formation and to discover drugs to treat these diseases, it is crucial to have a clear understanding on kinase-mediated cell signaling networks. A current gap in the kinase biology field is a lack of tools to identify which kinase phosphorylates which protein substrate inside …

Acetoacetate Enhances Oxidative Metabolism And Response To Toxicants Of Cultured Renal Cells, Trudi Maria Denoon

Theses and Dissertations

In vitro methods can be cost effective and facilitate large scale drug screening in the pharmaceutical industry. Renal proximal tubular cells have been a prime target for drug induced toxicity. LLC-PK1 cells possess many transport functions of the proximal tubule epithelia, however, its energy metabolism differs from the highly oxidative tubule metabolism in vivo. This may impact its reliability in predicting toxicity. Modulating the growth media composition has been shown to shift cultured liver and muscle cells from high glycolytic activity to increased oxidative metabolism, but few studies have been done on renal cells. This study is designed to test …

Structure-Guided Molecular Grafting Of A Complex Broadly Neutralizing Viral Epitope, G. Bajic, M. J. Maron, T. M. Caradonna, M. Tian, Adam Mermelstein , '21, Daniela Fera, G. Kelsoe, M. Kuraoka, A. G. Schmidt

Chemistry & Biochemistry Faculty Works

Antigenic variation and viral evolution have thwarted traditional influenza vaccination strategies. The broad protection afforded by a “universal” influenza vaccine may come from immunogens that elicit humoral immune responses targeting conserved epitopes on the viral hemagglutinin (HA), such as the receptor-binding site (RBS). Here, we engineered candidate immunogens that use noncirculating, avian influenza HAs as molecular scaffolds to present the broadly neutralizing RBS epitope from historical, circulating H1 influenzas. These “resurfaced” HAs (rsHAs) remove epitopes potentially targeted by strain-specific responses in immune-experienced individuals. Through structure-guided optimization, we improved two antigenically different scaffolds to bind a diverse panel of pan-H1 and …

Sorting Of Cargo Proteins Within The Regulated Secretory Pathway: The Peripheral Membrane Protein Hid-1 As A Sorting And Vesicle Biogenesis Factor, Blake H. Hummer

Electronic Theses and Dissertations

Large dense core vesicles (LDCVs) form at the trans-Golgi network (TGN) and mediate the regulated release of neuropeptides and peptide hormones. Despite their central role to physiology, the mechanisms controlling biogenesis and sorting to LDCVs is not well understood. Optimizing the retention using selective hooks (RUSH) method in neuroendocrine cells, we show it is possible to visualize sorting to the constitutive and regulated secretory pathways in real-time and that the bulk of transmembrane LDCV cargoes do not sort directly onto LDCVs, but exit the TGN into non-regulated vesicles to be incorporated to LDCVs at a later step. Additionally, we characterize …

Ototoxicity Of Cisplatin, Pyriplatin, And Phenathriplatin In The Auditory Hybridoma Cell Line, Hei-Oc1, Alexandra Johnston

Mahurin Honors College Capstone Experience/Thesis Projects

Cisplatin is an anti-cancer drug which is effective against several cancers, but also causes harmful side-effects, including ototoxicity and hearing loss. While cisplatin is a bifunctional compound that forms coordinate covalent bonds with both strands of DNA, recently investigated monofunctional platinum(II) compounds bind to only one DNA strand, and may activate different cell-death mechanisms. As several monofunctional platinum(II) compounds have anti-cancer properties, but could target different cell-death pathways, they could potentially have different and reduced side-effects. In this study, the HEI-OC1 auditory hybridoma cell line was used to investigate the ototoxicity of cisplatin and two monofunctional platinum(II) compounds, phenanthriplatin and …

Variations On A Theme: Intricacies Of Unanchored Poly-Ubiquitin Signaling And Toxicity, Jessica Renee Blount-Pacheco

Wayne State University Dissertations

Ubiquitin is an 8.5 kDa post-translational modifier involved in essentially all eukaryotic cellular processes. Through a process called ubiquitination, ubiquitinating enzymes chemically attach ubiquitin to substrate proteins to control their fates, resulting in anything from their recruitment into signaling pathways to their proteasomal degradation, with a plethora of possibilities in between. Ubiquitin molecules can also be attached to one another, resulting in poly-ubiquitin chains with various effects depending on the number of ubiquitin molecules and the specific amino acid residues used to link them together. While most poly-ubiquitin in the cell exists as conjugated species, there are also untethered poly-ubiquitin …

Use Of Alkoxyamines To Quantify And Map Uracils And To Kill B Cell Cancers, Madusha Lakshani Watuthanthrige Perera

Wayne State University Dissertations

Majority of B-cell lymphoma and leukemia originates in germinal centers and over express AID resulting high levels of uracils and eventually AP sites. Treating cells with a small alkoxyamine AA3 with a terminal alkyne group specifically killed B cell lymphoma cell lines without killing normal human B cells and non-hematological cancers. To study the cytotoxicity of AA3, I designed and synthesized a series of AA3 analogs with and without the alkyne group. Using cytotoxicity assays I demonstrated that the alkyne functionality plays a major role in AA3 cytotoxicity in B-cell cancers and further showed that the effectiveness is modulated by …

Computational Analysis Of Oxidative Stress In Endothelial Dysfunction: Insights On The Role Of Tetrahydrobiopterin, Ascorbate And Glutathione, Sheetal Kedar Panday

Wayne State University Dissertations

Oxidative stress and endothelial dysfunction are reported in the cardiovascular and neurovascular diseases. Oxidative stress is caused due to an increase in the generation of reactive oxygen (ROS) and nitrogen species (RNS) and incapacity of antioxidant systems to eliminate ROS and RNS. Endothelial dysfunction is characterized by a reduction in nitric oxide (NO) bioavailability. NO is constitutively produced by enzyme endothelial nitric oxide synthase (eNOS). A reduction in tetrahydrobiopterin (BH4), which is an essential cofactor of eNOS, can lead to eNOS uncoupling. There is complex interplay between the ROS/RNS and antioxidant system underlying pathophysiologies of vascular diseases, however our quantitative …