Biochemistry, Biophysics, and Structural Biology Commons^™

Assessing Functional Visual Recovery After Retinal Regeneration In Zebrafish Using The Optokinetic Response (Okr), Yeganeh Tabatabaei Alavi, Yeganeh Tabatabaei Alavi

Medical Student Research Symposium

Zebrafish have a unique ability to regenerate retinal neurons through Müller glia-mediated reprogramming, a process not found in mammals. While structural regeneration has been extensively documented, the degree to which regenerated photoreceptors restore functional vision remains unclear. The optokinetic response (OKR), a reflexive eye-tracking behavior triggered by moving visual stimuli, provides a quantitative, non-invasive method to assess visual performance. This study aims to determine whether photoreceptor regeneration following acute and chronic light-induced injury results in functional restoration of vision. Adult zebrafish will be divided into three groups: uninjured control, acute injury (short-term high-intensity light), and chronic injury (long-term low-intensity light). …

Structural Impacts Of Canonical And Non-Canonical Oligonucleotide Modifications, Deepak Shrestha

Wayne State University Dissertations

Nucleic acids, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acids), are the fundamental building blocks of living organisms. The events that cause modification of DNA/RNA are of interest because they impact the nucleic acid function. Targeting DNA is important for treating diseases such as cancer. Cisplatin (cisPt) is a well-studied drug that targets DNA and blocks replication, but its use is limited by adverse side effects and cellular resistance. Prevoius research addressed some of these challenges through development of platinum analogues such as carboplatin and oxaliplatin. Amino acid-linked platinum analogues (AAPt) are the focus of this thesis work. AAPt complexes …

Mass Spectrometry Combined With Chemical Biology To Study Human Ailments, Eric Thomas Jacquerio Davis

Wayne State University Dissertations

Post-translational modifications (PTMs) are essential as chemical changes that regulate protein function, activity, stability, interactions, and localization after protein synthesis. PTMs, such as phosphorylation, acetylation, and ubiquitination, can activate or deactivate proteins, influence their interactions with other molecules, and determine their cellular location. This regulation is vital for numerous biological processes, such as cell cycle control, signal transduction, and gene expression. Abnormal PTMs are linked to various diseases in humans, including parasitic infection, blood disorders, cancers, and neurodegenerative diseases, making their study crucial for understanding disease mechanisms and developing targeted therapies. In the Pflum lab, we are interested in the …

Development And Application Of Enhanced Sampling Techniques For Investigating Protein Interactions And Drug Discovery, Timothy Hasse

Wayne State University Dissertations

Understanding the dynamics of protein–protein and protein–ligand interactions is critical for elucidating biological function and guiding drug discovery. However, many such processes involve rare events and high energy barriers that are difficult to capture with conventional molecular dynamics (MD). Enhanced sampling techniques extend the reach of MD, enabling access to biologically relevant transitions and thermodynamic properties. In the first project, Gaussian accelerated MD (GaMD) was used to study the MEIG1–PACRG complex, a key regulator of sperm flagellar assembly. Simulations of wildtype and mutant MEIG1 (W50A, Y68A) revealed mutation-specific disruptions to the binding interface and identified druggable pockets, particularly on PACRG. …

The Role Of Fruiting Bodies In Protecting A Community Of Myxococcus Xanthus Spores From The Environment, Dave Lall

Wayne State University Dissertations

Environmental microorganisms have evolved a variety of strategies to survive fluctuations in environmental conditions, including the production of biofilms and differentiation into spores. Myxococcus xanthus are ubiquitous soil bacteria that produce starvation-induced multicellular fruiting bodies filled with environmentally resistant spores (a specialized biofilm). Isolated spores have been shown to be more resistant than vegetative cells to heat, ultraviolet radiation, and desiccation. The evolutionary advantage of producing spores inside of fruiting bodies is not clear. Here, we examine a hypothesis that the fruiting body provides additional protection from environmental insults. We developed a high-throughput method to compare the recovery (outgrowth) of …

