Biochemistry, Biophysics, and Structural Biology Commons^™

Mach: A Model For Explaining Molecular And Cellular Mechanisms, Caleb M. Trujillo

Open Access Dissertations

Biologists use mechanistic explanations to understand behaviors of the immense complexity of molecular and cellular systems. In undergraduate biology courses, students are expected to explain molecular and cellular mechanisms, but teaching this skill presents many challenges due to the highly abstract, intangible nature of the cellular world, the influence of everyday language, and the tendency of students to overestimate how much they can explain. Therefore, across three studies this dissertation addresses these obstacles to teach undergraduate biology students to explain molecular and cellular mechanisms. ^ The first step was to model how biology experts explain molecular and cellular mechanisms, and …

The Therapeutic Targeting Of Folate Receptor Alpha Positive Tumors Via Folate Receptor Selective Novel 5- And 6- Substituted Pyrrolo [2,3-D]Pyrimidine Antifolates", Shermaine Kimberly Mitchell-Ryan

Wayne State University Dissertations

Ovarian Cancer is the fifth leading cause of cancer-related death of women in the United States. Epithelial Ovarian Cancer (EOC) constitutes 85-90% of malignancies within the ovary, with an alarming majority of these cases diagnosed at advanced stage. While most patients are initially highly responsive to the current treatment standard, there is a very high probability that they will recur with a drug resistant fatal disease. Currently there is no validated comprehensive model of disease progression for ovarian cancer, although tremendous progress has been made in understanding the origin of this disease and a putative precursor lesion has been identified …

Photodynamic Therapy As An Effective Therapeutic Approach In Mame Models Of Triple Negative And Inflammatory Breast Cancers, Neha Aggarwal

Wayne State University Dissertations

Introduction: Photodynamic therapy (PDT) is a minimally invasive, FDA approved therapy for

treatment of several indications including endobronchial and esophageal cancers that are

accessible to light. Triple negative breast cancer (TNBC) and inflammatory breast cancer (IBC)

are aggressive and lethal subtypes of breast cancer that spread to chest wall and dermal

lymphatics, respectively, sites that would be accessible to light. Both TNBC and IBC patients

have a relatively poor survival rate due to lack of targeted therapies. Use of PDT is

underexplored for breast cancers but has been proposed for treatment of subtypes for which a

targeted therapy is unavailable. …

Proteasome Inhibition As A Potential Anti-Breast Cancer Therapy: Mechanisms Of Action And Resistance-Reversing Strategies, Rahul Rajesinh Deshmukh

Wayne State University Dissertations

AMPK activation and Ubiquitin Proteasome System (UPS) inhibition have gained great attention as therapeutic strategies for the treatment of certain types of cancers. While AMPK serves as a master regulator of cellular metabolism, UPS regulates protein homeostasis. Although the crosstalk between them is suggested, the relationship between these two important pathways is not very clear. We observed that proteasome inhibition leads to AMPK activation in human breast cancer cells. We report that a variety of proteasome inhibitors activate AMPK in all of the tested cancer cell lines. Our data using Liver Kinase B1 (LKB1)-deficient cancer cells suggests that proteasome inhibitor-induced …

Design And Synthesis Of Isatin-Based Caspase Inhibitors For Ruthenium Caging Applications, Kasun Chinthaka Ratnayake

Wayne State University Theses

ABSTRACT

DESIGN AND SYNTHESIS OF ISATIN BASED CASPASE INHIBITORS FOR RUTHENIUM CAGING APPLICATIONS

by

KASUN CHINTHAKA RATNAYAKE

August 2015

Advisor: Jeremy J. Kodanko, Ph.D.

Major: Chemistry (Organic)

Degree: Master of Science

Apoptosis is the energy dependent programmed cell death. Improper function of apoptosis could lead to diseases such as cancers, strokes, Alziemer’s disease. Caspases are the enzymes involved in the later stage of this process. Peptidyl and non-peptidyl caspase inhibitors have been synthesized recently. These non-peptidyl compound classes which consist of pyrrolidinyl-5-sulfo isatins have showed a greater potency against executioner caspases, caspase-3 and -7. According to literature and for further …

The Structural Heterogeneity And Dynamics Of Base Stacking And Unstacking In Nucleic Acids, Ada Anna Sedova

Legacy Theses & Dissertations (2009 - 2024)

