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Butler, Thomas. "Nanopore analysis of nucleic acids /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/9674.
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Horowitz, Eric D. "Intercalator-mediated assembly of nucleic acids." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/33937.
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The RNA World hypothesis suggests that RNA, or a proto-RNA, existed in an early form of life that had not yet developed the ability to synthesize protein enzymes. This hypothesis, by some interpretations, implies that nucleic acid polymers were the first polymers of life, and must have therefore spontaneously formed from simple molecular building blocks in the "prebiotic soup." Although prebiotic chemists have searched for decades for a process by which RNA can be made from plausible prebiotic reactions, numerous problems persist that stand in the way of a chemically-sound model for the spontaneous generation of an RNA World (e.g., strand-cyclization, heterogeneous backbones, non-selective ligation of activated nucleotides). The Molecular Midwife hypothesis, proposed by Hud and Anet in 2000, provides a possible solution to several problems associated with the assembly of the first nucleic acids. In this hypothesis, nucleic acid base pairs are assembled by small, planar molecules that resemble molecules which are known today to intercalate the base pairs of nucleic acid duplexes. Thus, the validity and merits of the Molecular Midwife hypothesis can be, to some extent, explored by studying the effects of intercalation on the non-covalent assembly of nucleic acids.
In this thesis, I explore the role of the sugar-phosphate backbone in dictating the structure and thermodynamics of nucleic acid intercalation by using 2′,5′-linked RNA intercalation as a model system of non-natural nucleic acid intercalation. The solution structure of an intercalator-bound 2′,5′ RNA duplex reveals structural and thermodynamic aspects of intercalation that provide insight into the origin of the nearest-neighbor exclusion principle, a principle that is uniformly obeyed upon the intercalation of natural (i.e. 3′,5′-linked) RNA and DNA. I also demonstrate the ability of intercalator-mediated assembly to circumvent the strand-cyclization problem, a problem that otherwise greatly limits the polymerization of short oligonucleotides into long polymers. Together, the data presented in this thesis illustrate the important role that the nucleic acid backbone plays in governing the thermodynamics of intercalation, and provide support for the proposed role of intercalator-mediated assembly in the prebiotic formation of nucleic acids.
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Chen, Baoshan. "Encapsidation of nucleic acids by cucumovirus coat proteins /." Title page, contents and summary only, 1991. http://web4.library.adelaide.edu.au/theses/09PH/09phc5183.pdf.
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Guo, Maolin. "Proteins and nucleic acids as targets for titanium(IV)." Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/13967.
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TiIV compounds have pronounced anticancer, antiviral and antibacterial activities, and titanocene dichloride (TDC) is currently on phase II clinical trials as an anticancer drug. However, the biological chemistry and mechanisms of action of TiIV are poorly understood. Proteins and nucleic acids are expected to be biological targets for TiIV. Human transferrin (hTF) is a bilobal serum glycoprotein (80 kDa) which transports FeIII to cells via receptor-mediated endocytosis. A structurally similar periplasmic iron binding protein (FBP, 34 kDa) is present in some pathogenic bacteria and is required for virulence. In this thesis, the aqueous coordination chemistry of TiIV with the phenolate ligand N,N'-ethylene-bis-(o-hydroxyphenylglycine)(H4ehpg) was investigated as a model for Ti-hTF (or FBP) interactions. TiIV forms 7-coordinae monomer (rac) and dimer (meso) complexes with H4ehpg (rac + meso) with novel stereo-selectivity. 1H and 31P NMR studies show that TDC binds selectivity to H4ehpg at neutral pH, but preferentially to adenosine triphosphate (APT) at pH values below 5; TiIV transfers from TiIV-ehpg to ATP at acidic pH values. The interactions of TDC with hTF and that of Ti2-hTF with ATP have characteristics which could allow transferrin to act as a mediator for titanium delivery to tumour cells. TDC reacts rapidly with apo-hTF under extra-cellular conditions and binds in the specific FeIII sites with release of the Cp and Cl ligands. TiIV is readily released from Ti2-hTF at endosomal pH (ca 5.0) and in the presence of ATP. Ti2hTF competes effectively for cell uptake of 59Fe-hTF into BeWo cancer cells. TDC binds strongly to the phosphate group of nucleotides in aqueous solution and TiIV binds to the phosphodiester groups of nucleotides in the less polar solvent N,N-dimethylformamide. This behaviour contrasts with that of the anticancer drug cisplatin which binds mainly to N-sites of nucleobases, and may account for the intracellular localisation behaviour of TiIV drugs.
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Forties, Robert A. "Applications of statistical mechanics to nucleic acids." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1311022751.
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Fernández, Estrabao César. "The NMR structure of proteins, nucleic acids and their complexes /." Zürich, 1999. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=13055.
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Klyashtornyy, Vladislav. "Principles of protein nucleic acid : recognition on the examples of the ribosomal protein L1 and the cold shock domain of YB-1 protein." Thesis, Evry-Val d'Essonne, 2012. http://www.theses.fr/2011EVRY0039/document.
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Cette thèse est une étude structurale sur l’interaction entre 2 protéines modèles et des acides nucléiques : la protéine L1 (navette ribosome/ARN messagers) et le sous domaine CSD de YB-1, protéine régulatrice de la transcription et traduction. Deux méthodes sont utilisées : i) diffraction des RX par des cristaux de complexes L1:ARN ribosomal ou messager et ii) modélisation et dynamique moléculaire pour l’interaction CSD avec des homo-ribo- ou homo-deoxyribo-nucléotides simple brin. Les méthodes sont décrites avec leurs forces et limites. Les résultats sur L1-rARN/ARN éclairent les mécanismes de régulation de la traduction en montrant des différences d’affinité pour l’ARN des sous domaines I et II de L1. L’analyse de mutants de L1 dans le site de liaison à l’ARN du sous domaine I éclairent la nature des liaisons non covalentes sous tendant l’affinité de ce sous domaine pour l’ARN et souligne l’importance de la structure de L1, de sa « complémentarité » avec l’ARN et de liaisons hydrogènes non accessibles au solvant. Les travaux de modélisation et de dynamique moléculaire sur l’interaction CSD:acides nucléiques montrent que la séquence nucléotidique module l’affinité de ce complexe, l’oligonucléotide de type oligoG donnant le complexe le plus stable suivi des séquences de type oligoU et oligoA puis des oligoT et oligoC. L’orientation du brin d’ARN par rapport au CSD impacte aussi la stabilité du complexe. Une analyse des surfaces d’interaction et de la nature des liaisons intermoléculaires, montre que des principes similaires guident l’interaction L1:ARN et CSD:acides nucléiques : géométrie complémentaire des partenaires et liaisons hydrogène protégées du solvantThis thesis is a structural study on the interaction between two model proteins and nucleic acids: the L1 protein (shuttle ribosome/mRNA) and the CSD subdomain of YB-1, a protein that regulates transcription and translation. Two methods are used: i) X-ray diffraction by crystals of L1:ribosomal or messenger RNA complexes and ii) molecular modeling and dynamics for the CSD interaction with homo-ribo or homo-deoxyribo- single-stranded nucleotide. The methods are described with their strengths and limitations. Results on L1-rARN/ARN enlighten the mechanisms regulating translation by showing differences in affinity for RNA of the subdomains I and II of L1. Analyses of L1 mutants in the RNA binding site from the subdomain I illuminate the nature of non-covalent bonds subtending the affinity of this subdomain for RNA and stress the importance of the structure of L1, its "complementarity" with RNA and of hydrogen bonds not accessible to the solvent. Molecular modeling and dynamics of the CSD:nucleic acids interaction shows that the nucleotide sequence modulates the affinity of the complex, oligoG giving the most stable complex followed by oligoU and then oligoA or oligoT and oligoC. The orientation of the RNA strand relative to the CSD also impacts the stability of the complex. An analysis of the interaction surfaces and of the nature of intermolecular bonds, shows that similar principles guide the L1: RNA and CSD: nucleic acids interactions, i.e. a complementary geometry between partners and presence of hydrogen bonds protected from the solvent
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Kalafut, Bennett Samuel. "Optical Tweezers studies of Nucleic Acids and their Interaction with Proteins." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/202969.
