Physical Sciences and Mathematics Commons^™

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, …

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 …

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 …

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 …

The Dynamic Nature And Biophysical Characterization Of Isu1, Fe-S Cluster Assembly Scaffold Protein In Saccharomyces Cerevisiae, And Its Significance To Human Disease, Brianne Elizabeth Lewis

Wayne State University Dissertations

Mitochondrial Fe-S cluster biosynthesis is accomplished within yeast utilizing the biophysical characteristics of the “Isu1” scaffold protein. As a member of a highly homologous protein family, Isu1 has sequence conservation with orthologs and a conserved ability to assemble [2Fe-2S] clusters. Regardless of species, scaffold orthologs can exist in both “disordered” and “structured” conformations and is directly related to conformations utilized during Fe-cofactor assembly. During assembly, the scaffold directs the delivery and the utilization of both Fe(II) and sulfide substrates in order to produce [2Fe-2S] clusters, however Zn(II) binding can alter the activity of the scaffold with stabilizing the protein in …

Functional Characterization Of Accessory Proteins And Novel Activities In Direct And Indirect Trna Aminoacylation, Udumbara Menike Rathnayake

Wayne State University Dissertations

Indirect tRNA aminoacylation is essential for most bacteria and archaea, particularly when these species do not have genes encoding asparaginyl- and/or glutaminyl-tRNA synthetase (AsnRS and GlnRS). In the absence of AsnRS, the first step in Asn-tRNAAsn synthesis involves misacylation of tRNAAsn with aspartate to produce Asp-tRNAAsn; this reaction is catalyzed by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS). Subsequently, in bacteria, an amidotransferase called GatCAB converts Asp-tRNAAsn to Asn-tRNAAsn. An analogous, two-step processes exist to produce Gln-tRNAGln. In this case, a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) misacylates tRNAGln to produce Glu-tRNAGln, which is then converted to Gln-tRNAGln by GatCAB. The central hub of …

Functional Study Of Smyd2 Glutathionylation In Cardiomyocytes, Dhanushka Nalin Perera Munkanatta Godage

Wayne State University Dissertations

Reactive oxygen species (ROS) are important signaling molecules that contribute to the etiology of multiple muscle-related diseases, including cardiomyopathy and heart failure. There is emerging evidence that cellular stress can lead to destabilization of sarcomeres, the contractile unit of muscle. However, it is not completely understood how cellular stress or ROS induce structural destabilization of sarcomeres or myofibrils. Protein glutathionylation is one of the major protein cysteine oxidative modifications that play an important role in redox signaling and oxidative stress. In this report, we used a clickable glutathione approach in a cardiomyocyte cell line, and found that SET and MYND …

Polarized Localization Microscopy (Plm) Detects Nanoscale Membrane Curvature And Induced Budding By Cholera Toxin Subunit B (Ctxb), Abir Kabbani

Wayne State University Dissertations

The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. Many proteins and lipids interact with diverse curvature sensing and curvature generating mechanisms. Deciphering the molecular mechanisms of toxin-membrane interactions has been limited by the resolution and drawbacks of conventional experimental techniques. This study reveals the inherent membrane bending capability of cholera toxin subunit B (CTxB) through the development and implementation of Polarized Localization Microscopy (PLM). PLM is a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics and molecular sorting.

PLM combines …

Carbohydrate-Based Inducers Of Cellular Stress For Targeting Cancer Cell Metabolism, Fidelis Ndombera

Wayne State University Dissertations

ABSTRACT

CARBOHYDRATE-BASED INDUCERS OF CELLULAR STRESS FOR TARGETING CANCER CELL METABOLISM

by

FIDELIS TOLOYI NDOMBERA

May 2018

Advisor: Dr. Young-Hoon Ahn

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Metabolic reprogramming and redox control of cancer cells is vital for their proliferation, but also provides selective strategies for treating cancer. Increased generation of reactive oxygen species (ROS) and an intricate control of redox status in cancer cells relative to normal cells provide a basis for designing ROS-inducing anticancer agents. In my work, I designed, synthesized and evaluated carbohydrate-based small molecules for ROS-generation, cytotoxicity and redox signaling and stress response. Our data …