Microbial Prevalence And Antibiotic Resistance Associated With Urban Food Production Systems, Nirosha Ruwani Amarasekara

Wayne State University Dissertations

Despite the increasing popularity of urban agriculture and farmers’ markets, there is a lack of scientific data on microbial prevalence and antibiotic resistance associated with this unique food production system. The overall goal of this research is to understand antibiotic resistance and microbial prevalence in soil and fresh produce associated with urban agriculture. Specifically, we aim to investigate ARG in association with resistance genes to metals and other compounds in urban agricultural soil as well as antibiotic resistome and Listeria contamination in fresh produce distributed in urban communities.

In the initial phase of this research, soil samples were collected from …

Uracil Landscape Of Mouse B Cell Genome Undergoing Class-Switch Recombination, Fathima Rukshana Mohamad Ramshan

Wayne State University Dissertations

Activation-induced cytosine deaminase (AID) is required for both somatic hypermutation (SHM) and class switch recombination (CSR) within mammalian antibody genes. Uracils created by AID in the Ig loci are recognized and processed by base-excision and mismatch repair pathways leading to antibody diversification. This involves a hypermutation process restricted to the V(D)J segments of the immunoglobulin genes, SHM, and an error-prone end-joining process that causes isotype switching, CSR, but the molecular details of these processes have remained obscure for the past 20+ years. Most studies of SHM and CSR have used AID-promoted mutations as a proxy for the uracils, but their …

Design, Synthesis, And Evaluation Of Class Iia Histone Deacetylase (Hdac) Inhibitors, Lauren Nicole Kotsull

Wayne State University Dissertations

Histone deacetylase (HDAC) proteins are linked to disease states, including cancer, and have become important targets for therapeutic development. Many inhibitors have been developed to target HDAC proteins, including FDA-approved inhibitors Vorinostat (SAHA), Belinostat, Romidepsin, and Panobinostat. These inhibitors non-specifically influence most of the 11 metal-dependent HDAC isoforms, which might contribute to the side effects that patients experience during treatment. The lack of selectivity also diminishes the use of HDAC inhibitors as biomedical research tools. To address these drawbacks, the development of selective inhibitors has been heavily studied. Yet, reported inhibitors are predominantly selective for class I HDAC isoforms (HDACs …

Benzene Exposure In Reproductive Biology: A Comprehensive Analysis Of Placental Response And Molecular Mechanisms In Trophoblast Function, Anthony Maxwell

Wayne State University Dissertations

The increase in environmental challenges and exposure to pollutants like benzene poses pressing concerns for reproductive health. This dissertation investigates the impact of benzene exposure during gestation on placental function, a key determinant for fetal development and pregnancy outcomes. Our work establishes that benzene exposure during pregnancy induces maternal immune activation, leading to increased rates of fetal resorptions, compromised fetal growth, and abnormal placental development. Intriguingly, we identify a sexual dimorphic response in male and female placentas to benzene exposure, attributable to inherent differences between male and female placental biology. On a molecular level, benzene is metabolized into hydroquinone in …

Dual Mechanisms Contributing To Pyruvate Dehydrogenase Activity Deficiency In A Barth Syndrome Cell Model, Zhuqing Liang

Wayne State University Dissertations

Barth syndrome (BTHS) is a rare genetic disease that results from mutations in the TAFAZZIN gene, which encodes the cardiolipin (CL) remodeling enzyme tafazzin (Taz). The mechanisms linking perturbation of CL remodeling and the pathological features of BTHS are not understood. We have recently reported that intermediary metabolism is perturbed in BTHS, and activity of the metabolic gatekeeper enzyme pyruvate dehydrogenase (PDH) is deficient in BTHS models. The mechanism whereby PDH is regulated by Taz is unknown, and this knowledge gap represents an obstacle to the development of therapeutics to treat BTHS. Using an established C2C12 myoblast model of BTHS, …

