Biochemistry, Biophysics, and Structural Biology Commons^™

Differential Transcription Of Cytochrome P450s And Glutathione S Transferases In Ddt-Susceptible And -Resistant Drosophila Melanogaster Strains In Response To Ddt And Oxidative Stress, Lijie Sun, Brandi Schemerhorn, Amber Jannasch, Kent R. Walters Jr., Jiri Adamec, William M. Muir, Barry R. Pittendrigh

Department of Biochemistry: Faculty Publications

Metabolic DDT resistance in Drosophila melanogaster has previously been associated with constitutive over-transcription of cytochrome P450s. Increased P450 activity has also been associated with increased oxidative stress. In contrast, over-transcription of glutathione S transferases (GSTs) has been associated with resistance to oxidative stress. However, little is known in regards to the impact of xenobiotics on induction of P450s and GSTs and if there exist differences in inducibility between the pesticide susceptible and resistant strains. Thus, we investigated the transcriptional expression of GSTs and P450s in DDT resistant (Wisconsin) and susceptible (Canton-S) Drosophila strains in response to …

A Cell Electrofusion Microfluidic Device Integrated With 3d Thin-Film Microelectrode Arrays, Ning Hu, Jun Yang, Shizhi Qian, Sang W. Joo, Xiaolin Zheng

Mechanical & Aerospace Engineering Faculty Publications

A microfluidic device integrated with 3D thin film microelectrode arrays wrapped around serpentine-shaped microchannel walls has been designed, fabricated and tested for cell electrofusion. Each microelectrode array has 1015 discrete microelectrodes patterned on each side wall, and the adjacent microelectrodes are separated by coplanar dielectric channel wall. The device was tested to electrofuse K562 cells under a relatively low voltage. Under an AC electric field applied between the pair of the microelectrode arrays, cells are paired at the edge of each discrete microelectrode due to the induced positive dielectrophoresis. Subsequently, electric pulse signals are sequentially applied between the microelectrode arrays …

Thiol Peroxidases Mediate Specific Genome-Wide Regulation Of Gene Expression In Response To Hydrogen Peroxide, Dmitri E. Fomenko, Ahmet Koc, Natalia Agisheva, Michael Jacobsen, Alaattin Kaya, Mikalai Malinouski, Julian C. Rutherford, Kam-Leung Siu, Dong-Yan Jin, Dennis R. Winge, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H₂O₂ response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to …

Differential Precipitation And Solubilization Of Proteins., Barry Ryan

Books/Book Chapters/ Proceedings

Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physico-chemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g. His-Tag, Size Exclusion etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during over-expression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilised so as to reform functional, correctly folded …

Collagen Matrices As An Improved Model For In Vitro Study Of Live Cells Using Raman Microspectroscopy, Franck Bonnier, Peter Knief, Aidan Meade, Jennifer Dorney, Kunal Bhattacharya, Fiona Lyng, Hugh Byrne

Conference papers

Due to its high lateral resolution, Raman microspectrsocopy is rapidly becoming an accepted technique for the subcellular imaging of single cells. Although the potential of the technique has frequently been demonstrated, many improvements have still to be realised to enhance the relevancy of the data collected. Although often employed, chemical fixation of cells can cause modifications to the molecular composition and therefore influence the observations made. However, the weak contribution of water to Raman spectra offers the potential to study live cells cultured in vitro using an immersion lens, giving the possibility to record highly specific spectra from cells in …

Role Of Notch Signaling In T Cell Polarization, Shilpa Keerthivasan

Dissertations

The differentiation of CD4+ T cells to different effector lineages in response to pathogenic stimuli is the core of the adaptive immune system. One of the effector subsets recently discovered is Thelper 17 (Th17) and it plays a predominant role in autoimmune diseases and inflammatory disorders.

In my thesis, I aimed to study the role of Notch cell surface receptors in Th17 differentiation. Using in vitro Th17 differentiation assays of human naïve CD4+ T cells, I have shown that Notch signaling, particularly Notch1, plays a crucial role in Th17 polarization. By using pharmacological inhibitors and specific knockdown of Notch1, I …

Characterization Of Poly (Rc) Binding Protein (Pcbp2) And Frataxin, Sudipa Ghimire-Rijal

Wayne State University Theses

Iron is a micronutrient that acts as a cofactor in many prosthetic groups involving itself in almost every biological process. Iron is the key component in our body fluid, flowing in our veins all the time. Iron deficiency disorders affects more than 9 million people worldwide. Similarly, a high level of iron is associated with various disorders which suggest that in order for body to function properly level of iron should be tightly regulated. Many iron binding proteins help in maintaining cellular iron homeostasis by keeping iron in reduced form.

