Biochemistry, Biophysics, and Structural Biology Commons^™

Regulation Of The Vacuolar Atpase Activity, Paulina Konarzewska

Dissertations, Theses, and Capstone Projects

In eukaryotes, V-ATPases play an essential role in cellular pH homeostasis as they transport hydrogen ions into the vacuoles. This assures an acidic vacuolar interior and normal physiological processes associated with this organelle. Studies show that mutations in any of the VMA genes encoding for subunits of the V-ATPase pump result in growth inhibition in the absence of inositol, suggesting that V-ATPases may play an important role in phospholipid biosynthesis or vice versa. It is not clear how VMA genes affect phospholipid biosynthesis and how the regulators of phospholipid biosynthetic genes may affect ATPase activity. Here, we employ biochemical and …

The Role Of Atp And Cleavage Factor Phosphorylation In Pre-Mrna 3' Cleavage Reaction, Asya Khleborodova

Dissertations, Theses, and Capstone Projects

The 3' end cleavage/polyadenylation (3' processing) is important in generating a functional messenger RNA (mRNA) transcript. It is long-known that ATP can significantly stimulate the in vitro cleavage of adenovirus type 2 L3 (Ad2L3) RNA substrate. Here, we used ATP analogs in structure-activity assays to show that the structural features of ATP and its analogs determine in vitro 3' cleavage efficiency. More specifically, we demonstrate that the structure of the nitrogenous base, the nucleotide sugar and the triphosphate group contribute to the efficiency of the nucleotide-stimulated in vitro 3' cleavage reaction.

It was found that AppNp, an analog of ATP …

Incipient Speciation In Freshwater Fish Species From Two Isolated Watersheds, Paula Gore Miller

Dissertations, Theses, and Capstone Projects

The process of speciation occurs as a result of restricted gene flow between segments of an interbreeding population occupying different geographic areas. This separation may result in isolated populations which undergo genetic and phenotypic changes. The Wisconsin glacial period, which ended approximately 17,500 years ago, dramatically altered the geography of North America. The glacier covered almost the entire North America as it advanced. Areas that were not covered with ice provided suitable habitats (refugia) for relict species that were previously widespread in the northern section of the continent. As the ice sheet retreated, animals and plants were able to return …

Biophysical Characterization Of A De Novo Elastin, Kelly Nicole Greenland

Dissertations, Theses, and Capstone Projects

Natural human elastin is found in tissue such as the lungs, arteries, and skin. This protein is formed at birth with no mechanism present to repair or supplement the initial quantity formed. As a result, the functionality and durability of elastin's elasticity is critically important. To date, the mechanics of this ability to stretch and recoil is not fully understood. This study utilizes de novo protein design to create a small library of simplistic versions of elastin-like proteins, demonstrate the elastin-like proteins, maintain elastin's functionality, and inquire into its structure using solution nuclear magnetic resonance (NMR).

Elastin is formed from …

Exploiting Cancer Cell Signaling And Metabolism: Implications For Therapeutic Approach, Suman Mukhopadhyay

Dissertations, Theses, and Capstone Projects

Over the last decade, metabolic dysregulation in cancer cells has stimulated a significant amount of interest in basic research. It has been established that cancer cells increase glucose uptake and alter the fate of glycolytic and tricarboxylic acid (TCA) cycle intermediates for the synthesis of biological molecules to accommodate high rates of cellular growth and proliferation. Moreover, it is more prominent that some metabolic dysregulations are specific to particular oncogenes. Exploiting the dysregulated metabolic dependency of cancer cells with therapeutic means could represent a novel approach for clinical aspect.

To meet the need of increased anabolic metabolism cancer cells engage …

Proton Pumping In Cytochrome C Oxidase, Jianxun Lu

Dissertations, Theses, and Capstone Projects

Cytochrome c oxidase (CcO) is a large trans-membrane protein, which is the final enzyme in the respiratory electron transport chain in mitochondria or aerobic bacteria. It implements proton pumping through the mitochondrial membrane against the electrochemical gradient, by utilizing the chemical energy released by reducing O₂ to water. The active site of the chemical reaction is called the Binuclear Center (BNC) that is made up of heme a₃, Cu_B, a Tyrosine residue and their ligands. The protein is reduced four times by electron from cytochromes c to reduce O₂ and to generate four different …

Conformational Heterogeneity And Exchange Within The Junction Of The Human U2-U6 Snrna Complex Measured By 19f Nmr Spectroscopy, Caijie Zhao