Design, Synthesis, And Reactivity Of Homo- And Heterobimetallic Complexes Bridged By A Xanthene Linker, Thilini Samangi Hollingsworth

Wayne State University Dissertations

Cooperative reactivity of bimettalics can be is observed in many different areas of chemistry and have been increasingly investigated because of the advantageous reactivity when compared to the corresponding mononuclear systems. The focus of my dissertation is on (1) investigation of the homobimetallic cooperativity in lactide polymerization catalysis; (2) investigation of the heterobimetallic cooperativity in the biomimetic studies of Mo-Cu carbon monoxide dehydrogenase (CODH) enzyme in order to make a functional model of its active site.

Three new main group bis(alkoxide) complexes Mg(OR)2(THF)2, Zn(Cl)(μ2-OR)2Li(THF) and In(OR)2(μ2-Cl)2Li(THF)2 featuring bulky alkoxide [OCtBu2Ph] were synthesized serve as metal alkoxide precursors for bimetallic lactide …

Differential Activation Of Dead Box Rna Helicases Rhlb And Rhle By Hfq/Srnas And Their Target Mrnas, Amit Kumar

Wayne State University Theses

Number of small RNA (sRNA) gene regulators have mounted in E. coli over the years whereas the number of validated protein partners has not changed considerably. Hfq has remained the only well studied global regulatory partner of sRNAs in E. coli. However, direct or indirect involvement of other protein partners has always been speculated. Study from Blasi lab has shown that CsdA, one of the five DEAD-box RNA helicases of E. coli, is required for the DsrA mediated upregulation of rpoS under cold stress condition. Previous study from our lab has identified two other DEAD-box RNA helicases, RhlB and RhlE, …

Design, Synthesis And Analysis Of Potential Photo-Activatable Cathepsin K Inhibitors, Khalin Evania Nisbett

Wayne State University Theses

Abstract

DESIGN, SYNTHESIS AND ANALYSIS OF POTENTIAL PHOTO-ACTIVATABLE CATHEPSIN K INHIBITORS

by

KHALIN NISBETT

May 2017

Advisor: Dr. Jeremy Kodanko

Major: Chemistry

Degree: Master of Science

Tightly regulated cysteine CA proteases play a major role in maintaining the homeostasis within cells. Subsequently, when these proteases are dysregulated and mislocalized they disrupt healthy cell dynamics and contribute to many life-threatening pathologies such arteriosclerosis, osteoporosis and cancer. As such many pharmaceutical companies and research teams are highly interested in these proteases as targets. One emergent strategy is the spatiotemporal control of biological processes. In relation to this, a series of spatiotemporally controlled …

The Development Of Chemical Methods To Discover Kinase Substrates And Map Cell Signaling With Gamma-Modified Atp Analog-Dependent Kinase-Catalyzed Phosphorylation, Dissanayaka Mudiyanselage Maheeka Madhubashini Embogama

Wayne State University Dissertations

Kinase-catalyzed phosphorylation plays an important role in cell physiology by regulating a myriad of cellular functions. Thus aberrant kinase activity is implicated in various diseases. Methods are needed to discover kinase substrates and map signaling pathways to explore biology and to help drug discovery. A few techniques are currently available to discover kinase substrate and map cell signaling. However, to augment kinase substrate discovery approaches, it is essential to develop alternative techniques. Pflum has recently discovered cosubstrate promiscuity of protein kinases with gamma-modified ATP analogs. Here, kinase-catalyzed biotinylation with ATP-biotin was used to develop novel tools to discover kinase substrates …

Structural Basis Of Membrane Protein Scaffolding And Signaling In Human Disease, Joshua Holcomb

Wayne State University Dissertations

Protein structural elucidation by means of X-ray crystallography is a powerful approach for both insight into a proteins biophysical properties and function. To date X-ray crystallography remains the gold standard in high resolution structural determination and serves as the basis for rational drug design for the purpose of combating a number of human diseases. Such an approach also allows for the exploitation of how various proteins interact with their substrates providing a molecular basis for their physiological function. In this dissertation, using X-ray crystallographic analysis along with other biophysical characterization methods, we seek to understand the mechanistic foundation for which …