Base stacking provides stability to nucleic acid duplexes, and base unstacking is involved in numerous biological functions related to nucleic acids, including replication, repair, transcription, and translation. The patterns of base stacking and unstacking in available nucleic acid crystal structures were classified after separation into their individual single strand dinucleotide components and clustering using a k-means-based ensemble clustering method. The A- and B-form proximity of these dinucleotide structures were assessed to discover that RNA dinucleotides can approach B-form-like structures. Umbrella sampling molecular dynamics simulations were used to obtain the potential of mean force profiles for base unstacking at 5'-termini for …

Francisella Tularensis Catalase Restricts Immune Function By Impairing Trpm2 Channel Activity, Nicole Lynn Flaherty

Legacy Theses & Dissertations (2009 - 2024)

As an innate defense mechanism, macrophages produce reactive species that weaken pathogens and serve as secondary messengers to modify signaling responses involved in immune function. The gram-negative bacterium F. tularensis utilizes its antioxidant armature to limit the host immune response but the mechanism behind this suppression has not been defined. Here we establish that F. tularensis limits Ca2+ entry thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild-type (LVS) or catalase deficient F. tularensis (∆katG) show distinct profiles in their H2O2 scavenging capacity, 1 pM/sec and 0.015 pM/sec, respectively. Murine alveolar macrophages infected with ∆katG display distinct …

Rna Aptamer Mediated Manipulation Of The 70 Kilodalton Heat Shock Protein Chaperone Machinery, Deepak Thirunavukarasu

Legacy Theses & Dissertations (2009 - 2024)

Protein quality control involves refolding of damaged proteins and facilitating degradation of irreparable proteins. Understanding the protein quality control mechanism is critical, since defects in it has been implicated in a number of age-related diseases like neurodegenerative diseases and also in cancer. A vast network of molecular chaperones and proteolytic systems collaborate to maintain protein quality control. The 70 kilodalton Heat shock protein (Hsp70) is a highly conserved and ubiquitous chaperone, which interacts with a variety of protein substrates including newly synthesized polypeptides, unfolded, partially misfolded and native proteins to maintain protein quality control. Hsp70 chaperone function is coupled to …

Novel Nmr Based Technologies To Study Macromolecular Structures, Subhabrata Majumder

Legacy Theses & Dissertations (2009 - 2024)

Nuclear Magnetic Resonance Spectroscopy (NMR) is one of the principle tools in structural biology to probe macromolecular structures and interactions. The atomic resolution afforded by this technique has been widely used to probe protein-protein, and protein-ligand interactions in-vitro. However, the natural milieu of the proteins is the living cell and the cellular cytoplasm is extremely heterogeneous. The NMR studies of folded protein in-cell, till now, have been limited by non-specific interactions of the cytosol. This thesis outlays a general methodology to study protein structure/interactions inside the living cells using NMR. In a closely related objective, it also describes the use …

Rna Aptamers For Molecular Chaperones Hsp27 And Hsp90, Sathishkumar Kumar Munusamy

Legacy Theses & Dissertations (2009 - 2024)

Hsp90 and Hsp27 are members of the heat shock protein family of chaperones that perform multiple roles in cellular maintenance through protein folding and inhibition of apoptosis. They are abundantly expressed in cells and are over-expressed during conditions of stress. Hsp90 requires ATP for its chaperone function while Hsp27 self-associates into higher order oligomers enclosing its substrate. Their ability to interact with other proteins or with themselves lies at the heart of their mechanisms. The specific consequences of each of their interactions on global cellular health have not yet been fully discovered. The sheer diversity of proteins that interact with …

Structure And Stability Of Amyloid Fibrils Studied By Advanced Vibrational Spectroscopy, Marudachalam Shanmugasundaram

Legacy Theses & Dissertations (2009 - 2024)

Protein misfolding often leads to the formation of refractory protein aggregates like amyloid fibrils. These fibrils possess a highly ordered structure and are implicated in over 25 severe diseases including Alzheimer’s, Parkinson’s and prion diseases. This work was focused on understanding the morphology and conformation of amyloid fibrils and their stability after formation. The deconstruction of fibrils as well as other aggregates like inclusion bodies under mild conditions was also investigated using Archaeal chaperones.