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Mechanics and biological function of nucleic acids are intimately coupled. The DNA double helix must be opened to allow base pairing of RNA during transcription; RNA must bend and fold in its many cellular functions. Presented in this dissertation are two investigations of mechanical deformations of nucleic acids, conducted with optical tweezers.In the introduction, the mechanical properties of DNA and RNA and their relevance to their cellular functions are introduced, to give the reader context for the results presented in the Chapters 2 and 3. This is followed by an introduction to the theory of semiflexible polymer elasticity. The optical tweezers instruments used in conducting these investigations are then presented, along with calibration procedures and a short introduction to optical trapping physics.Chapter 2 presents an investigation of the effect of downstream DNA tension on initiation by T7 RNA polymerase. A hidden Markov model is fit to force-dependent lifetimes obtained from optical tweezers experiments, allowing us to identify which steps in initiation are force-dependent and estimate rates and transition state distances. We find that 1-2 pN of tension is sufficient to turn o gene expression by causing transcription bubble collapse and destabilizing the bound state. Our force-dependence scheme and estimated transition distances provide independent supportfor the \scrunching" model of initiation.The effects of cation binding and screening on single-stranded helix formation in poly(A) RNA are presented in Chapter 3. Magnesium and calcium bind to poly(A), stabilize the helix, and change its mechanical properties. A new model of helix-coil transitions is presented and used to estimate energetics and mechanical properties.Chapter 4 presents the first fully objective algorithm for use in analyzing the noisy staircaselike data that is often produced by single-molecule fluorescence experiments. A test based on the SIC (BIC) statistic is used in conjunction with a progressive step-placement scheme to locate changepoints (steps) in noisy data. Its performance is compared to other step detection algorithms in use by biophysicists by repeating tests performed in a recent review.Experimental protocols and computer codes used in these investigations are presentedin detail in the appendices.
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Furman, Jennifer Lynn. "IN VITRO AND IN VIVO DETECTION OF NUCLEIC ACIDS AND PROTEINS USING SPLIT-PROTEIN REASSEMBLY." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/195828.
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The ability to directly monitor the presence of specific proteins or nucleic acids in a variety of in vitro and in vivo contexts has great utility for understanding biology as well as for the development of diagnostic agents. Herein I describe several methodologies, utilizing split-protein reassembly, which provides potentially general strategies for sequence-specific detection of DNA and RNA sequences as well as poly(ADP-ribose). I also provide a new split-protein approach for the direct detection of native proteins, such as the cancer marker HER2.The green fluorescent protein (GFP) provides a convenient sensor for reporting on a variety of cellular events. A series of spectroscopically distinct GFP variants was developed for sequence-specifically reporting on DNA. Each of these variants was demonstrated to provide a sensitive readout for the presence of a particular DNA sequence. Furthermore, utilizing a method of mixed split-protein complementation, I was able to simultaneously report on the presence of two distinct DNA sequences in the same solution.To provide a general solution for reporting on the presence of particular RNA sequences, a method was developed that utilized elements from a hybridization-based detection strategy coupled with split-protein reassembly. Specifically, DNA guide sequences complementary to an intended target were attached to hairpin sites that served as binding sites for high-affinity zinc fingers. Localization of the zinc fingers allowed for reassembly of the attached split enzyme, providing a sensitive readout for the presence of potentially any RNA sequence of interest. This methodology was applied to the detection of mRNA encoding VEGF, hDM2, and HER2, each of which may be overexpressed in cancer.A method was established for reporting on the presence of modifications to DNA and proteins that are elicited in response to DNA damage. Specifically, sensors were designed, which incorporated endogenous damage-recognition domains, to report on the global presence of particular DNA modifications, including the formation of 8-oxoguanine and pyrimidine dimers. Furthermore, to provide a technique for monitoring the general accrual of DNA damage and to interrogate the DNA damage response in cells, a sensor was developed which reported on the accrual of a posttranslational protein modification, poly(ADP-ribosyl)ation.Finally, I describe advances toward the adaptation of our protein-based biosensors for use in living cells, utilizing both GFP-based approaches for live cell imaging as well as luminescent-based strategies for reporting on proteins and nucleic acids following cell lysis.
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Pérez, González Daniel Cibrán. "Single-molecule studies of nucleic acid folding and nucleic acid-protein interactions." Thesis, University of St Andrews, 2017. http://hdl.handle.net/10023/12039.
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Nucleic acids and proteins, some of the building blocks of life, are not static structures but highly dynamic entities that need to interact with one another to meet cellular demands. The work presented in this thesis focuses on the application of highly sensitive fluorescence methods, both at ensemble and single-molecule level, to determine the dynamics and structure of specific biomolecular interactions with nanometer resolution and in temporal scales from nanoseconds to minutes, which includes most biologically relevant processes. The main aims of my PhD can be classified in three areas: i) exploring new fluorescent sensors with increased specificity for certain nucleic acid structures; ii) understanding how some of these nucleic acids sense the presence of small molecules in the cellular environment and trigger gene regulation by altering their structure; and iii) understanding how certain molecular machines, such as helicase proteins, are able to unwind the DNA double helix by using chemical energy in the form of ATP hydrolysis.
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Streu, Kristina. "Structure, Thermodynamics, and Dynamical Properties of Nucleic Acids, Proteins, and Glass-Forming Liquids." Thesis, Boston College, 2016. http://hdl.handle.net/2345/bc-ir:107098.
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Thesis advisor: Udayan MohantyThe stabilization of particular conformations of protein and nucleic acid structure is believed to play an important role in many important biological functions. In chapter one, the α -helical conformation and structural stability of single and double stapled all- hydrocarbon cross-linked p53 peptides when bound and unbound to MDM2 are investigated. Our study provides a comprehensive rationalization of the relationship between peptide stapling strategy, the secondary structural stability, and the binding affinity of p53-MDM2 complex. In chapter two, we study counterion-mediated collapse of a strongly charged model polyelectrolyte chain by Group-II divalent metal cations using coarse-grained Brownian dynamics simulations. Polyelectrolyte effects govern the association of counterions with the chain. Large ions are less effective in counterion condensation than small ions. However, upon counterion condensation, the reduction of the backbone charge is independent of size of the metal cations. Above a threshold value of Coulomb strength parameter, counterion release entropy drives the formation of counterion-induced compact states. In chapter three, the nature of surface tension in the random first order theory of supercooled liquid is analyzed within the framework of Landau-Lifshitz fluctuation theory. We show that the surface tension of a droplet satisfies the differential equation 4πr2(dσ)+ 8πrσ(r)− Br1/2 = 0 , where B/ T = 12πkBcv , T is temperature, kB is dr Boltzmann constant, and cv is heat capacity. A consequence is that the slope of the relaxation time at the glass transition temperature, i.e., the fragility index, is expressed as the square of the ratio of heat capacity and configurational entropy of the supercooled liquid. When backbone extended nucleosides are incorporated into a double helix, a unique helical structure is formed. In chapter four, we find that the predicted stability of modified backbone DNA strands in aqueous solution is in good agreement with experimental melting temperature data. The incorporation of extended backbone nucleosides into a duplex results in elongation of the end-to-end chain distance due to the distortion of the B-DNA conformation at the mutated base-pair insertion. We also find that the modified backbone helical twist is approximately 40 degrees, larger than B-DNA helical twist and closer to the twist angle predicted for D-form DNA. The folding of RNA tertiary structure has been described as an equilibrium between partially folded I (intermediate) states, and the fully folded native conformation, or N state. RNA is highly sensitive to the ionic environment due to its negative charge, and tertiary structures tend to be strongly stabilized by Mg2+. There is a need for models capable of describing the ion atmosphere surrounding RNA with quantitative accuracy. In chapter 5, we present a generalized Manning condensation model of RNA electrostatics for studying the Mg2+-induced RNA folding of the 58mer ribosomal fragment
Thesis (PhD) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Ogawa, Atsushi. "Development of new in vitro display/selection methods for proteins and nucleic acids." 京都大学 (Kyoto University), 2006. http://hdl.handle.net/2433/143988.