Dissertations, Theses, and Capstone Projects

Pre-mRNA splicing, the process by which non-coding sequences are removed and coding sequences are ligated, plays an important role in the maturation of precursor messenger RNA molecules prior to their translation into proteins. In eukaryotes, splicing is catalyzed by the spliceosome, a large and dynamic nuclear macromolecular "machine" that comprises five small nuclear (sn)RNA molecules and more than one hundred proteins. Among the RNA components, the complex formed between the U2 and U6 snRNA molecules is implicated in catalytic activity. In this dissertation, I have used ¹⁹F NMR techniques to characterize the conformation ("fold") and dynamics of a protein-free …

De Novo Design And Engineering Of Functional Metal And Porphyrin-Binding Protein Domains, Bernard Howard Everson

Dissertations, Theses, and Capstone Projects

In this work, I describe an approach to the rational, iterative design and characterization of two functional cofactor-binding protein domains. First, a hybrid computational/experimental method was developed with the aim of algorithmically generating a suite of porphyrin-binding protein sequences with minimal mutual sequence information. This method was explored by generating libraries of sequences, which were then expressed and evaluated for function. One successful sequence is shown to bind a variety of porphyrin-like cofactors, and exhibits light- activated electron transfer in mixed hemin:chlorin e6 and hemin:Zn(II)-protoporphyrin IX complexes. These results imply that many sophisticated functions such as cofactor binding and electron …

Using Multiconformation Continuum Electrostatics Model For Pka And Proton Transfer Pathway Calculations In Protein, Xuyu Zhu

Dissertations, Theses, and Capstone Projects

Residue protonation state changes and proton transfer reactions in protein are basic and ubiquitous reactions in biological chemistry. The Multiconformation Continuum Electrostatics (MCCE) program is applied here to calculate the pKa of residues in the specially engineered protein staphylococcal nuclease (SNase) and further modified to investigate the unidirectional proton transfer mechanism in the proton pump protein bacteriorhodopsin. The pKa$_s$ are obtained by Monte Carlo sampling of coupled side chain protonations and positions as a function of pH in MCCE. The pKa$_s$ of 96 acids and bases introduced into buried sites in the SNase protein were calculated and the results compared …

Functional Characterization Of The Plant 15-Cis-Zeta-Carotene Isomerase Z-Iso, Jesus Alonso Beltran Zambrano

Dissertations, Theses, and Capstone Projects

Vitamin A deficiency is a widespread health issue in the tropics. To solve this issue, efforts are underway to increase provitamin A carotenoids such as β-carotene in staple crops which can be achieved by breeding, metabolic engineering or a combination of both approaches. However, rational strategies to improve carotenoid content in crops require sufficient knowledge of pathway regulation. Therefore, to better understand how plants synthesize provitamin A and to guide metabolic engineering strategies in crops such as maize, the functional characterization of the new ζ-carotene isomerase (Z-ISO) is of significant importance.

Z-ISO was recently discovered in maize and Arabidopsis (Chen …

A Role Of Yeast Adhesin Amyloids In Force-Dependent Adhesion And Biofilm Formation, Cho Xiao Juan Chan

Dissertations, Theses, and Capstone Projects

Candida albicans adhesins have amyloid-forming sequences (Ramsook et al. 2010, Otoo et al. 2008). Similarly, Tango and Waltz predicted that amyloid-forming sequences are also present in Saccharomyces cerevisiae flocculins, Flo1p and Flo11p. The cell surface of Flo1p- and Flo11p-expressing cells have ordered domains that are thioflavin T fluorescent and Congo red birefringent, two hallmarks of amyloids. Flo1p and Flo11p amyloids were important for activities of the flocculins including cell-to-cell adhesion, cell-to-substrate adhesion, and agar invasion. In addition, amyloid-perturbing dyes thioflavin S and Congo red inhibited aggregation, biofilm formation and agar invasion.

Force-induced formation and propagation of adhesion nanodomains in Als5p-expressing …

The Role Of The Striatal Neuropeptide Neurotensin In The Methamphetamine-Induced Neural Injury In Mice, Qingkun Liu

Dissertations, Theses, and Capstone Projects

Methamphetamine (METH) is a widely abused psychostimulant that induces neurotoxicity to several brain regions, including the striatum. Similar to dopamine (DA) in chemical structure, METH can be transported into DA pre-synaptic terminals, evoking the neurodegeneration in DA terminals and post-synaptic striatal neurons. Despite the critical role of DA in METH-induced neurodegeneration, no pharmaceutical therapeutics has been approved for these conditions. It is therefore essential to investigate the endogenous factors regulating the dopaminergic system. The neuropeptide neurotensin has emerged as a potential modulator of METH-induced striatal neurodegeneration mainly due to its intimate interactions with dopamine in the striatum.