Design, Synthesis And Biological Evaluation Of Histone Deacetylase (Hdac) Inhibitors: Saha (Vorinostat) Analogs And Biaryl Indolyl Benzamide Inhibitors Display Isoform Selectivity, Ahmed Negmeldin

Wayne State University Dissertations

HDAC proteins have emerged as interesting targets for anti-cancer drugs due to their involvement in cancers, as well as several other diseases. Several HDAC inhibitors have been approved by the FDA as anti-cancer drugs, including SAHA (suberoylanilide hydroxamic acid, Vorinostat). Unfortunately, SAHA inhibits most HDAC isoforms, which limit its use as a pharmacological tool and may lead to side effects in the clinic. In this work we were interested in developing isoform selective HDAC inhibitors, which may decrease or eliminate the side effects associated with non-selective inhibitors treatment. In addition, isoform selective HDAC inhibitors can be used as biological tools …

Development Of Chemical Tools To Investigate Protein S-Glutathionylation In Response To Metabolic Alteration, Kusal Theekshana Gayan Samarasinghe

Wayne State University Dissertations

Oxidative stress is a common characteristic of age-related diseases such as vascular diseases, diabetes and cancer. Many diseases are known to be regulated by glutathionylation. Glutathionylation is referred to as the formation of disulfide bond between a protein cysteine and a glutathione. To understand the molecular mechanisms behind the disease initiation and progression, identification of such glutathionylated proteins is important. Even though existing methods have been widely used, several limitations of these methods hinder the identification of such proteins in disease conditions. Therefore, we developed a versatile chemical method that generates clickable glutathione inside the cells. In this method, we …

Real-Time Investigation Of Bulky Lesion Bypass By Y-Family Dna Polymerase, Dpo4, Using Single Molecule Fret, Pramodha Liyanage

Wayne State University Dissertations

DNA is constantly exposed to various DNA damaging agents that are generated by various internal and external sources. Some of this damage may not be able to be repaired by cellular machineries causing DNA replication to be blocked. Once the replication fork is blocked by a DNA adduct, damage tolerance DNA polymerases, mainly Y-family, are able to restore the DNA replication by synthesizing past the DNA adduct. Benzo[a]pyrene (B[a]P) is one of the most studied environmental carcinogens. It is known to make covalent DNA adducts after metabolic activation and the bulkiness of the B[a]P adducts impose a strong barrier to …

Ligand Binding Studies Of A Peptide Targeting Helix 69 Of 23s Rrna In Bacterial Ribosomes, Hyosuk Seo

Wayne State University Dissertations

In the development of finding a peptide targeting H69 of 23S rRNA in bacterial ribosomes, phage display was employed at pH 5.5, a buffer condition previously reported of H69 preferring a closed conformation. After sequencing, several peptides were chosen through sequence alignment, followed by preparation using solid-phase peptide synthesis. The peptides were characterized using MALDI-TOF and purified with HPLC. A truncated peptide TARHIY was selected from FID assay. Through binding studies using ESI-MS, SPR, BLItz, and NMR, the binding properties of the peptide to H69 were determined, such as binding affinity, stoichiometry, and interaction site. The peptide exhibited moderate binding …

Development Of Gamma-Modified Atp Analogs To Study Kinase-Catalyzed Phosphorylations, Ahmed Eid Fouda

Wayne State University Dissertations

Kinase-catalyzed protein phosphorylation is one of the most important post-translational modifications that controls cascades of biochemical reactions. Irregularities in phosphorylation result in many diseases, such as diabetes mellitus, Parkinsons, and cancer. The development of new methods to monitor kinase-catalyzed phosphorylation is needed to decipher details of normal and diseased cell signaling. The Pflum lab recently developed several -modified ATP analogs to study kinase catalyzed phosphorylation reactions. The -modified ATP analogs have different tags, such as biotin for substrate labeling or aryl-azide for kinase substrates identification. Unfortunately, use of -modified ATP analogs was limited to in vitro studies due to the …