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Kyoto University (京都大学)0048
新制・課程博士
博士(工学)
甲第12291号
工博第2620号
新制||工||1369(附属図書館)
24127
UT51-2006-J284
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 青山 安宏, 教授 今中 忠行, 教授 濵地 格
学位規則第4条第1項該当
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Ye, Hong. "X-ray diffraction, neutron diffraction and circular dichroism studies of nucleic acids and proteins." Thesis, Keele University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245601.
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Eltoukhy, Ahmed Atef. "Development of polymer and lipid materials for enhanced delivery of nucleic acids and proteins." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/81668.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013.Cataloged from PDF version of thesis.
Includes bibliographical references.
The development of synthetic vectors enabling efficient intracellular delivery of macromolecular therapeutics such as nucleic acids and proteins could potentially catalyze the clinical translation of many gene and protein-based therapies. However, progress has been hindered by a lack of safe and effective materials and by insufficient insight into the relationship between key delivery properties and efficacy. Accordingly, working with a promising class of cationic, degradable gene delivery vectors, poly(-amino ester)s (PBAEs), we develop novel, hydrophobic PBAE terpolymers that display dramatically increased gene delivery potency and nanoparticle stability. We then develop a technique based on size-exclusion chromatography that enables the isolation of well-defined, monodisperse PBAE polymer fractions with greater transfection activities than the starting polymer. This technique also allows us to elucidate the dependence of gene delivery properties on polymer molecular weight (MW). Subsequently, we examine the cellular uptake and trafficking mechanisms of PBAE/DNA polyplexes, and demonstrate that polyplex internalization and transfection depend on a key endo/lysosomal cholesterol transport protein, Niemann-Pick C1 (Npcl). Finally, working with cationic lipids termed lipidoids, which have shown exceptional potency for the delivery of RNAi therapeutics, we develop these materials for intracellular delivery of proteins using a simple and novel approach in which nucleic acids serve as a handle for protein encapsulation and delivery. Preliminary in vivo experiments suggest the potential application of this approach toward lipidoid-mediated delivery of protein-based vaccines. Taken together, the work presented here advances the development of polymer and lipid materials for the safe and effective intracellular delivery of DNA and protein therapeutics.
by Ahmed Atef Eltoukhy.
Ph.D.
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Bury, Charles S. "Investigation of X-ray induced radiation damage in proteins, nucleic acids and their complexes." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:f62abc16-aed1-469c-aa71-7e36813e5218.
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Macromolecular X-ray crystallography (MX) is currently the dominant technique for the structural eluci- dation of macromolecules at near atomic resolution. However, the progression and deleterious effects of radiation damage remains a major limiting factor in the success of diffraction data collection and subsequent structural solution at modern third generation synchrotron facilities. For experiments conducted at 100 K, protein specific damage to particular amino acids has been widely reported at doses of just several MGy, before any observable decay in average diffraction intensities. When undetected, such artefacts of X-ray irradiation can lead to significant modelling errors in protein structures, and ultimately the failure to derive the correct biological function from a model. It is thus vital to develop tools to help MX experimenters to detect and correct for such damage events. This thesis presents the development of an automated program, RIDL, which is designed to objectively quantify radiation-induced changes to electron density at individual atoms, based on Fobs,n â Fobs,1 Fourier difference maps between different dose states for a single crystal. The high-throughput RIDL program developed in this work provides the ability to systematically investigate a wide range of macromolecular systems. To date, damage to the broad class of nucleic acids and nucleoprotein complexes has remained largely uncharacterised, and it is unclear how radiation damage will disrupt the validity of such models derived from MX experiments. This thesis presents the first systematic investigations on a range of nucleic acid, protein-RNA and protein-DNA complex case studies. In general, it is concluded that nucleic acids are highly robust to radiation damage effects at 100K, relative to control protein counterparts across the tested systems. For protein crystals at 100K, cleavage of the phenolic C-O bond in tyrosine has disseminated through the MX radiation damage literature as a dominant specific damage event at 100K, despite the absence of any energetically favourable cleavage mechanism. To clarify the radiation susceptibility of tyrosine, this thesis presents a systematic investigation on radiation damage to tyrosine in a wide range of MX protein radiation damage series retrieved from the Protein Data Bank. It is concluded that the tyrosine C-O bond remains intact following X-ray irradiation, however the aromatic side-group can undergo radiation-induced displacement. This thesis also presents further applications of the RIDL program. A protocol is introduced to calculate explicit half-dose values for the electron density at individual atoms to decay to half of their initial value at zero absorbed dose. In addition, a methodology is developed to detect radiation-induced changes to electron density occurring over the course of the collection of a single MX dataset of diffraction images, all of which are required for structural solution. These protocols aim to advise experimenters of when previously-undetected site-specific damage effects may have corrupted the quality of their macromolecular model. Overall, the work in this thesis is highly applicable to both the future understanding of radiation damage in macromolecular structures, as well as of interest to the wider crystallographic community.
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Demharter, Samuel. "Novel applications for hierarchical natural move Monte Carlo simulations : from proteins to nucleic acids." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:c0ef3ba5-4fe0-4684-a0ce-202003cd79a5.
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Biological molecules often undergo large structural changes to perform their function. Computational methods can provide a fine-grained description at the atomistic scale. Without sufficient approximations to accelerate the simulations, however, the time-scale on which functional motions often occur is out of reach for many traditional methods. Natural Move Monte Carlo belongs to a class of methods that were introduced to bridge this gap. I present three novel applications for Natural Move Monte Carlo, two on proteins and one on DNA epigenetics. In the second part of this thesis I introduce a new protocol for the testing of hypotheses regarding the functional motions of biological systems, named customised Natural Move Monte Carlo. Two different case studies are presented aimed at demonstrating the feasibility of customised Natural Move Monte Carlo.
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Killian, Tobias Friedrich [Verfasser], and Reinhard [Akademischer Betreuer] Sterner. "Development, characterization and synthesis of multi-specific proteins for targeted delivery of nucleic acids and nucleic acid derivatives / Tobias Friedrich Killian ; Betreuer: Reinhard Sterner." Regensburg : Universitätsbibliothek Regensburg, 2019. http://d-nb.info/1192974778/34.
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Guarnaccia, Corrado. "Interaction of RGG and HTH motifs with nucleic acids : a study with rationally designed synthetic and recombinant polypeptides." Thesis, Open University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368806.
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Plyte, Simon Edward. "The biochemical and biophysical characterisation of the P11 gene 5 protein and its complex with nucleic acids." Thesis, University of Portsmouth, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.277240.
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Welderufael, Zenawi Teklay. "J-coupling predictor function building and large-scale NMR simulations of proteins and nucleic acids." Thesis, University of Southampton, 2016. https://eprints.soton.ac.uk/409733/.
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This thesis discusses large scale quantum mechanical NMR simulations and fitting works, and deals with biomolecular spin systems. Chapter one introduces the theoretical background that underpins the simulation work. It describes spin and it properties, the interactions of spin with the magnetic field, the environment and it self and the Hamiltonians that describe those interactions. It also covers the Bloch equations that is normally used to predict the longitudinal and transverse relaxations. It further briefly describes the common relaxation mechanisms that are available for a spin system. It also included NMR experiments that are often used in the study of protein and nucleic acid systems. Chapter 2 summarizes the building of J-coupling predictor function. It provides the detail on how the estimator function was built. The function is required for NMR simulations and incorporates the functionality of J-coupling estimation, which provides J-coupling values for proteins and nucleic acids. After building the estimator function NOESY spectra simulation of ubiquitin and RNA stem loops spin systems was performed. Chapter 3 then discusses our simulation method, treating the system quantum mechanically along with an advanced and detailed description of NOE using Redfield relaxation theory. Chapter 4 reports on large–scale simulations and fitting of an exchanging 1H-15N HSQC spectra of calmodulin (PDB: 1CLL) upon stepwise addition of a ligand, that was analysed using a iii formalism that operates in the direct product of spin state space and chemical state space. It also presents the least squares technique for the fitting procedure and the Nelder-Mead simplex minimization technique used in the process. The chapter also highlights the data collated from the fitting work. Finally, chapter 5 concludes the thesis with the simulation of NOESY stem loops RNA spin system and fitting with respect to correlation time and frequency offset. It also explains how we fitted the data from theory to those from the experiment and why the diagonal peaks were eliminated from the fitting.