Biological Adaptations Leading To Metastatic Potential In Ovarian Cancer, Hussein Chehade

Wayne State University Dissertations

Ovarian cancer induces most deaths from gynecological cancers. Patients succumb due to heavy metastatic tumor burden, which accounts for 90% of cancer related deaths and is characterized by the dissemination of malignant cells from primary tumor to secondary site(s), partaking in cancer progression. Ovarian cancer progression is an extremely demanding process that requires cancer cells to be able to survive anoikis conditions and be metabolically flexible to survive microenvironmental changes during transit to a secondary site. Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1) is a bi-organellar protein primarily present in the mitochondria by which it can prevent apoptosis and/or enhance mitochondrial …

From Culture To Metagenomics: A Comprehensive Evaluation Of The Female Reproductive Tract Microbiome In Pregnancy, Jonathan Panzer

Wayne State University Dissertations

The advent of Next Generation Sequencing technologies prompted novel investigations of the microbiome. These revealed both increased diversity of known microbiomes such as those of the gut, oral cavity and vagina, but also bacterial DNA in tissues previously considered sterile such as the blood, liver, brain, and placenta. Thus, previous paradigms of sterility, based primarily on culture data, were called into question. However, interpretation of bacterial DNA signals from low-microbial biomass tissues is complex due to the strong potential for contamination and interference from the surrounding environment. Yet, many new methodologies are being developed to help isolate valid bacterial DNA …

Analyzing The Impact Of Zip Subfamily Ii Mutations On The Zinc And Iron Transport Of Zupt From Escherichia Coli, Bradley Vandecar

Wayne State University Theses

The disfunction or misregulation of the ZIP family of metal ion transporters has been linked to a plethora of diseases and cancers in humans. Zn2+ is the primary substrate for ZIPs and is transported through the M1 metal binding site, though this activity is stimulated through the binding of Fe2+ in the M2 metal binding site. The members of ZIP Subfamily II are predicted to have an inactive M2 site due to the lack of Fe2+ transport and from a previously generated homology model. This work utilizes homology modelling, whole cell metal ion uptake assays, and growth studies in minimal …

In Silico Drug Discovery For Sars-Cov-2 Papain-Like Protease, Olivia Haskin

Wayne State University Theses

SARS-CoV-2 Papain-like protease is an essential viral protein for viral replication. In our lab, we hypothesize that successful inhibition of this protease will stop viral replication and allow for clearance of the infection by the host. The work described here aimed to design and execute an in silico drug discovery workflow for successfully selecting PLpro inhibitors from the Mcule purchasable compound database. This work reports such a workflow and nine potential compounds for in vitro testing. This work also describes structural activity relationship analysis of a proprietary set of PLpro inhibitors synthesized by medicinal chemistry collaborators and makes recommendations for …

Expression And Purification Of E. Coli Yoaa, A Putative Helicase, Mark Gregory, Vincent Sutera Mr., Susan Lovett Dr.

Medical Student Research Symposium

All cells must maintain their genomic integrity to survive, which they achieve through several repair mechanisms that necessitate unwinding the damaged DNA by helicases. In Escherichia coli (E. coli), YoaA has been genetically shown to be involved in DNA repair and shares conserved sequences with helicase DinG. The goal of our study was to purify YoaA for further biochemical characterization. For expression, YoaA was fused to a His tag and overexpressed in MG1655 E.coli under the lacZ or T7 promoters for 2 hours, 4 hours, or overnight at 24oC, 30oC or 37oC. For purification, crude lysate was applied to a …

Kinase-Catalyzed Labeling To Identify Kinase-Substrate Pairs Using Γ-Phosphate Modified Atp Analogs, Rachel Beltman