In this study, …

Design And Optimization Of A De Novo Protein Charge Separation Dyad, Andrew C. Mutter

Dissertations, Theses, and Capstone Projects

The ever-increasing demand for cheap, plentiful energy to fuel the needs of a growing population requires research into alternative clean energy. Solar irradiation has the potential to power the planet many times over; the challenge is efficient capture and conversion of this energy source. Nature has already solved this problem with photosynthesis, which harvests solar irradiation converting it to stored chemical energy and is the source of the energy for life. The goal of my dissertation is to use de novo designed protein to mimic the charge separation system in photosynthesis. A stable protein scaffold will be designed and used …

Development Of Cell-Active Inhibitors And Activity-Based Probe Of Cysteine Cathepsins, Dibyendu Dana

Dissertations, Theses, and Capstone Projects

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. Still however, many of their cellular function remain poorly understood. Chemical biology approaches employing small molecules can be utilized for this purpose. Unfortunately small molecule probes that are cell-permeable and non-peptidyl in nature are scarcely available.

In this work, first a library of sulfonyloxiranes is synthesized. From this library, 2-(2-ethylphenylsulfonyl)oxirane is identified as a selective inhibitor of cysteine cathepsins. Cell-based study reveals that 2-(2-ethylphenylsulfonyl)oxirane is a cell-permeable, covalent, and irreversible inhibitor of cathepsin B with …

Identification And Characterization Of Protein Kinase C Substrates In Human Breast Cells, Xin Zhao

Dissertations, Theses, and Capstone Projects

Aberrations in PKC signaling can lead to the development of multiple human diseases and the most prominent association of PKC with disease has been in tumor growth and metastasis. PKC and its related pathways have been recognized as promising targets for blocking the malignancy of breast cancer cells. To better understand PKC-mediated pathway in breast cancer cells, it is important to identify the cellular substrates of PKC. The main focus of this work is to identify physiologically relevant cellular substrates of PKC in human breast cells and to characterize their roles in cancer-related phenotypes. The work to be described consists …

Fabrication And Characterization Of Sol-Gel Based Nanoparticles For Drug Delivery, Reeta Yadav

Dissertations, Theses, and Capstone Projects

Nanogels are cross linked polymeric sol-gel based nanoparticles that offer an interior network for incorporation and protection of biomolecules, exhibiting unique advantages for polymer based delivery systems. We have successfully synthesized stable sol-gel nanoparticles by means of [a] silicification reactions using cationic peptides like polylysine as gelating agents, and [b] lyophilization of sol-gels. Macromolecules such as Hemoglobin and Glucose Oxidase and small molecules such as Sodium Nitroprusside (SNP) and antibiotics were encapsulated within the nanogels. We have used transmission electron microscopy, dynamic light scattering, zeta potential analysis, and spectroscopy to perform a physicochemical characterization of the nanogels resulting from the …

Venomic Characterization Of The Terebridae And Novel Terebrid Neuropeptides, Mary Elizabeth Wright

Dissertations, Theses, and Capstone Projects

Unravelling the complex mixture of neuropeptides produced by the terebrid venom duct holds the promise of discovering tomorrow's therapeutics. Cone snails have already demonstrated the potential found in the venom of these unusual marine organisms, through the commercial approval of drugs for pain and other indications. Terebrids, as the sister family to the cone snails, have been much less investigated, but have a species richness that makes them very attractive in the search for novel neuropeptides. The venomics research described in this work encompasses the first comprehensive characterization of the terebrid venom duct transcriptomes of two species, Cinguloterebra anilis and …

Recruitment Of The Ribosomal 40s Subunit To The 3'Untranslated Region Of A Viral Mrna, Via The Eif4 Complex, Facilitates Cap-Independent Translation, Sohani Das Sharma