The Development Of Peptide Ligands To Target H69 Rrna, Danielle Nicole Dremann

Wayne State University Dissertations

ABSTRACT

THE DEVELOPMENT OF PEPTIDE LIGANDS TO TARGET H69

by

DANIELLE NICOLE DREMANN

December 2015

Advisor: Prof. Christine S. Chow

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

In the development of peptide ligands to target H69, SPPS and ESI MS was used to determine if 1) peptides could bind to modified H69 and 2) if increased affinity for the target RNA could be enhanced with modification. An alanine and arginine scan was synthesized and tested for this determination. Selected peptides were then tested using biophysical techniques such as circular dichroism and isothermal titration calorimetry. An assay was also designed to …

Interaction Forces And Reaction Kinetics Of Ligand-Cell Receptor Systems Using Atomic Force Microscopy, Anwesha Sarkar

Wayne State University Dissertations

Atomic Force Microscopy (AFM) provides superior imaging resolution and the ability to measure forces at the nanoscale. It is an important tool for studying a wide range of bio-molecular samples from proteins, DNA to living cells. We developed AFM measurement procedures to measure protein interactions on live cells at the single molecular level. These measurements can be interpreted by using proper statistical approaches and can yield important parameters about ligand-receptor interactions on live cells. However, the standard theory for analyzing rupture force data does not fit the experimental rupture force histograms. Most of the experimental measurements of rupture force data …

Dna Aptamers Selected Against Wild-Type Helix 69 Ribosomal Rna And Their Implications In Combating Antibiotic Resistance, Sakina Miriam Hill

Wayne State University Dissertations

Outbreaks of advanced antibiotic-resistant strains of microbes have hastened the need to identify new viable molecular targets for the development of novel anti-infectives. For this purpose, helix 69 (H69, or m3a 19-nucleotide (nt) hairpin motif that is highly conserved throughout phylogeny and rich in modified nucleotides, including pseudouridine () and 3-methylpseudouridine (m3) was chosen as a potential target. Helix 69, which is located in domain IV of Escherichia coli 23S ribosomal RNA (rRNA), undergoes conformational changes when in close proximity to the decoding region of 16S rRNA and transfer RNAs (tRNAs) in the peptidyl-transferase center (PTC). Functionally, the exact biological …

Dynamics Of Biopolymers On Nanomaterials Studied By Quasielastic Neutron Scattering And Mdsimulations, Gurpreet Dhindsa

Wayne State University Dissertations

Neutron scattering has been proved to be a powerful tool to study the dynamics of biological systems under various conditions. This thesis intends to utilize neutron scattering techniques, combining with MD simulations, to develop fundamental understanding of several biologically interesting systems. Our systems include a drug delivery system containing Nanodiamonds with nucleic acid (RNA), and two specific model proteins, β-Casein and Inorganic Pyrophosphatase (IPPase).

RNA and nanodiamond (ND) both are suitable for drug-delivery applications in nano-biotechnology. The architecturally flexible RNA with catalytic functionality forms nanocomposites that can treat life-threatening diseases. The non-toxic ND has excellent mechanical and optical properties and …

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 …

Effects Of Statin Drugs And Tocotrienol Rich Fraction Supplementation In Chronic Hemodialysis Patients And Metabolomic Profile, Eno Latifi

Wayne State University Theses

Chronic kidney disease (CKD) is known as a heterogeneous disorder which currently is on the rise and lately has been classified as a public health issues in the United State and worldwide. CKD is an irreversible and progressive disease which can lead to kidney failure, and this is depicted by the advanced stage of the disorder when it reaches the point, that is classified as end stage of renal disease (ESRD) (Stage 5 of CKD) (eGRF <15 mL/min/ 1.73 m2 working capacity), where both organs are in a total or permanent kidney failure. End-Stage renal disease patients, on hemodialysis have been associated to experience an accelerated form of atherosclerosis, which is induced by inflammation, impairment of antioxidant system and elevated oxidative stress. Since the problem effecting ESRD patients is multifactorial, the objective of this investigation is to explore and look at the effects of supplementing with vitamin E-tocotrienol rich fraction (TRF), a micronutrient which has anti-inflammatory, antioxidant, and lipid lower capabilities into tackling these comorbid conditions experienced by this population. Therefore the aims of this investigation will be to explore changes in lipid profiles, inflammatory markers, and oxidative status, as well as look at any changes in metabolomic profiles. It was hypothesized that by supplementing with TRF a vitamin E, for 16 weeks in ESRD patients undergoing hemodialysis, it may help reverse and/or improve, oxidative status, inflammatory markers, increase antioxidants status and improve lipid profiles.