Wayne State University Dissertations

Post-translational modifications (PTMs) are responsible for a variety of cellular processes. One such PTM is protein phosphorylation, which is catalyzed by kinases. Kinase enzymes play important roles in cellular signaling pathways, but dysregulation of kinase-mediated events results in the formation of diseases, which make kinases favorable drug targets. To uncover the role kinases play in the development of diseases, kinase-mediated cellular events need to be better understood. The current gap in the field is the lack of tools available to identify the kinase that is responsible for specific phosphorylation events within the cell. To improve the gap in the field, …

The Importance Of Protein Context In Spinocerebellar Ataxia Type 3, Sean Luis Johnson

Wayne State University Dissertations

Spinocerebellar Ataxia Type 3 (SCA3) is a member of the family of polyglutamine (polyQ) neurodegenerative disorders that includes Huntington's Disease and several other SCAs. SCA3, the most common dominant ataxia in the world, is caused by polyQ tract expansion in the protein, ataxin-3. How SCA3 occurs and how to treat it remain unresolved issues. The primary culprit of toxicity in all polyQ diseases is the glutamine repeat: its abnormal expansion leads to neuronal dysfunction and death. With that said, there is indisputable evidence that the way polyQ-dependent toxicity presents—areas impacted, cellular processes perturbed—is predicated in large part on regions outside …

Study Of Zyomogen Granule Movement Along Actin Filaments Using A Single Beam Optical Trap, Justin James Raupp

Wayne State University Dissertations

Zymogen granules are enzymatic vesicles in the pancreas. The surface of these zymogen granules (ZGs) has several different kinds of myosin molecules, such as myosin 1c, 6, 5c, and 7b. These molecular motors may contribute to ZG transportation in cells. To understand the molecular motors involved in the vesicle trafficking, we observed the in vitro motility of purified ZGs from rat pancreas and examined the stepping behavior and force that is generated using a single beam optical trap. To be involved in trafficking, molecular motors have certain characteristics, a high duty ratio and the ability to move continuously along actin …

Extending Plasma Proteome Coverage To Enable Biomarker Discovery, Jordan Bruce Burton

Wayne State University Dissertations

Two features make plasma a valued source of health information: First, plasma contains secreted proteins from all tissues in the body and can, therefore, provide information about the health status of all tissues. Second, plasma is available in large quantity using a minimally invasive procedure that is well tolerated. The status of plasma as a valued source of health information is matched by the difficulty in extracting proteome information from the samples. Using state of the art instrumentation a standard analysis of plasma yields quantitative information on approximately 300 proteins. Extreme fractionation strategies coupled with increased mass spectrometry time extends …

Increased Cd11b+ Immune Cells Contribute To Thoracic Aortic Aneurysm Rupture, Lucynda Pham

Wayne State University Theses

A life-threatening clinical manifestation of Marfan Syndrome (MFS) is the development of thoracic aortic aneurysms (TAA) which can lead to dissection and or rupture. MFS is caused by mutations in extracellular matrix (ECM) protein, Fibrillin-1 (FBN1) which forms the structural component of elastic microfibrils. It is unclear how mutations in FBN1 cause aneurysm development and progression. In recent years, inflammation and immune cells are being considered an important biological process that contributes to TAA. However, the exact roles of the immune cells are unknown. Here we find increased CD11b+ cells recruited and infiltrated into the vessel wall and their spatial …

Identification Of Glutathionylated Cysteines Regulating Cell Migration, Dhanushika Kukulage

Wayne State University Dissertations

Cell migration is vital in many physiological processes, such as wound repair and metastasis. Impaired cell migration in wound healing may result in chronic wounds, by which 6.7 million Americans are affected, and they suffer from bacterial infections, amputations, increased mortality, and depression. Reactive oxygen species (ROS) or hydrogen peroxide (H2O2) play an essential role as a second messenger in redox signaling. Evidence has been found that H2O2 takes part in regulating cell migration as well. A primary mechanism of ROS regulating redox signaling is oxidative protein modifications. Protein S-glutathionylation, one of the oxidative modifications, is the conjugation of glutathione …