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Shedge, Hemangi Y. "Specific and non-specific binding of proteins and nucleic acids on chemically modified reticulated vitreous carbon electrodes." Connect to this title online, 2009. http://etd.lib.clemson.edu/documents/1246559605/.
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Long, Kimberly Renee. "Identification and Characterization of Agv1, a Pre-Metazoan Arf GAP: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/339.
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Human immunodeficiency virus type 1 (HIV-1) is a member of the lentivirus subfamily of retroviruses. HIV-1 expresses multiple genes from a single provirus by alternative splicing. Early in viral expression, fully spliced 2-kb viral RNA is exported from the nucleus and encodes the viral regulatory protein, Rev, which is essential for nuclear transport of partially spliced and unspliced genomic-length RNA. Rev binds to an RNA structural element called the Rev response element (RRE) and mediates nuclear export through the leucine-rich nuclear export signal (NES) pathway. The human Rev Interacting Protein (hRIP) interacts specifically with the Rev NES. Rev NES mutants that are unable to export Rev-dependent RNAs are also unable to bind to hRIP. The hRIP cDNA encodes a 562 amino acid protein containing an N-terminal zinc finger with homology to Arf GAP domains, a central serine and threonine rich region, and C-terminal phenylalanine-glycine (FG) repeats characteristic of nucleoporins.
To identify an hRIP ortholog in a genetically tractable organism, we performed database searches using the N-terminal zinc finger of hRIP. Using this approach, we identified a novel gene in Schizosaccharomyces pombe. Alignment of the entire reading frame of the putative ortholog with hRIP indicates similarity with the serine/threonine rich region and with the FG repeats, suggesting that S. pombecould be a good model system to study the cellular function of hRIP.
We find that the S. pombe ORF is an essential gene, which encodes a 483 amino acid protein that is also able to interact with the NES of HIV-1 Rev. Based on being an essential gene, and the presence of a putative Arf GAP domain, the ORF was named an Arf GAP essential for viability, agv1+. We show that Agv1 is not directly involved in the nuclear export of poly(A+) RNA or 5S rRNA, nuclear export of leucine-rich NES-containing proteins, or nuclear import of nuclear localization signal (NLS)-containing proteins. However, Agv1 does appear to play a role in the cytoplasmic localization of 5S rRNA.
We demonstrate that loss of Agv1 alters the localization of endoplasmic reticulum (ER) membrane and Golgi membrane resident proteins, accumulates intracellular membrane, and blocks processing of carboxypeptidase Y. Furthermore, the S. cerevisiae ADP-ribosylation factor (Arf) GTPase activating protein (GAP) Glo3, but not a catalytically inactive Glo3 mutant [R59K], is able to partially compensate for the loss of Agv1 function in temperature sensitive strains, indicating that Agv1 is an S. pombe Arf GAP with some functional features similar to S. cerevisiae Glo3.
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Salomon, William E. "Single-Molecule Imaging Reveals that Argonaute Re-Shapes the Properties of its Nucleic Acid Guides: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/804.
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Small RNA silencing pathways regulate development, viral defense, and genomic integrity in all kingdoms of life. An Argonaute (Ago) protein, guided by a tightly bound, small RNA or DNA, lies at the core of these pathways. Argonaute uses its small RNA or DNA to find its target sequences, which it either cleaves or stably binds, acting as a binding scaffold for other proteins. We used Co-localization Single-Molecule Spectroscopy (CoSMoS) to analyze target binding and cleavage by Ago and its guide. We find that both eukaryotic and prokaryotic Argonaute proteins re-shape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization: a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Counter to the rules of nucleic acid hybridization alone, we find that mouse AGO2 and its guide bind to microRNA targets 17,000 times tighter than the guide without Argonaute. Moreover, AGO2 can distinguish between microRNA-like targets that make seven base pairs with the guide and the products of cleavage, which bind via nine base pairs: AGO2 leaves the cleavage products faster, even though they pair more extensively.
This thesis presents a detailed kinetic interrogation of microRNA and RNA interference pathways. We discovered sub-domains within the previously defined functional domains created by Argonaute and its bound DNA or RNA guide. These sub-domains have features that no longer conform to the well-established properties of unbound oligonucleotides. It is by re-writing the rules for nucleic acid hybridization that Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than that of RNA or DNA. Taken altogether, these studies further our understanding about the biology of small RNA silencing pathways and may serve to guide future work related to all RNA-guided endonucleases.
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Fitzgerald, Amanda Ann. "Folding and Assembly of Multimeric Proteins: Dimeric HIV-1 Protease and a Trimeric Coiled Coil Component of a Complex Hemoglobin Scaffold: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/341.
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Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology.
The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed.
These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function.
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Levine, Kara B. "Identification of the Human Erythrocyte Glucose Transporter (GLUT1) ATP Binding Domain: A Dissertation." eScholarship@UMMS, 1999. https://escholarship.umassmed.edu/gsbs_diss/247.
Full textAbstract:
The human erythrocyte glucose transport protein (GLUT1) interacts with, and is regulated by, cytosolic ATP. This study asks the following questions concerning ATP modulation of GLUT1 mediated sugar transport. 1) Which region(s) of GLUT1 form the adenine nucleotide-binding domain? 2) What factors influence ATP modulation of sugar transport? 3) Is ATP interaction with GLUT1 sufficient for sugar transport regulation?
The first question was addressed through peptide mapping, n-terminal sequencing, and alanine scanning mutagenesis of GLUT1 using [32P]-azidoATP, a photoactivatable ATP analog. We then used a combination of transport measurements and photolabeling strategies to examine how glycolytic intermediates, pH, and transporter oligomeric structure affect ATP regulation of sugar transport. Finally, GLUT1 was reconstituted into proteoliposomes to determine whether ATP is sufficient for the modulation of GLUT1 function in-vitro.
This thesis presents data supporting the hypothesis that residues 332-335 contribute to the efficiency of adenine nucleotide binding to GLUT1. In addition, we show that AMP, acidification, and conversion of the transporter to its dimeric form antagonize ATP regulation of sugar transport. Finally, we present results that support the proposal that ATP interaction with GLUT1 is sufficient for transport modulation.
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Maderazo, Alan Baer. "A Study on the Cellular Localization of Factors Involved in Yeast Nonsense-Mediated mRNA Decay and their Mechanisms of Control on Nonsense mRNA Translation: a Dissertation." eScholarship@UMMS, 2000. https://escholarship.umassmed.edu/gsbs_diss/105.
Full textAbstract:
Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance mechanism conserved in eukaryotes. This thesis explores several interesting aspects of the NMD pathway. One important aspect of NMD which is presently the subject of intense controversy is the subcellular localization of NMD. In one set of experiments, the decay kinetics of the ade2-1 and pgk1 nonsense mRNAs (substrates for NMD) were investigated in response to activating the NMD pathway to determine if cytoplasmic nonsense mRNAs are immune to NMD in the yeast system. The results of these studies demonstrated that activation of NMD caused rapid and immediate degradation of both the ade2-1 and the early nonsense pgk1 steady state mRNA populations. The half lives of the steady state mRNA populations for both ade2-1 and pgk1 (early nonsense) were shortened from >30 minutes to approximately 7 minutes. This was not observed for pgk1mRNAs that contained a late nonsense codon demonstrating that activation of NMD specifically targeted the proper substrates in these experiments. Therefore, in yeast, nonsense mRNAs residing in the cytoplasm are susceptible to NMD. While these findings are consistent with NMD occurring in the cytoplasm, they do not completely rule out the possibility of a nuclear-associated decay mechanism.