Myoepithelial Cell Morphogenesis And Differentiation In The Mouse Submandibular Salivary Gland In Development And Disease, Elise Marie Gervais

Legacy Theses & Dissertations (2009 - 2024)

Organogenesis is the process by which tissues organize, gain considerable size, and undergo cellular differentiation or specialization to form fully functional organs. To study the processes involved in organogenesis of branched organs, the mouse submandibular salivary gland is frequently used as a model system, as it can undergo morphogenesis and differentiation and be genetically manipulated ex vivo. The mouse submandibular salivary gland undergoes a specific process of outgrowth and invagination known as branching morphogenesis which allows for the significant increase in gland size and complexity, as well as maximization of surface area for secretion of saliva. Surrounding the mouse submandibular …

Genome-Scale Analyses Of Transcription And Transcriptional Regulation In Bacteria, Devon Marie Fitzgerald

Legacy Theses & Dissertations (2009 - 2024)

The textbook model of bacterial transcription regulation posits that promoters occur immediately upstream of genes and that transcription factors (TFs) modulate transcription through promoter-proximal binding. However, the recent application of unbiased genome-wide approaches, such as ChIP-seq and RNA-seq, has revealed a much more complex picture, including TF binding and transcription initiation occurring in unexpected locations. This dissertation describes the use of deep sequencing-based approaches to evaluate the genome-wide binding of transcription-related proteins and identify locations of transcription initiation. I have assessed the genome-wide binding of three Escherichia coli TFs and an alternative σ factor. Additionally, I have analyzed genome-wide patterns …

Vitamin D Regulates Metabolic Gene Expression, Glutamate And Glutamine Utilization, And Mitochondrial Function In Human Mammary Epithelial Cells, Sarah Beaudin

Legacy Theses & Dissertations (2009 - 2024)

Exposure to 1,25-Dihydroxyvitamin D (1,25D) decreases proliferation and induces differentiation in telomerase-immortalized human mammary epithelial (hTERT-HME1) cells. The studies described here addressed the mechanisms by which these effects are exerted. Microarray experiments were used to identify a subset of metabolic genes and pathways that are altered by 1,25D. In particular, genes involved in glutamate and glutamine utilization, including SLC1A1 and GLUL, were studied. Interestingly, qPCR analysis in a panel of six cell lines, representing either normal epithelial tissue or breast cancer, demonstrated diverse gene expression responses to 1,25D. In an isogenic model of mammary cell transformation, 1,25D altered gene expression …

Using A Novel Multiplexing Method To Track Cell Populations And Cytodifferentiation During Development Of The Submandibular Salivary Gland, Charles Thomas Manhardt

Legacy Theses & Dissertations (2009 - 2024)

The development of submandibular salivary glands is complex and requires coordination of specific signaling events. Submandibular salivary glands originate as an epithelial invagination into the adjacent mesenchyme that leads to a single stalk and end bud; this end bud will go through a clefting process. Numerous rounds of clefting will lead to a fully developed salivary gland by this process, which is known as branching morphogenesis. As the gland undergoes morphogenesis, specific cues leading to differentiation of multiple cell types and even epithelial sub classes are required. By the later stages of development the glands are fully innervated, have an …

Nosh-Aspirin (Nbs-1120), A Dual Nitric Oxide And Hydrogen Sulfide-Releasing Hybrid, Reduces Inflammatory Pain, Miriam D. Fonseca, Fernando Q. Cunha, Khosrow Kashfi, Thiago M. Cunha

Publications and Research

The development of nitric oxide (NO)- and hydrogen sulfide (H2S)-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) has generated more potent anti-inflammatory drugs with increased safety profiles. A new hybrid molecule incorporating both NO and H2S donors into aspirin (NOSH-aspirin) was recently developed. In the present study, the antinociceptive activity of this novel molecule was compared with aspirin in different models of inflammatory pain. It was found that NOSH-aspirin inhibits acetic acid-induced writhing response and carrageenan (Cg)-induced inflammatory hyperalgesia in a dosedependent (5–150 lmol/kg, v.o.) manner, which was superior to the effect of the same doses of aspirin. NOSH-aspirin’s antinociceptive effect was also …

Trends In Midwinter Counts Of Bald Eagles In The Conterminous United States, 1986-2005, Karen Steenhof, Laura Bond, Leah L. Dunn

Biomedical Research Institute Publications and Presentations

Each January, several hundred individuals count eagles along standard, non-overlapping survey routes as part of a nationwide Midwinter Bald Eagle survey. Nationwide counts of eagles were coordinated by the National Wildlife Federation from 1979 until 1992, when the Bureau of Land Management’s Raptor Research and Technical Assistance Center assumed responsibility for overseeing the count. Responsibility for count coordination shifted to the National Biological Survey (1993-1996) and later to the U.S. Geological Survey (USGS), Forest and Rangeland Ecosystem Science Center, Snake River Field Station.