The study was double-blinded, randomized, parallel, placebo-controlled design trial, of 81 adult patients undergoing chronic hemodialysis at Great Lake Dialysis Clinic, Detroit MI, where …

Biophysical Studies Of Mutant H69 Rna Constructs, Daya Nidhi Kharel

Wayne State University Theses

The importance of helix 69 (H69) of the large ribosomal subunit is emphasized by its location in the ribosome and key contacts with protein factors and tRNAs during different steps of protein synthesis. The presence of three highly conserved pseudouridine modifications in H69 has a significant impact on modulation of the ribosome conformations. Specific nucleotides of H69 may be necessary for the function of this helix-loop RNA, as some mutations are deleterious to bacterial growth. In this study, we investigated the effects of single nucleotide mutations in a 19-nucleotide H69 construct with and without pseudouridines on the RNA stability and …

Genetic And Biochemical Studies Of Human Apobec3h Enzyme, Thisari Sachithra Aluthgama Guruge

Wayne State University Theses

The AID/APOBEC enzymes are DNA/RNA cytosine deaminases with important functions in innate and adaptive immunity. APOBEC3 enzymes play a crucial role in restricting the replication of exogenous retroviruses such HIV-1 and endogenous retrotransposition events. In particular, APOBEC3 enzymes have evolved in humans by gene duplication to compose seven members. APOBEC3H is highly diverse in its allelic sequence and the distribution is population-specific. It has evolved under strong positive selection over millions of years. To study the catalytic mechanism, structure, function and the underlying cause of its high diversity, a high resolution crystal or NMR structure is required are not yet …

Hiv-1 Rna Dimerization At Single Molecule Level, Hansini R. Mundigala

Wayne State University Dissertations

The Dimerization Initiation Sequence (DIS) is a conserved hairpin-loop motif on the 5' UTR of the HIV-1 genome. It plays an important role in genome dimerization through formation of a "kissing complex" intermediate between two homologous DIS sequences. This bimolecular kissing complex ultimately leads to the formation of an extended RNA duplex. Understanding the kinetics of this interaction is key to exploiting DIS as a possible drug target against HIV. We wish to report a novel study that makes an important contribution to understanding the dimerization mechanism of HIV-1 RNA in vitro. Our work has employed single-molecule fluorescence resonance energy …

Investigation Into The Binding Interactions Of Klenow Fragment To Dna Modified With Carcinogens Af And Aaf Using Surface Plasmon Resonance, Ashley M. Floyd

Wayne State University Dissertations

The two major forms of DNA adducts from the carcinogen N-acetoxyacetyl-2-aminofluorene, N-(deoxygunanonsin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), are both known to impede replication, though in different ways. AAF is a strong block to replication leading to frameshift mutations, while the AF adduct is more easily bypassed, causing base substitutions. Surface plasmon resonance (SPR) was used to study the binding of exonuclease deficient E. coli polymerase I, Klenow fragment (KF), to DNA modified with AF or AAF at two locations: as a templating base or in the last formed base pair. KF binding to the modified DNA bases was also monitored to …

Characterization Of Initial Iron Binding Location And The Structure/Iron Binding Site On S.Cerevisiae Isu And On D.Melanogaster Frataxin, Andria V. Rodrigues

Wayne State University Dissertations

Iron-induced free radical damage has been implicated in the pathology of diseases of iron overload such as Friedreich's Ataxia, a genetic disorder characterized by an accumulation of iron in actively metabolizing tissues ultimately leading to cardio- and neuro- degeneration and cell death. It is caused by an inability to synthesize the mitochondrial protein, frataxin. Frataxin has been shown by numerous groups to be a part of the iron-sulfur cluster (ISC) multicomplex, where it functions in the capacity of a potential iron provider and an allosteric modulator of both the cysteine desulfurase and scaffold protein ISU. My research has been focused …