To investigate the involvement of the nucleus in NMD, the putative nuclear targeting sequence identified in Nmd2p (one of the trans-acting factors essential for NMD) was characterized. Subcellular fractionation experiments demonstrated that the majority of Nmd2p localized to the cytoplasm with a small proportion detected in the nucleus. Specific mutations in the putative nuclear localization signal (NLS) of Nmd2p were found to have adverse effects on the protein's decay function. These effects on decay function, however, could not be attributed to a failure in nuclear localization. Therefore, the residues that comprise the putative NLS of Nmd2p are important for decay function but do not appear to be required for targeting the protein to the nucleus. These results are in accordance with the findings above which implicate the cytoplasm as an important cellular compartment for NMD.
This thesis then investigates the regulatory roles of the trans-acting factors involved in NMD (Upf1p, Nmd2p, and Upf3p) using a novel quantitative assay for translational suppression, based on a nonsense allele of the CAN1 gene (can1-100). Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and promoted can1-100 nonsense suppression. Changes in mRNA levels were not the basis of suppression, however, since deletion of DCP1 or XRN1 or high-copy can1-100 expression in wild-type cells caused mRNA stabilization similar to that obtained in upf/nmd cells but did not result in comparable suppression. can1-100 suppression was highest in cells harboring a deletion of UPF1, and overexpression of UPF1 in cells with individual or multiple upf/nmd mutations lowered the level of nonsense suppression without affecting the abundance of the can1-100 mRNA. These findings indicate that Nmd2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRNA decay.
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Yigit, Erbay. "The Argonaute Family of Genes in Caenorhabditis Elegans: a Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/328.
Full textAbstract:
Members of the Argonaute family of proteins, which interact with small RNAs, are the key players of RNAi and other related pathways. The C. elegans genome encodes 27 members of the Argonaute family. During this thesis research, we sought to understand the functions of the members of this gene family in C. elegans. Among the Argonaute family members, rde-1 and alg-1/2have previously been shown to be essential for RNAi and development, respectively. In this work, we wanted to assign functions to the remaining members of this large family of proteins.
Here, we describe the phenotype of 31 deletion alleles representing all of the previously uncharacterized Argonaute members. In addition to rde-1, our analysis revealed that two other Argonaute members csr-1 and prg-1 are also essential for development. csr-1 is partially required for RNAi, and essential for proper chromosome segregation. prg-1, a member of PIWI subfamily of Argonaute genes, exhibits reduced brood size and temperature-sensitive sterile phenotype, implicating that it is required for germline maintenance.
Additionally, we showed that RDE-1 interacts with trigger-derived sense and antisense siRNAs (primary siRNAs) to initiate RNAi, while several other Argonaute proteins, SAGO-1, SAGO-2, and perhaps others, functioning redundantly, interact with amplified siRNAs (secondary siRNAs) to mediate downstream silencing. Moreover, our analysis uncovered that another member of Argonaute gene family, ergo-1, is essential for the endogenous RNAi pathway.
Furthermore, we built an eight-fold Argonaute mutant, MAGO8, and analyzed its developmental phenotype and sensitivity to RNAi. Our analysis revealed that the genes deleted in the MAGO8 mutant function redundantly with each other, and are required for RNAi and the maintenance of the stem cell totipotency.
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Ghosh, Shubhendu. "Cross-Talk Between Factors Involved in mRNA Translation and Decay: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/454.
Full textAbstract:
The proper workings of an organism rely on the accurate expression of genes throughout its lifetime. An important determinant for protein production is the availability of template mRNA molecules, the net effect of which is governed by their rates of synthesis vs. their rates of degradation. Normal mRNAs are proposed to be relatively stable in the cytoplasm while present in a protective, circularized conformation – the closed loop – through eIF4G-bridged interactions with 3’-bound poly(A) binding protein (Pab1p) and 5’-bound eIF4E. Introduction of a premature nonsense codon into an otherwise normal mRNA results in its rapid destabilization in cells, suggesting that not all stop codons behave the same, and events at premature termination events that lead to accelerated degradation of nonsense-containing mRNAs likely differ from those at normal termination, in which normal decay rates are maintained. The enhanced degradation observed for nonsense-containing mRNAs occurs through an evolutionarily conserved pathway involving the products of the UPF1, UPF2/NMD2, and UPF3 genes, the precise biochemical roles of which have remained elusive. We have developed a yeast cell-free translation system that allows us to assay biochemical events occurring at premature termination codons, compare them to those occurring at normal terminators, and study the role of Upf1p in these events. We find that premature termination is an inefficient process compared to normal termination and that one outcome of termination at a premature termination codon (PTC) is reinitiation at a nearby start codon. This in vitro post-termination reinitiation phenotype is dependent on the presence of Upf1p, a finding we have recapitulated in vivo. We also developed biochemical assays to define a role for Upf1p in translation following premature termination in vitro and find that Upf1p is involved in post-termination ribosome dissociation and reutilization. Supporting this idea are our findings that Upf1p predominantly cosediments with purified 40S ribosomal subunits. Finally, using our in vitro translation/toeprinting system, we have further characterized events leading to the formation of the mRNA closed loop structure and find that two states of the closed loop exist. The first requires the preinitiation 48S complex and includes Pab1p, eIF4G, eIF4E, and eIF3, whereas the second is formed after 60S joining and additionally requires the translation termination factors eRF1 and eRF3.
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Sparks, Cynthia A. "Cloning and Cell Cycle Analysis of NuMA, a Phosphoprotein That Oscillates Between the Nucleus and the Mitotic Spindle." eScholarship@UMMS, 1995. https://escholarship.umassmed.edu/gsbs_diss/35.
Full textAbstract:
The overall objective of this study was to identify novel proteins of the nuclear matrix in order to contribute to a better understanding of nuclear structure and organization. To accomplish this, a monoclonal antibody specific for the nuclear matrix was used to screen a human λgt11 expression library. Several cDNAs were isolated, cloned, sequenced, and shown to represent NuMA, the nuclear mitotic spindle apparatus protein. Further characterization of the gene and RNA was undertaken in an effort to obtain information about NuMA. The NuMA gene was present at a single site on human chromosome 11q13. Northern and PCR analysis of NuMA mRNA showed a major 7.2 kb transcript and minor forms of 8.0 and 3.0 kb. The minor forms were shown to be alternatively spliced although their functional significance is not yet understood. Immunofluorescence microscopy demonstrated that NuMA oscillates between the nucleus and the microtubule spindle apparatus during the mitotic cell cycle. NuMA appeared as a 200-275 kDa protein detectable in all mammalian cells except human neutrophils. To determine whether NuMA's changes in intracellular distribution correlated with post-translational modifications, the protein's phosphorylation state was examined through the cell cycle using highly synchronized cells. NuMA was a phosphoprotein in interphase and underwent additional phosphorylation events in mitosis. The mitotic phosphorylation events occurred with similar timing to lamin B (G2/M transition) and were concomitant with NuMA's release from the nucleus and its association with the mitotic spindle. However, the mitotic phosphorylation occurred in the absence of spindle formation. Dephosphorylation of NuMA did not correlate with reassociation with the nuclear matrix but occurred in two distinct steps after nuclear reformation. Based on the timing of these events, phosphorylation may playa role in nuclear processes. In conclusion, the work in this dissertation identified NuMA, a nuclear matrix protein and showed that it is phosphorylated during the cell cycle and may be important for nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G1/S.
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Belk, Jonathan Philip. "A Characterization of Substrates and Factors Involved in Yeast Nonsense-Mediated mRNA Decay: A Dissertation." eScholarship@UMMS, 2002. https://escholarship.umassmed.edu/gsbs_diss/65.