Initial objectives of the survey were to establish an index to the total wintering Bald Eagle population …

Clique Topology Reveals Intrinsic Geometric Structure In Neural Correlations, Chad Giusti, Eva Pastalkova, Carina Curto, Vladimir Itskov

Department of Mathematics: Faculty Publications

Detecting meaningful structure in neural activity and connectivity data is challenging in the presence of hidden nonlinearities, where traditional eigenvalue-based methods may be misleading. We introduce a novel approach to matrix analysis, called clique topology, that extracts features of the data invariant under nonlinear monotone transformations. These features can be used to detect both random and geometric structure, and depend only on the relative ordering of matrix entries. We then analyzed the activity of pyramidal neurons in rat hippocampus, recorded while the animal was exploring a 2D environment, and confirmed that our method is able to detect geometric organization using …

Relationship Between Heat-Labile Enterotoxin Secretion Capacity And Virulence In Wild Type Porcine-Origin Enterotoxigenic Escherichia Coli Strains, Prageeth Wijemanne, Jun Xing, Emil M. Berberov, David H. Francis, Rodney A. Moxley

School of Veterinary and Biomedical Sciences: Faculty Publications

Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system(T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme- linked immunosorbent assay tomeasure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had …

Temperatures Of Storage Areas In Large Animal Veterinary Practice Vehicles In The Summer And Comparison With Drug Manufacturers’ Storage Recommendations, Jeff D. Ondrak, Meredyth L. Jones, Virginia R. Fajt

School of Veterinary and Biomedical Sciences: Faculty Publications

Background: Large animal veterinarians carry drugs in their practice vehicles in storage areas that are not typically refrigerated. The most common upper limits of manufacturers’ storage temperatures for United States (U.S.)-approved non-refrigerated drugs are 25 or 30 °C. Because ambient temperatures in many locations in the U.S. exceed these temperatures during the summer, we measured storage area temperatures over 4 months in the summer of 2013 to evaluate the extent to which labeled storage temperatures are exceeded.

Methods: A convenience sample of 12 vehicles from 5 central Texas practices and 12 vehicles from 4 south central Nebraska practices was …

Screening Of Natural Products For Their Effect On Kallikrein-Kinin System: Potential Implications In The Treatment Of Hereditary Angioedema, Hassan Abdu Madkhali

Electronic Theses and Dissertations

Plasma prekallikrein (PK) activation exerts both physiological and pathological effects within the cardiovascular system and central nervous system (CNS). Activated PK (kallikrein) controls cytokine release from human mononuclear cells and controls both the intrinsic blood coagulation and the alternative complement cascades. The uncontrolled activation of these cascades results in the development of pathological pain and angioedema. Increased plasma kallikrein levels are associated with hereditary angioedema (HAE) and myocardial infarction, whereby kallikrein amplifies the generation of activated factor XII (FXIIa) and bradykinin (BK) release from high molecular weight kininogen (HMWK). The primary objective of this study was to screen natural products …

Hiv Integrase Mechanisms Of Resistance To Raltegravir, Elvitegravir, And Dolutegravir, Kyla Nicole Ross

Wayne State University Theses

ABSTRACT

HIV INTEGRASE MECHANISMS OF RESISTANCE TO RALTEGRAVIR, ELVITEGRAVIR, AND DOLUTEGRAVIR

by

KYLA ROSS

December 2015

Advisor: Dr. Ladislau Kovari

Major: Biochemistry and Molecular Biology

Degree: Master of Science

HIV-1 integrase (HIV-1 IN or IN) is a multimeric enzyme that integrates the HIV-1 genome into the chromosomes of infected CD4+ T-cells. Currently there are three FDA approved HIV-1 IN strand transfer inhibitors (INSTIs) used in clinical practice: raltegravir (RAL), elvitegravir (ELV), and dolutegravir (DTG). The [Q148H], [Q148H, G140S], [Q148R], [Q148R, G140A] and [N155H, E92Q] mutations decrease IN susceptibility to RAL and ELV and may result in therapeutic failure. As an …

Synergetic Effect Of Permethrin And Dmh On Anti-Oxidation And Damage Response Genes, Karim Mohamed

Wayne State University Theses

The objective of this study was to evaluate the Synergetic Effect of Permethrin and 1,2-Dimethylhydrazine (DMH) on Anti-oxidation and Damage Response genes. The animal models used for this study were 8 week old C57/blk6 female mice. The mice were fed a Folate Adequate (FA) diet. At 8 weeks, the mice were intraperitoneally injected with pesticides. Three 8-week old female mice were injected intraperitoneally with a mix of cis and trans permethrin (75%:25% respectively) at 30% median lethal dose (96 mg/kg body weight). Permethrin was dissolved in corn oil and injected 24 hours before sacrifice. In three other mice, DMH was …

Therapeutic Targeting Of Bmp2 In Nf1-Deficient Malignant Peripheral Nerve Sheath Tumors (Mpnsts), Sidra Ahsan