Dissertations, Theses, and Capstone Projects

Translation of uncapped plant viral RNAs can be facilitated by either an internal ribosomal entry site (IRES) in the 5' untranslated region (UTR) or a cap-independent translation element (CITE) in the 3' UTR. Barley yellow dwarf virus (BYDV) mRNA, which lacks both cap and poly(A) tail, has a translation element (3'BTE) in its 3' UTR that is essential for efficient translation initiation at the 5'-proximal AUG. This mechanism requires binding of the eukaryotic initiation factor 4G (eIF4G) subunit of the heterodimer eIF4F to the 3'BTE and base pairing between the 3'BTE and the 5' UTR. Here we investigate how this …

Interactions Of Eukaryotic Translation Initiation Factors And 3' Untranslated Region Of Barley Yellow Dwarf Virus Mrna During Protein Synthesis: A Study Of Equilibrium Binding, Kinetics And Thermodynamics, Bidisha Banerjee

Dissertations, Theses, and Capstone Projects

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley Yellow Dwarf Virus (BYDV) employs a cap-independent mechanism of translation initiation which is mediated by a structural element BTE (BYDV translation element) located in the 3’ UTR of its mRNA. eIF4F bound the BTE and a translational inactive mutant with high affinity; thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5-164% compared to WT were studied. Using fluorescence anisotropy …

Structure And Function In Bacteriophage Phi6, James Carpino

Dissertations, Theses, and Capstone Projects

The present study of bacteriophage Phi6 has been preceded by a great number of exploratory studies of its structure and function, and these studies have formed a basis for Phi6's development into a model organism. In this study, two aspects of the model organism have been examined. 1. There are several uncharacterized and presumed untranslated regions (UTRs) in Phi6's 13.3 kilobase-pair dsRNA genome. I examined the impact of specific modification to the 3' UTR of the small segment of bacteriophage Phi6. I determined that modification to the purported UTR of the small segment resulted in severe fitness costs, supporting a …

Mechanisms Of Regulation Of Mrna 3' Processing By P53 Pathway, Emral Cakmak Devany

Dissertations, Theses, and Capstone Projects

Although the p53 network has been intensively studied, genetic analyses long hinted at the existence of components that remained elusive. This dissertation focuses on the study of the regulation of mRNA 3' processing during DNA damage response (DDR) by the p53 pathway and the regulation of p53 expression by the mRNA 3' processing machinery. The results in this dissertation revealed new roles of tumor suppressor p53 in mRNA 3' processing. In Chapter II, I showed that p53 inhibits the cleavage step of polyadenylation reaction and that cells with different levels of p53 expression have different mRNA processing profiles. As part …

Role Of The Polyadenylation Factor Cstf-50 In Regulating The Brca1/Bard1 E3 Ubiquitin (Ub) Ligase Activity, Danae Fonseca

Dissertations, Theses, and Capstone Projects

The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. The studies presented in this dissertation focus on the determination and characterization of the role of mRNA processing factor CstF-50 and escort protein p97 in the regulation of the BRCA1/BARD1 E3 ubiquitin (Ub) ligase activity during the DNA damage response (DDR).

As part of the studies presented in Chapter II, I determined that the polyadenylation factor CstF plays a direct role in DDR, specifically in transcription-coupled repair (TCR), and that it localizes with RNA polymerase II (RNAP II) and BARD1 to sites of …

Polymerase Alpha Components Associate With Telomeres To Mediate Overhang Processing, Raffaella Diotti

Dissertations, Theses, and Capstone Projects

Telomeres consist of TTAGGG repeats, which end with a 3' G-overhang and are bound by a six-protein complex, known as Shelterin. In humans, telomeres shorten at each cell division, unless telomerase is expressed and able to add telomeric repeats to the 3' G-overhang. However, for effective telomere maintenance, the DNA strand complementary to that made by telomerase must be synthesized. In this study, I focused on the Polα/primase complex, in particular the subunits p68 (POLA2, the regulatory subunit) and p180 (Polα, the catalytic subunit), and their potential roles at telomeres. I was able to detect p180, p68 and OBFC1, a …

Mechanism Of Phospho-Alpha-Tubulin-Driven Motility In Human Breast Epithelial Cells, Shatarupa De

Dissertations, Theses, and Capstone Projects

Protein kinase C (PKC), an enzyme important in signaling pathways that give rise to various cell phenotypes, has been closely associated with metastatic phenotypes of breast cancer. Tissues from patients of varying degrees of tumorgenicity have shown an elevated expression of the PKCα isoform as well as other diacylglycerol (DAG)-sensitive isoforms. Moreover, independent studies with highly invasive breast cell lines, such as MDA-MB-231 cells, have also shown an elevated level of PKCα. Therefore, PKCα has long been a therapeutic target for breast cancer. Among its known substrates (recently identified by our laboratory), α6-tubulin will, upon phosphorylation by PKC, enhance migration …