Working on the hypothesis that Poly (rC) Binding Protein family …

Preparation And Crystal Structures Of Chiral And Non-Chiral Mixed Ligand Copper Complexes Containing N-Methyl Imidazole And Various N-Phthaloylalanines, Hiral Patel

Theses, Dissertations and Culminating Projects

A quasiracemate is the cocrystallization of two similarly shaped but chemically distinct molecules of opposite handedness. There has been extensive work done in the field of organic quasiracemates by Dr. Kraig A. Wheeler. Limited work has been done in inorganic quasiracemates. The goal of this study was to prepare various metal based enantiomers, racemates, and quasiracemates using A-phthaloylalanines, and TV-methylimidazole as ligands bound to copper arranged in a centrosymmetric pattern. In this study, we developed a method to prepare A-phthaloylalanines and its copper complexes. Three complexes have yielded crystal structures: a racemate, bis(7V-methylimidazole) bis(D,L-3-chloro-7V-phthaloylalanato)copper(II); a quasiracemate, bis(A-methyhmidazole)(L-3-chloro-iV-phthaloylalanato)(D-3-methyl-7V-phthaloylalanato) copper(II); and an …

Single Cell Analysis For The Characterization Of Cell Populations Using A Live Cell Array, Maureen Ann Walling

Legacy Theses & Dissertations (2009 - 2024)

In the past decade, the shift from whole cell population analyses towards single cell measurement methods and techniques is based on experimental results that reveal significant levels of non-genetic heterogeneity in clonal cell populations. This heterogeneity manifests in multiple aspects of cell activity and is, in part, a result of stochastic noise in processes leading to gene expression, namely transcription and translation. The growing understanding of this occurrence has led to the development of methods to monitor and analyze heterogeneity for a more thorough description of cell populations and overall activity.

Structural Interactions And Dynamics Of Disease Related Proteins By Using Nmr Spectroscopy, Shadakshara Swamy Puttamadappa

Legacy Theses & Dissertations (2009 - 2024)

Nuclear Magnetic Resonance (NMR) is a powerful spectroscopic technique to study the structure, molecular interactions, and dynamics of proteins. Modern NMR instrumentation, advancements in experimental techniques and revolutionary developments in recombinant DNA technology have made NMR a versatile and very convenient tool for biomolecule characterization.

In-Cell And In Vitro Studies Of Disease Related Protein-Protein Interactions Using Nmr-Spectroscopy, Andres Yudiel Maldonado

Legacy Theses & Dissertations (2009 - 2024)

The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule that plays a central role in the etiology of diabetes, inflammation, and neurodegeneration. The cytoplasmic domain of RAGE, ctRAGE, is critical for RAGE-dependent signal transduction. As the most membrane proximal event, mDia1 binds to ctRAGE and is essential for RAGE ligand-stimulated phosphorylation of AKT and cell proliferation/migration. We show that ctRAGE contains an unusual alpha-turn that mediates the mDia1-ctRAGE interaction and is required for RAGE dependent signaling. The results establish a novel mechanism through which an extracellular signal initiated by RAGE ligands regulates RAGE signaling in …

Distinct Functions Of The Fshr Ck2 Consensus Sequence And Ck2 Kinase Activity In Fsh Action, Kerri S. Kluetzman

Legacy Theses & Dissertations (2009 - 2024)

Human reproduction in the female is a complex process requiring coordinated control of gonadotropic hormone signaling in a temporal, spatial and contextually defined manner. Paracrine and autocrine signaling between granulosa cells and oocytes of the developing ovarian follicle result in production of steroids, inhibin and growth factors that dictate maturation, ovulation or atresia. Follicular stimulating hormone (FSH) stimulation of the FSH receptor (FSHR), a G-protein coupled receptor (GPCR) is a key regulator of these processes. Recent developments in GPCR molecular and structural biology have helped define activation and signaling mechanisms that will aid in further defining the etiology of infertility …

The Role Of Trm9 In Stress Responses, Ashish Ravindra Patil

Legacy Theses & Dissertations (2009 - 2024)