Full textAbstract:
Many intricate and highly conserved mechanisms have evolved to safeguard organisms against errors in gene expression. The nonsense-mediated mRNA decay pathway (NMD) exemplifies one such mechanism, specifically by eliminating mRNAs containing premature translation termination codons within their protein coding regions, thereby limiting the synthesis of potentially deleterious truncated polypeptides. Studies in Saccharomyces Cerevisiae have found that the activity of at least three trans-acting factors, known as UPF1, UPF2/NMD2, and UPF3is necessary for the proper function of the NMD pathway. Further research conducted in yeast indicates that the degradation of substrates of the NMD pathway is dependent on their translation, and that the sub-cellular site of their degradation in the cytoplasm.
Although most evidence in yeast suggests that substrates of the NMD pathway are degraded in the cytoplasm while in association with the translation apparatus, some mammalian studies have found several mRNAs whose decay appears to occur within the nucleus or before their transport to the cytoplasm has been completed. In addition, study of the mammalian TPI mRNA found that this transcript was unavailable as a substrate for the NMD pathway once it had been successfully exported to the cytoplasm, further supporting the notion that the degradation of mammalian substrates of the NMD pathway occurs in association with the nucleus, or during export from the nucleus to the cytoplasm.
To determine if yeast cytoplasmic nonsense-containing mRNA can become immune to the NMD pathway we examined the decay kinetics of two NMDS substrate mRNAs in response to repressing or activating the NMD pathway. Both the ade2-1 and pgk1-UAG-2nonsense-containing mRNAs were stabilized by repressing this pathway, while activation of NMD resulted in the rapid and immediate degradation of each transcripts. These findings demonstrate that nonsense-containing mRNAs residing in the nucleus are potentially susceptible to NMD at each round of translation.
The remainder of this thesis utilizes protein overexpression studies to gain understanding into the function of factors related to the processes of nonsense-mediated mRNA decay and translation in Saccharomyces cerevisiae. Overexpression of a C-terminal truncated form of Nmd3p was found to be dominant-negative for cell viability, translation and the normal course of rRNA biogenesis.
Overexpression studies conducted with mutant forms of the nonsense-mediated mRNA decay protein Upf1p, found that overexpression of mutants in the ATP binding and ATP hydrolysis region ofUpflp were dominant-negative for growth in an otherwise wild-type yeast strain. Furthermore, overexpression of the ATP hydrolysis mutant of Upf1p (DE572AA), resulted in the partial inhibition of NMD and a general perturbation of the translation apparatus. These results support previous studies suggesting a general role for Upf1p function in translation.
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Manalastas-Cantos, Karen Katrina [Verfasser], and Dmitri [Akademischer Betreuer] Svergun. "Development and applications of small-angle scattering-based structure modeling tools for proteins and nucleic acids / Karen Katrina Manalastas-Cantos ; Betreuer: Dmitri Svergun." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1219303151/34.
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Manalastas-Cantos, Karen [Verfasser], and Dmitri [Akademischer Betreuer] Svergun. "Development and applications of small-angle scattering-based structure modeling tools for proteins and nucleic acids / Karen Katrina Manalastas-Cantos ; Betreuer: Dmitri Svergun." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1219303151/34.
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Melanson, Vanessa R. "Characterization of the Interaction Between the Attachment and Fusion Glycoproteins Required for Paramyxovirus Fusion: a Dissertation." eScholarship@UMMS, 2005. https://escholarship.umassmed.edu/gsbs_diss/24.
Full textAbstract:
The first step of viral infection requires the binding of the viral attachment protein to cell surface receptors. Following binding, viruses penetrate the cellular membrane to deliver their genome into the host cell. For enveloped viruses, which have a lipid bilayer that surrounds their nucleocapsids, entry into the host cell requires the fusion of viral and cellular membranes. This process is mediated by viral glycoproteins located on the surface of the virus. For many enveloped viruses, such as influenza, Ebola, and human immunodeficiency virus, the fusion protein is responsible for mediating both attachment to cellular receptors and membrane fusion.
However, paramyxoviruses are unique among fusion promoting viruses because their receptor binding and fusion activities reside on two separate proteins. This unique distribution of functions necessitates a mechanism by which the two proteins can transmit the juxtaposition of the viral and host cell membranes, mediated by the attachment protein (HN/H), into membrane fusion, mediated by the fusion (F) protein. This mechanism allows for paramyxoviruses to gain entry into and spread between cells, and therefore, is an important aspect of virus infection and disease progression.
Despite the conservation of receptor binding activity among members of the Paramyxovirinaesubfamily, for most of these viruses, including Newcastle disease virus (NDV), heterologous HN proteins cannot complement F in the promotion of fusion; both the HN and F proteins must originate from the same virus. This is consistent with the existence of a virus-specific interaction between the two glycoproteins. Thus, one or more domains on the HN and F proteins is thought to mediate a specific interaction between them that is an integral part of the fusion process.
Therefore, the primary focus of this thesis is the identification of the site(s) on HN that directly contacts F in the HN-F interaction. The ectodomain of the HN protein consists of a stalk and a terminal globular head. Analysis of the fusion activity of chimeric paramyxovirus HN proteins indicates that the stalk region of HN determines its F protein specificity. The first goal of this research was to address the question of whether the stalk not only determines F-specificity, but does so by directly mediating the interaction with F. To establish a correlation between the amount of fusion and the extent of the HN-F interaction, a specific and quantitative co-immunoprecipitation assay was used that detects the HN-F complex at the cell surface.
As an initial probe of the role of the HN stalk in mediating the interaction with F, N-glycans were individually added at several positions in the region. N-glycan addition at positions 69 and 77 in the stalk specifically and completely block both fusion and the HN-F interaction without affecting either HN structure or its other activities. However, though they also prevent fusion, N-glycans added at other positions in the stalk also modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein as indicated by sucrose gradient sedimentation analyses. These additional N-glycans likely indirectly affect fusion, perhaps by interfering with changes in the conformation of HN that link receptor binding to the fusion activation of F.
To address the issue of whether N-glycan addition at any position in HN would abolish fusion, an N-glycan was added in another region at the base of the globular head of HN (residues 124-152), which was previously predicted by a peptide-based analysis to mediate the interaction with F. HN carrying this additional N-glycan exhibits significant fusion promoting activity, arguing against this site being part of the F-interactive domain in HN. These data support the idea that the F-interactive site on HN is defined by the stalk region of the protein.
Site-directed mutagenesis was used to begin to explore the role of individual residues in the stalk in the interaction with F. The characteristics of the F-interactive domain in the stalk of HN are that it is a conserved motif with enough sequence heterogeneity to account for the specificity of the interaction. One such region that meets these requirements is the intervening region (IR) (residues 89-95); a non-helical domain situated between two conserved heptad repeats. Several amino acid substitutions for a completely conserved proline residue in this region impair not only fusion and the HN-F interaction, but also decrease neuraminidase activity in the globular domain and alter the structure of the protein, suggesting that the substitutions indirectly affect the HN-F interaction. Substitutions for L94 also interfere with fusion, but have no significant effect on any other HN function or its structure. Amino acid substitutions at two other positions in the IR (A89 and L90) also modulate only fusion. In all cases, diminished fusion correlates with a decreased ability of the mutated HN protein to interact with F at the cell surface. These findings indicate that the IR is critical to the role of HN in the promotion of fusion and are consistent with its direct involvement in the interaction with the homologous F protein. These are the first point mutations in the HN protein for which a correlation has been demonstrated between the extent of the HN-F interaction and the amount of fusion. This argues strongly that the co-IP assay is an accurate reflection of the HN-F interaction.