Genetic Analysis Of Vibrio Cholerae During Its Hyperinfectious State In Danio Rerio (Zebrafish), Ravipaul Singh

Wayne State University Theses

ABSTRACTGENETIC ANALYSIS OF VIBRIO CHOLERAE DURING ITS HYPERINFECTIOUS STATE IN DANIO RERIO (ZEBRAFISH) by RAVIPAUL SINGH AUGUST 2022 Advisor: Dr. Jeffrey Withey Major: Biochemistry and Molecular Biology Degree: Master of Science Vibrio cholerae is a gram-negative, comma-shaped, monotrichous bacillus and the causative agent of the diarrheal disease cholera. V. cholerae is a natural resident of aquatic environments such as rivers and estuaries and is a highly motile bacterium. Upon entering the human host, V. cholerae travels to its site of colonization in the small intestine and upregulates its virulence factors. Upon escape from the host, V. cholerae can enter a …

Effect Of Allosteric Site Binding On Smyd2 Conformational Ensemble, Jacob Daniel Sobota

Wayne State University Theses

Molecular dynamics simulation allows for the simulation of proteins with precise control over what ligands are present at what sites. The conformational ensemble of a protein can be obtained from MD simulation with and without binding at an allosteric site and compared between to gain insight at what effects binding at an allosteric site has on a protein’s structure. SMYD2, a critical regulator of cellular processes, was recently found to have a novel secondary binding site with allosteric effects on its activity. The discovery of an allosteric site has implications for drug design in the inhibition of SMYD2 to treat …

Characterizing The Post-Translational Modifications Of The Pro-Oncogenic Type Ii Transmembrane Serine Protease Tmprss13, Carly Elizabeth Martin

Wayne State University Dissertations

TMPRSS13, a type II transmembrane serine protease discovered at the turn of the century, has recently been shown to be significantly overexpressed in both breast cancer (BCa) and colorectal cancer (CRC), and to mediate chemoresistance in cell lines from both cancer types. Furthermore, loss of TMPRSS13 in a genetic model of mouse mammary carcinoma significantly reduced tumor burden and growth rate, and increased overall tumor-free survival. Its location on the cell surface as well as its low expression in normal breast and colon make TMPRSS13 an attractive candidate as an oncogenic biomarker and therapeutic target in cancer. However, little is …

Deconstructing Brown Adipocyte Neogenesis In Brown And White Adipose Tissue, Rayanne Burl

Wayne State University Dissertations

Global incidence of Type 2 Diabetes (T2D) has reached epidemic proportions, and increasing evidence indicates that dysfunctional adipose tissue is an important contributor to the pathogenesis of T2D. Expanding brown adipocyte (BA) populations within adipose tissues through adrenergic activation improves energy balance and insulin sensitivity. In order to exploit this remodeling of adipose tissue for therapeutic benefit, we need to understand the mechanisms by which adrenergic signaling expand populations of BAs in vivo. These studies utilized single-cell RNA-sequencing and transgenic mouse models, in combination with single-molecule fluorescence in situ hybridization (smFISH) and immunoistochemical analysis, to study BA neogenesis in vivo. …

Vaginal Microbiota Of Recurrent Bacterial Vaginosis Patients Under Oral Metronidazole Therapy: Prognosis Based On Specific Species Of Gardnerella And Other Bacterial Vaginosis Associated Species In Association With The Vaginal Microbiome, Ashomathi Ashok Sudhamathi Mollin

Wayne State University Theses

Bacterial Vaginosis (BV) is a state of vaginal dysbiosis in which BV-associated species displace the typically dominant species of Lactobacillus. This urogenital disease elevates the risk for complications such as pre-term birth, STDs and HIV. Currently, there is no consensus on the etiology of BV. Standard of care treatment is oral metronidazole; however, therapy has high rates of recurrence. Our previous qPCR study of recurrent BV patients (3+ episodes/year) found strong associations of Gardnerella_Gsp07, G. swidsinksii, and G. leopoldii with refractory and recurrent responses, and a robust recovery of Lactobacillus in remission patients. However, these findings were not applicable across …