Cells need to respond appropriately to environmental changes in order to maintain homeostasis. The cellular response to an environmental stress is regulated at transcriptional, translational and post translational levels. The tRNA, which acts as an adaptor molecule between the mRNA and the protein, plays an important role in the translational regulation of cellular responses to stress and is one of the most heavily modified biomolecules. In Saccharomyces cerevisiae , the wobble uracil of the tRNA(3'-UCU-5') Arg, tRNA(3'-UUC-5') Glu and certain other specific tRNAs are modified to 5-methoxycarbonylmethyluridine (mcm5U) and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) residues by the tRNA methyltransferase 9 (Trm9). Modifications at …

Regulation Of The Aryl Hydrocarbon Receptor And The Signal Transducer And Activator Of Transcription 1 By Long-Term Estrogen Exposure In Breast Carcinoma Cells, Neal Englert

Legacy Theses & Dissertations (2009 - 2024)

The risk of developing breast cancer is known to be associated with a woman's lifetime exposure to estrogens, both endogenous and exogenous. Increased exposure to estrogens stimulates cellular proliferation, which is a widely accepted theory of estrogen receptor positive mammary carcinogenesis. The molecular mechanisms of gene expression regulation in response to long-term estrogen exposure (LTEE) of MCF-7 breast cancer cells were addressed in this study, with a focus on the aryl hydrocarbon receptor (AHR) and signal transducer and activator of transcription 1 (STAT1).

Advanced Statistical Methods For Biochemical And Forensic Applications Of Raman Spectroscopy, Vitali Sikirzhytski

Legacy Theses & Dissertations (2009 - 2024)

This work is dedicated to the development and application of advanced statistical methods for biochemical and forensic applications of Raman spectroscopy. We developed a new concept of multidimensional Raman signatures for forensic identification of body fluid traces, which was successfully applied to the study of dried blood, semen, saliva, sweat and vaginal fluid stains. Several alternative advanced chemometric techniques were also successfully tested for the problems of pure body fluids discrimination and mixture analysis. Studies of body fluid mixtures with low blood and semen concentrations indicated that the detection limit of a minor contributor was as low as a few …

Structure And Function Of Coding And Non-Coding Rna Domains, Fei Liu

Legacy Theses & Dissertations (2009 - 2024)

As a versatile molecule, RNA exhibits an astonishing variety of functional activities, which is typically attributed to its particular structure-forming capacity. There are an increasing number of established roles and systems where RNA structures, in particular, RNA pseudoknots, affect specific biological processes based on their structural features. The structure-function-relationships of RNA in three systems has been invesgated: the telomerase RNA pseudoknot domain from S. cerevisiae, a poteintial unusual H-type pseudoknot forming region near the 3'-splice-site in the influenza virus NS1 mRNA, and the 5'-untranslated-region of the CC16 mRNA where mutations in the wildtype sequence have been related to asthma. UV-monitored …

Design And Application Of Composite Rna Aptamers, Shengchun Wang

Legacy Theses & Dissertations (2009 - 2024)

aptamers are being developed as an essential tool in many fields of biological research. Their utility is not limited to being protein inhibitors; a lot of novel functions can be realized. However, in vivo application of RNA aptamers still faces many challenges. The aim of this dissertation is to design and apply composite aptamers in multiple expression and delivery systems to address some critical issues, such as correct folding, high level production, degradation by nucleases, excessive consumption of cellular resource and potential toxic effect.

Homologous And Heterologous Crystallin Protein Interactions As The Molecular Basis Of Inherited Cataract, Priya Ranjan Banerjee

Legacy Theses & Dissertations (2009 - 2024)

Age-related cataract is the most common cause of blindness worldwide. Nearly fifty percent of Americans above the age of 75 are diagnosed with this disease (http://www.nei.nih.gov/eyedata/pbd_tables.asp), and surgical intervention is the sole method of treatment at present (http://www.nei.nih.gov/healthyvision/objective/cataracts.asp). In the developing world, even this treatment is not readily available. These are compelling reasons to search for better treatments to delay, prevent or arrest cataract formation. Recent evidence suggests that age-related cataracts also have a genetic component. Therefore, determining the mechanisms underlying genetic cataracts with a known association to a protein-mutation is one important strategy towards understanding the molecular basis for …