The second goal of this research was to address the HN-F interaction from the perspective of the F protein by investigating the relationship between receptor binding, the HN-F interaction, and fusion using a highly fusogenic form of the F protein. It has previously been shown that an L289A substitution in NDV F eliminates the requirement for HN in the promotion of fusion and enhances HN-dependent fusion above wild-type (wt) levels. Here, it was shown that the HN-independent fusion exhibited by L289A-F in Cos-7 cells cannot be duplicated in BHK cells. However, when L289A-F is co-expressed with wt HN, enhanced fusion above wt levels is observed in BHK cells. Additionally, when L289A-F is co-expressed with IR-mutated HN proteins previously shown to promote low levels of fusion with wt F, a 2.5-fold increase in fusion was observed. However, similar to wt F, an interaction between L289A-F and the IR-mutated HN proteins was not detected. These results imply that the attachment function of HN, as well as the conformational change in L289A-F, are necessary for the enhanced level of fusion exhibited by HN proteins co-expressed with L289A-F. Indeed, two MAbs detected a conformational difference between L289A-F and the wt F protein. These findings support the idea that the L289A substitution converts F to a form that is less dependent on an interaction with HN for conversion to the fusion-active form.
The last goal of this research was to address the cellular site of the HN-F interaction, still a controversial issue based on conflicting data from studies of different paramyxoviruses, using various approaches. This is a particular point of interest, as it speaks to the mechanism by which the HN-F interaction regulates fusion. Thus, NDV HN and F were successfully retained intracellularly with a multiple arginine or KK motif, respectively. The results of Endoglycosidase H resistance and F cleavage studies indicate that the mutated proteins, HN-ER and F-ER, are retained in a compartment prior to the medial-Golgi apparatus and that they are unable to interact with a high enough affinity to co-retain or even cause reduced transport of their wt partner glycoproteins. This is consistent with the HN-F interaction occurring at the cell surface, possibly triggered by receptor binding.
In conclusion, this thesis presents evidence to argue that the IR in the stalk of the NDV HN protein directly mediates the interaction with the F protein that is necessary for fusion. Overall, the data presented in this thesis extend the current knowledge of the mechanism by which the paramyxovirus attachment protein can trigger the F protein to initiate membrane fusion. A clear understanding of this process has the potential to identify new anti-viral strategies, such as small molecule inhibitors, aimed at controlling paramyxovirus infection by interfering with early steps in the virus infection cycle.
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Laliberte, Jason P. "Role of Host Cellular Membrane Raft Domains in the Assembly and Release of Newcastle Disease Virus: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/360.
Full textAbstract:
Newcastle disease virus (NDV) belongs to the Paramyxoviridae, a family of enveloped RNA viruses that includes many important human and animal pathogens. Although many aspects of the paramyxovirus life cycle are known in detail, our understanding of the mechanisms regulating paramyxovirus assembly and release are poorly understood. For many enveloped RNA viruses, it has recently become apparent that both viral and host cellular determinants coordinate the proper and efficient assembly of infectious progeny virions.
Utilizing NDV as a model system to explore viral and cellular determinants of paramyxovirus assembly, we have shown that host cell membrane lipid raft domains serve as platforms of NDV assembly and release. This conclusion was supported by several key experimental results, including the exclusive incorporation of host cell membrane raftassociated molecules into virions, the association of structural components of the NDV particle with membrane lipid raft domains in infected cells and the strong correlation between the kinetics of viral protein dissociation from membrane lipid raft domains and incorporation into virions. Moreover, perturbation of infected cell membrane raft domains during virus assembly resulted in the disordered assembly of abnormal virions with reduced infectivity. These results further established membrane raft domains as sites of virus assembly and showed the integrity of these domains to be critical for the proper assembly of infectious virions.
Although specific viral protein-protein interactions are thought to occur during paramyxovirus assembly, our understanding of how these interactions are coordinated is incomplete. While exploring the mechanisms underlying the disordered assembly of non-infectious virions in membrane raft-perturbed cells, we determined that the integrity of membrane raft domains was critical in the formation and virion incorporation of a complex consisting of the NDV attachment (HN) and fusion (F) proteins. The reduced virus-to-cell membrane fusion capacity of particles released from membrane raft-perturbed cells was attributed to an absence of the HN – F glycoprotein-containing complex within the virion envelope. This result also correlated with a reduction of these glycoprotein complexes in membrane lipid raft fractions of membrane raft-perturbed cells. Specifically, it was determined that the formation of newly synthesized HN and F polypeptides into the glycoprotein complex destined for virion incorporation was dependent on membrane lipid raft integrity.
Finally, a novel virion complex between the ribonucleoprotein (RNP) structure and the HN attachment protein was identified and characterized. Unlike the glycoprotein complex, the detection of the RNP – HN protein-containing complex was not affected by membrane raft perturbation during virus assembly in the cell. The biological importance of this novel complex for the proper assembly of an infectious progeny virion is currently under investigation.
The results presented in this thesis outline the role of host cell membrane lipid raft domains in the assembly and release processes of a model paramyxovirus. Furthermore, the present work extends our understanding of how these particular host cell domains mechanistically facilitate the ordered assembly and release of an enveloped RNA virus.
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Sirois, Cherilyn M. "Nucleic Acid Sensing by the Immune System: Roles For the Receptor For Advanced Glycation End Products (RAGE) and Intracellular Receptor Proteins: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/551.
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As humans, we inhabit an environment shared with many microorganisms, some of which are harmless or beneficial, and others which represent a threat to our health. A complex network of organs, cells and their protein products form our bodies’ immune system, tasked with detecting these potentially harmful agents and eliminating them. This same system also serves to detect changes in the healthy balance of normal functions in the body, and for repairing tissue damage caused by injury. Immune recognition of nucleic acids, DNA and RNA, is one way that the body detects invading pathogens and initiates tissue repair. A number of specialized receptor proteins have evolved to distinguish nucleic acids that represent “threats” from those involved in normal physiology. These proteins include members of the Toll-like receptor family and diverse types of cytosolic proteins, all of which reside within the confines of the cell. Few proteins on the cell surface have been clearly characterized to interact with nucleic acids in the extracellular environment. In this dissertation, I present collaborative work that identifies the receptor for advanced glycation end products (RAGE) as a cell surface receptor for nucleic acids and positions it as an important modulator of immune responses. Molecular dimers of RAGE interact with the sugar-phosphate backbones of nucleic acid ligands, allowing this receptor to recognize a variety of DNA and RNA molecules regardless of their nucleotide sequence. Expression of RAGE on cells promotes uptake of DNA and enhances subsequent responses that are dependent on the nucleic acid sensor Toll-like receptor 9. When mice deficient in RAGE are exposed to DNA in the lung, the predominant site of RAGE expression, they do not mount a typical early inflammatory response, suggesting that RAGE is important in generating immune responses to DNA in mammalian organisms. Further evidence suggests that RAGE interacts preferentially with multimolecular complexes that contain nucleic acids, and that these complexes may induce clustering of receptor dimers into larger multimeric structures. Taken together, the data reported here identify RAGE as an important cell surface receptor protein for nucleic acids, which is capable of modulating the intensity of immune responses to DNA and RNA. Understanding of and intervention in this recognition pathway hold therapeutic promise for diseases characterized by excessive responses to self nucleic acids, such as systemic lupus erythematosus, and for the pathology caused by chronic inflammatory responses to self and foreign nucleic acids.
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Paschal, Bryce M. "Structure and Function of Cytoplasmic Dynein: a Thesis." eScholarship@UMMS, 1992. https://escholarship.umassmed.edu/gsbs_diss/82.
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In previous work I described the purification and properties of the microtubule-based mechanochemical ATPase cytoplasmic dynein. Cytoplasmic dynein was found to produce force along microtubules in the direction corresponding to retrograde axonal transport. Cytoplasmic dynein has been identified in a variety of eukaryotes including yeast and human, and there is a growing body of evidence suggesting that this "molecular motor" is responsible for the transport of membranous organelles and mitotic chromosomes.