Wayne State University Dissertations

Neurofibromatosis type I (NF1)-deficient malignant peripheral nerve sheath tumor (MPNST) is an aggressive tumor for which the standard treatment is surgical removal with wide margins, often leaving behind cancer cells needing chemotherapy. RAS-GRD is the most widely studied functional target of NF1 implicated in tumorigenesis, however, therapeutic interventions targeting RAS activity have met with limited success. Using gene expression profiling, our lab identified BMP2-SMAD1/5/8 signaling pathway as a therapeutic target in MPNSTs, independent of the NRAS and MEK1/2 regulation. The overall goal of my research was to validate the significance of BMP2 in MPNSTs in novel cellular models, study the …

Expression And Functional Analysis Of Lipids And Glycolipids From The Mammal-Dwelling Stages Of Trypanosoma Cruzi, Felipe Gazos Lopes

Open Access Theses & Dissertations

Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine …

Role Of Small Molecules In Rescuing Protein Folding Under Oxidative Stress, Mahmoud Fawzi Megahed Helal Khalil

Open Access Theses & Dissertations

Increased levels of nitrosative stress intracellularly within the endoplasmic reticulum is a key factor involvled in the pathogenesis of both Parkinson's (PD) and Alzheimer's (AD) diseases. Previous in-vitro studies in our lab showed that increased levels of nitrosative stress lead to aggregation of misfolded proteins and formation of Lewy Bodies, the main biomarker of PD and AD diseases. Although this was mainly through nitrosylation of Protein Disulfide Isomerase (PDI), the chief endoplasmic reticulum (ER) resident oxidoreductase chaperone responsible for maturation of disulfide-bond-containing proteins, we demonstrate in this project that increased levels of nitrosative stress has an additional direct effect on …

Using Antenna Tile-Assisted Substrate Delivery To Improve Detection Limits Of Deoxyribozyme, Amanda J. Cox

HIM 1990-2015

One common limitation of enzymatic reactions is the diffusion of a substrate to the enzyme active site and/or the release of the reaction products. These reactions are known as diffusion –controlled. Overcoming this limitation may enable faster catalytic rates, which in the case of catalytic biosensors can potentially lower limits of detection of specific analyte. Here we created an artificial system to enable deoxyribozyme (Dz) 10-23 based biosensor to overcome its diffusion limit. The sensor consists of the two probe strands, which bind to the analyzed nucleic acid by Watson-Crick base pairs and, upon binding re-form the catalytic core of …

Development Of Novel Fluorescent Tools For Investigating Virulence Factors And Drug Susceptibility In Mycobacterium Tuberculosis, Kaley Wilburn

HIM 1990-2015

Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), a life-threatening disease primarily affecting the lungs that infects about one third of the world's population and causes 1.3 million deaths annually. It is estimated that TB has been infecting humans for around 70,000 years and has killed more people than any other infectious disease. The highly effective, persistent, and multifaceted virulence strategies that have allowed Mtb to continue to spread and thrive for so long are still poorly understood at the molecular level. This lack of knowledge contributes to ongoing challenges to curing TB. Although drugs capable of killing …

Detection Of Drug-Resistance Conferring Snps In Mycobacterium Tuberculosis Using Binary Dnazymes, Marina Addario

HIM 1990-2015

Mycobacterium tuberculosis (Mtb) is the pathogen that causes Tuberculosis (TB) and is responsible for an average of 1.5 million deaths annually. Although a treatment regimen does exist, Multi-Drug Resistant (MDR-TB) and eXtremely Drug Resistant (XDR-TB) TB strains are becoming a more prevalent concern partly due to failure of patient compliance with the current six to nine month drug treatment regimen. The current diagnostic methods are not able to identify these MDR and XDR-TB strains efficiently therefore more effective point-of-care (POC) diagnostics and drug susceptibility testing (DST) are urgently needed to detect drug resistance and facilitate prompt, appropriate treatment plans. In …

Elucidating The Molecular Pathway Of Atypical Plasmodium Falciparum Kinases Through Substrate Characterization, Daniel Segarra

HIM 1990-2015

Plasmodium falciparum, the organism responsible for the most prevalent and most virulent cases of malaria in humans, poses a major burden to the developing world. The parasite is increasingly developing resistance to traditional therapies, such as chloroquine, so the need to determine novel drug targets is more prevalent than ever. One such method involves targeting proteins unique to the malarial proteome that do not have homologues in humans. An especially promising group of targets are protein kinases, which are involved in many different biochemical pathways within the cell. Eukaryotic cell cycle progression is moderated by a family of protein kinases …