Biophysical Investigations Of The Molecular Basis Of Cataract Associated With The R76s Mutation In Human Gammad-Crystallin, Vurghun Ahmadov

Legacy Theses & Dissertations (2009 - 2024)

Cataract disease results when the eye lens becomes opaque and scatters a significant part of the incoming light into the eye. The lens contains very high concentrations of the lens proteins, called crystallins, which are present at concentrations comparable to those found in protein crystals (about 400-600 mg/mL). Chemical modifications of the crystallins, such as oxidation and deamidation, or genetic mutations are known to result in increased light-scattering in vitro, and are implicated in cataract formation in vivo. Here we present the in vitro work on a mutant protein of human gammaD crystallin (HGD), namely R76S (i.e. Arg 76 to …

Tca Cycle Inactivation In Staphylococcus Aureus Alters Nitric Oxide Production In Raw 264.7 Cells, Chandirasegaran Massilamany, Arunakumar Gangaplara, Donald J. Gardner, James M. Musser, David J. Steffen, Greg A. Somerville, Jay Reddy

Jay Reddy Publications

Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In this study, it was observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, the authors observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts …

Avoiding Proteolysis During Protein Chromatography., Barry Ryan

Books/Book Chapters/ Proceedings

All cells contain proteases which effect catalytic hydrolysis of the peptide bond between amino acids in the protein backbone. Typically, proteinases are prevented from non-specific proteolysis by regulation and physical separation into different sub-cellular compartments; however, this segregation is not retained during cell lysis to release a protein of interest. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach; firstly inhibition of proteolysis in situ, followed by the separation of the protease from the protein of interest via chromatographical purification. Proteinase inhibitors are routinely used to limit the effect of the proteinases before they are …

The Impact Of Tricaine Methanesulfonate, 2-Phenoxyethanol, And Carvone-Methyl Salicylate On The Innate Immune Response Of Zebrafish (Danio Rerio), Charles R. Wulff

Honors Theses

Anesthesia plays a vital role in the maintenance of aquaculture species, where it is used to minimize stress during complex handling tasks such as transport, assessment, and harvesting. However, anesthetics have been shown to suppress the innate immune response, which could impact immunity and increase risk of infection. Tricaine methanesulfonate (MS-222) and 2-Phenoxyethanol (2-PE) represent two of the most commonly used anesthetics in aquaculture, with R-(+)-carvone, in the form of carvone-methyl salicylate (CMS) has recently been proposed as an alternative anesthetic for food fish. These three anesthetics were used to assess the influence of anesthetics on the immune system of …

The Role Of Zip Superfamily Of Metal Transporters In Chronic Diseases, Purification & Characterization Of A Bacterial Zip Transporter: Zupt., Iryna King

Wayne State University Theses

In mammals zinc is the second most abundant essential trace metal. Since Zn² is a small, hydrophilic, and a highly charged ion, it cannot be transported across the plasma or intracellular organelle membrane by passive diffusion. Different types of cells require a different constant concentration of zinc at all times. Presence of excess free Zn ions can be toxic to the cell. All cells must have tightly regulated homeostatic mechanisms in order to preserve healthy levels and proper compartmentalization of zinc. This is accomplished through the actions of specialized proteins that facilitate zinc uptake, efflux and compartmentalization. If the …

Effects Of Dietary Fat Saturation On Lipoprotein Metabolism In Rodents And Humans, Deepinder Kaur

Wayne State University Dissertations

Consumption of trans-fatty acids (tFA) and saturated fatty acids (SFA) have been associated with higher incidence of coronary heart disease. Experimental and observational data suggests that consumption of PHVO containing tFA, like SFA leads to increased total cholesterol and LDL-cholesterol (LDL-C) but compared to tFA, SFA raises HDL-cholesterol (HDL-C). An attempt to eliminate PHVO from the food supply has resulted in the need to find alternatives which despite their higher dietary fat saturation as compared to naturally occurring vegetable oils, may be preferable to PHVO. However, amongst SFA, the effects on plasma lipoproteins are variable. Thus, we hypothesized that specific …

Synthesis, Self-Assembly And Biological Properties Of Self-Folding Cu(Ii) Coordination Polymers, Alejandro Arzola-Rubio