Abhd5 Induced Morphological Changes On Model Membrane Systems, Nasser S. Junedi

Honors College Theses

Proper regulation of neutral lipid storage (lipogenesis) and release (lipolysis) are critical molecular processes localized to an organelle called the Lipid Droplet (LD). The LD consists of a core with neutral lipids such as triacylglycerols (TAGs) and sterol esters surrounded by a phospholipid monolayer. Dysregulation of the processes localized to the LD are involved in the pathology of various diseases such as Neutral Lipid Storage Disease, diabetes, stroke and cancer. The non-enzymatic protein ABHD5 (α-β Hydrolase Domain-Containing Protein 5), is thought to play a key role in the process of lipolysis by forming homo-oligomers on the surface of the LD …

Lysine 53 Acetylation Of Cytochrome C In Prostate Cancer: Warburg Metabolism And Evasion Of Apoptosis, Viktoriia Bazylianska, Hasini A. Kalpage, Junmei Wan, Asmita Vaishnav, Gargi Mahapatra, Alice A. Turner, Dipanwita Dutta Chowdhury, Katherine Kim, Paul T. Morse, Icksoo Lee, Joseph S. Brunzelle, Lisa Polin, Prabal Subedi, Elisabeth I. Heath, Izabela Podgorski, Katrin Marcus, Brian Fp Edwards, Maik HüTtemann

Biochemistry and Molecular Biology Faculty Publications

Prostate cancer is the second leading cause of cancer-related death in men. Two classic cancer hallmarks are a metabolic switch from oxidative phosphorylation (OxPhos) to glycolysis, known as the Warburg effect, and resistance to cell death. Cytochrome c (Cytc) is at the intersection of both pathways, as it is essential for electron transport in mitochondrial respiration and a trigger of intrinsic apoptosis when released from the mitochondria. However, its functional role in cancer has never been studied. Our data show that Cytc is acetylated on lysine 53 in both androgen hormone-resistant and -sensitive human prostate cancer xenografts. To characterize the …

Effects Of Cocaine And/Or Heroin Use On Resting Cardiovascular Function, Shabber Syed Bs, Lina A. Shkokani Bs, Leslie H. Lundahl Phd, Renato S. Roxas Md, Philip D. Levy Md, Mark K. Greenwald Phd

Medical Student Research Symposium

Background: Regular cocaine and/or heroin use is associated with major health risks, especially cardiovascular disease (CVD), but confounded by other factors.

Objectives: We examined effects of chronic (years of regular use) and recent (past-month) use of cocaine and heroin, controlling for other factors, on resting cardiovascular function.

Methods: In a sample of cocaine and/or heroin users (N=292), we obtained data on demographics, body mass index (BMI), history of substance use, and electrocardiogram, heart rate (HR) and blood pressure (BP). Following bivariate correlations, three-block (1: demographics, BMI; 2: tobacco, alcohol, marijuana; 3: cocaine, heroin) regression analyses were …

Substrate Profiling Of The Epigenetic Erasers Hdac1 And Lsd1, Herath Mudiyansela Gedara Kavinda Eranga Herath

Wayne State University Dissertations

Regulators of chromatin structure have emerged as a key driver of transcriptional responses inside the cell. Two such groups of regulators, histone writers and erasers; the proteins, that add or remove histone post translational modifications (PTMs), have become the central players in chromatin structure. Thus, the aberrant expression of writers and erasures is a hallmark in human diseases. For example, overexpression of the erasures histone deacetylase1 (HDAC1) and lysine specific demethylase1 (LSD1) had been reported in many cancers. Currently, HDAC inhibitors have been used successfully for cancer treatment and several inhibitors targeting LSD1 are in clinical trial. To date, apart …