Mechanisms Of Deadenylation Regulation Under Different Cellular Conditions, Xiaokan Zhang

Dissertations, Theses, and Capstone Projects

Control of gene expression by regulating mRNA stability after DNA damage has the potential to contribute to the cells rapid response to stress. The main focus of this dissertation is to elucidate the role(s) of nuclear PARN deadenylase in controlling mRNA stability, hence gene expression, of factors in the p53 signaling pathway during the DNA damage response (DDR). Understanding the mechanisms of these regulatory pathways will provide new insights on how the control of gene expression upon DNA damage decides cellular fate, offering new opportunities for therapeutics. In Chapter II, I presented evidence that PARN along with the cleavage factor …

Computational Insights Into The Oxygen Evolving Complex Of Photosystem Ιι, Muhamed Amin

Dissertations, Theses, and Capstone Projects

The Oxygen Evolving Complex (OEC) of Photosystem II (PSII) is a unique Mn4O5Ca2+ cluster that catalyzes the photoactivated water splitting reaction. The OEC is a model system for bio-inspired artificial systems to use solar energy to pull electrons from water to produce fuel. The OEC goes through a cycle of 5 S states storing 4 holes, via electron transfer to P680+, the primary electron donor in PSII to generate a high valence S4 state that oxidizes water. The key questions are what controls the order of oxidation and deprotonation of the OEC complex and how does the PSII protein modulate …

Roles Of Igh Intronic Enhancer Eμ In Clonal Selection At The Pre-B To Immature B Cell Transition And In The Elimination Of Autoreactive B Cells, Cheng Peng

Dissertations, Theses, and Capstone Projects

The immunoglobulin heavy chain locus (Igh ) intronic enhancer, Eμ, enhances transcription of recombined Igh genes. We have previously shown that in mice with an Eμ-deficient Igh allele (V _H δ^a ), Igμ is expressed at half of the wild-type levels in pre-B cells. We also described an Eμ-dependent "check-point", operating at the pre-B to immature B cell transition, for heavy chain allelic exclusion. We now show that deletion of Eμ results in a smaller immature B cell compartment, and the pre-BCR/BCR signaling is diminished in pre-B cells as a result of the reduced Igμ levels, making it …

Conformational Features Of The Human U2-U6 Snrna Complex, Ravichandra Bachu

Dissertations, Theses, and Capstone Projects

The splicing of precursor messenger (pre-m) RNA, during which noncoding intervening sequences are excised and flanking coding regions ligated, is an integral reaction of gene expression. In eukaryotes, it is carried out by a dynamic RNA-protein complex called the spliceosome, in which five small nuclear (sn) RNA components are actively involved in recognition and chemical aspects of the process. A complex formed between U2 and U6 snRNAs is implicated in the chemistry of pre-mRNA splicing. The catalytic activity of the U2-U6 snRNA complex is dependent on the presence of Mg2+ ions, and the complex has been shown to have several …

Iron Regulates Mrna Translation Initiation Through Rna Iron Responsive Element (Ire), Jia Ma

Dissertations, Theses, and Capstone Projects

The non-coding IRE-RNA structure, a 30 nt stem loop structure, regulates synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA binding with iron regulatory protein (IRP) proteins inhibits ribosome accessing mRNA. Increasing iron concentration decreases IRP binding with IRE-RNA. Previous models of IRE-mRNA translation regulation concentrate on Fe-S binding to IRP and IRP degradation after release from IRE-RNA. These models lack information on the details of decreasing IRE-RNA/IRP protein binding with iron concentration elevation. This research shows 1. Eukaryotic initiation factor 4F (eIF4F) binds to IRE-RNA with high affinity and works as a positive control element …

The Sex Of The Cell Dictates Its Response, Carlos Ganesh Penaloza

Dissertations, Theses, and Capstone Projects

Male and female differences in frequency of occurrence in disease have perplexed scientists for some time. This in part derives from limitations in the systems in which one can evaluate sex differences. At the organismal level, differences can be hidden by a myriad of extensive and complex factors. Additional limitations exist since most biomedical studies are performed almost exclusively on male subjects, as the female hormonal milieu is intrinsically more variable and too troublesome for routine inclusion in research protocols. Research documenting sex differences continues to grow, and while most researchers suggests that sex hormones are at the core of …