Open Access Theses & Dissertations

The synthesis of materials via molecular self-assembly is a powerful bottom-up approach for fabricating matter in the nano- and micro-meter scales. This methodology involves the spontaneous and reversible organization of small molecules that interact with one another to create larger structures driven by non-covalent interactions such as hydrogen bonding, hydrophobic forces and metal-ligand coordination interactions. In this thesis, the design, synthesis and characterization of a new set of molecules that spontaneously self-organize in water to form three-dimensional nanoscale toroidal structures was investigated. These supramolecular structures were designed with an intrinsic affinity to bind and condense DNA strands with the intent …

Hydrolysis Of Amelogenin By Matrix Metalloprotease-20 Accelerates Mineralization In Vitro, Vuk Uskoković, Feroz Khan, Haichuan Liu, Halina Ewa Witkowska, Li Zhu, Wu Li, Stefan Habelitz

Pharmacy Faculty Articles and Research

In the following respects, tooth enamel is a unique tissue in the mammalian body: (a) it is the most mineralized and hardest tissue in it comprising up to 95 wt% of apatite; (b) its microstructure is dominated by parallel rods composed of bundles of 40 – 60 nm wide apatite crystals with aspect ratios reaching up to 1:10,000 and (c) not only does the protein matrix that gives rise to enamel guides the crystal growth, but it also conducts its own degradation and removal in parallel. Hence, when mimicking the process of amelogenesis in vitro, crystal growth has to …

Surface Entropy Reduction To Increase The Crystallizability Of The Fab-Rna Complex, Priyadarshini Palaniandy Ravindran

Electronic Theses and Dissertations

Crystallizing RNA has been an imperative facet and a challenging task in the world of RNA research. Assistive methods such as Chaperone Assisted RNA Crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by increasing the crystal contacts and providing initial phasing information. Using this technology the crystal structure of [delta]C209 P4-P6 RNA (an independent folding domain of the self-splicing Tetrahymena group I intron) complexed to Fab2 (high affinity binding Fab) has been resolved to 1.95 Å (1). Although the complexed class I ligase ribozyme has also been crystallized using CARC …

An Upper Limit For Macromolecular Crowding Effects, Andrew C. Miklos, Congang Li, Courtney D. Sorell, L. Andrew Lyon, Gary J. Pielak

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Background: Solutions containing high macromolecule concentrations are predicted to affect a number of protein properties compared to those properties in dilute solution. In cells, these macromolecular crowders have a large range of sizes and can occupy 30% or more of the available volume. We chose to study the stability and ps-ns internal dynamics of a globular protein whose radius is similar to 2 nm when crowded by a synthetic microgel composed of poly(N-isopropylacrylamide-co-acrylic acid) with particle radii of similar to 300 nm.

Results: Our studies revealed no change in protein rotational or ps-ns backbone dynamics and only mild …

Biomimetic Precipitation Of Uniaxially Grown Calcium Phosphate Crystals From Full-Length Human Amelogenin Sols, Vuk Uskoković, Wu Li, Stefan Habelitz

Pharmacy Faculty Articles and Research

Human dental enamel forms over a period of 2 – 4 years by substituting the enamel matrix, a protein gel mostly composed of a single protein, amelogenin with fibrous apatite nanocrystals. Self-assembly of a dense amelogenin matrix is presumed to direct the growth of apatite fibers and their organization into bundles that eventually comprise the mature enamel, the hardest tissue in the mammalian body. This work aims to establish the physicochemical and biochemical conditions for the synthesis of fibrous apatite crystals under the control of a recombinant fulllength human amelogenin matrix in combination with a programmable titration system. The growth …

14-3-3Γ Binds To Cam Kkα And Blocks Estrogen Signaling In Mcf-7 Cells, John M. Schmitt, Amanda P. Ankeny

Faculty Publications - Department of Biological & Molecular Science

Abstract Extracellular Signal-Regulated Kinase (ERK) is activated by estrogen (E2) downstream of CaM KK leading to cell growth in MCF-7 breast cancer cells. Previous studies have shown that ERK activation may be inhibited by cAMP and PKA. PKA has numerous cellular targets including CREB, Src, Raf-1, arrestins, and CaM KK. CaM KK is inhibited by direct PKA phosphorylation and the subsequent interaction with 14-3-3γ . Agonists that activate cAMP and PKA may block CaM KK activation of ERK and cell proliferation. Our goal was to evaluate the ability of cAMP and PKA to antagonize the effects of E2 on MCF-7 …