The first part of this thesis investigates the molecular basis of microtubule-activation of the cytoplasmic dynein ATPase. By analogy with other mechanoenzymes, this appears to accelerate the rate-limiting step of the cross-bridge cycle, ADP release. Using limited proteolysis, site-directed antibodies, and N-terminal microsequencing, I identified the acidic C-termini of α and β-tubulin as the domains responsible for activation of the dynein ATPase.
The second part of this thesis investigates the structure of the 74 kDa subunit of cytoplasmic dynein. The amino acid sequence deduced from cDNA clones predicts a 72,753 dalton polypeptide which includes the amino acid sequences of nine peptides determined by microsequencing. Northern analysis of rat brain poly(A) revealed an abundant 2.9 kb mRNA. However, PCR performed on first strand cDNA, together with the sequence of a partially matching tryptic peptide, indicate the existence of three isoforms. The C-terminal half is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70 kDa subunit of flagellar outer arm dynein. Based on what is known about the Chlamydomonas70 kDa subunit, I suggest that the 74 kDa subunit is responsible for targeting cytoplasmic dynein to membranous organelles and kinetochores of mitotic chromosomes.
The third part of this thesis investigates a 50 kDa polypeptide which co-purifies with cytoplasmic dynein on sucrose density gradients. Monoclonal antibodies were produced against the 50 kDa subunit and used to show that it is a component of a distinct 20S complex which contains additional subunits of 45 and 150 kDa. Moreover, like cytoplasmic dynein, the 50 kDa polypeptide localizes to kinetochores of metaphase chromosomes by light and electron microscopy. The 50 kDa-associated complex is reported to stimulate cytoplasmic dynein-mediated organelle motility in vitro. The complex is, therefore, a candidate for modulating cytoplasmic dynein activity during mitosis.
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van, Wijnen Andre John. "Transcriptional Control of Human Histone Gene Expression: Delineation and Regulation of Protein/DNA Interactions: A Thesis." eScholarship@UMMS, 1991. https://escholarship.umassmed.edu/gsbs_diss/237.
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Transcriptional regulation of cell cycle controlled genes is fundamental to cell division in eukaryotes and a broad spectrum of physiological processes directly related to cell proliferation. Expression of the cell cycle dependent human H4, H3 and H1 histone genes is coordinately regulated at both the transcriptional and posttranscriptional levels. We have systematically analyzed the protein/DNA interactions of the immediate 5'regions of three prototypical cell cycle controlled histone genes, designated H4-F0108, H3-ST519 and H1-FNC16, to define components of the cellular mechanisms mediating transcriptional regulation.
Multiple biochemically distinct protein/DNA interactions were characterized for each of these genes, and the binding sites of several promoter-specific nuclear DNA binding activities were delineated at single nucleotide resolution using a variety of techniques. These findings were integrated with results obtained by others and revealed that the in vitro factor binding sites in H4, H3 and H1 histone promoters coincide with genomic protein/DNA interaction sites defined in vivofor the H4-F0108 and H3-STS19 genes, and with evolutionarily conserved cis-acting sequences shown to affect the efficiency of histone gene transcription. Specifically, we have defined binding sites for Sp1, ATF, CP1/NF-Y, HiNF-D, HiNF-M, HiNF-P and HMG-I related factors. Based on sequence-similarities and cross-competition experiments, we postulate that most of these protein/DNA interaction elements are associated with more than one class of histone genes. Thus, the protein/DNA interactions characterized in this study may represent components of a cellular mechanism that couples transcription rates of the various histone gene classes.
Regulation of the protein/DNA interactions involved in transcriptional control of these H4, H3 and H1 histone genes was investigated in a spectrum of cell types using several distinct in vitro cell culture models for the onset of differentiation and quiescence, as well as cell cycle progression. Moreover, we studied control of histone gene associated DNA binding activities during hepatic development from fetus to adult in transgenic mice reflecting the onset of differentiation and quiescence in vivo. We show that the H4 histone promoter protein/DNA interaction mediated by factor HiNF-D is selectivelymodulated, and directly at the level of DNA binding activity, during the entry into, progress through and exit from the cell cycle in normal diploid cells, as well as during hepatic development. The regulation of this protein/DNA interaction occurs in parallel with analogous interactions occurring in H3 and H1 histone genes. Moreover, these proliferation-specific protein/DNA interactions are collectively deregulated during the cell cycle in four distinct cell types displaying properties of the transformed phenotype. Hence, the cellular competency to coordinately transcribe distinct classes of histone genes during the cell cycle may be mediated by the intricate interplay of constitutively expressed general transcription factors and temporally regulated, cell growth controlled nuclear factors interacting specifically with cell cycle dependent histone genes.
Finally, we show that HiNF-D is represented by two electrophoretically distinct species. The ratio of these forms of HiNF-D fluctuates dramatically during the cell cycle of normal diploid cells, but remains relatively constant in tumor cells. Total HiNF-D binding activity embodied by both HiNF-D species is negatively influenced in vitro by incubation with exogenous phosphatase activity. These observations provide a first indication for the hypothesis that HiNF-D may exist in distinct post-translationally modified forms that are subject to a stringent cell growth control mechanism involving protein kinases and phosphatases. Such a cellular post-translational modification mechanism, which directly impinges on (or activates) the DNA binding activity of a key factor controlling histone genes, would provide a highly efficient means by which to influence the rate of transcription in rapid response to intra-cellular requirements for histone mRNA and extra-cellular cues signalling the onset and cessation of cell proliferation.
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Ghildiyal, Megha. "Endogenous Small RNAs in the Drosophila Soma: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/459.
Full textAbstract:
Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNAs have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, modes of target regulation and in the biological pathways they regulate.
Historically, siRNAs were believed to arise only from exogenous double-stranded RNA triggers in organisms lacking RNA-dependent RNA polymerases. However, the discovery of endogenous siRNAs in flies expanded the biological significance of siRNAs beyond viral defense. By high throughput sequencing we identified Drosophila endosiRNAs as 21 nt small RNAs, bearing a 2´-O-methyl group at their 3´ ends, and depleted in dicer-2 mutants.
Methylation of small RNAs at the 3´ end in the soma, is a consequence of assembly into a mature Argonaute2-RNA induced silencing complex. In addition to endo-siRNAs, we observed certain miRNAs or their miRNA* partners loading into Argonaute2. We discovered, that irrespective of its biogenesis, a miRNA duplex can load into either Argonaute (Ago1 or Ago2), contingent on its structural and sequence features, followed by assignment of one of the strands in the duplex as the functional or guide strand. Usually the miRNA strand is selected as the guide in complex with Ago1 and miRNA* strand with Ago2.
In our efforts towards finding 3´ modified small RNAs in the fly soma, we also discovered 24-28nt small RNAs in certain fly genotypes, particularly ago2 and dcr-2mutants. 24-28nt small RNAs share many features with piRNAs present in the germline, and a significant fraction of the 24-28nt small RNAs originate from similar transposon clusters as somatic endo-siRNAs. Therefore the same RNA can potentially act as a precursor for both endo-siRNA and piRNA-like small RNA biogenesis. We are analyzing the genomic regions that spawn somatic small RNAs in order to understand the triggers for their production. Ultimately, we want to attain insight into the underlying complexity that interconnects these small RNA pathways.
Dysregulation of small RNAs leads to defects in germline development, organogenesis, cell growth and differentiation. This thesis research provides vital insight into the network of interactions that fine-tune the small RNA pathways. Understanding the flow of information between the small RNA pathways, a great deal of which has been revealed only in the recent years, will help us comprehend how the pathways compete and collaborate with each other, enabling each other’s optimum function.
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Bassell, Gary J. "Development and Application of Ultrastructural in Situ Hybridization to Visualize the Spatial Organization of mRNA: a Dissertation." eScholarship@UMMS, 1992. https://escholarship.umassmed.edu/gsbs_diss/153.
Full textAbstract:
It has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this thesis was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems and structures involved. This work required the development of novel in situ hybridization methods for use with electron microscopy. This allowed resolution to visualize single mRNA molecules and individual filaments.
The development of a silver enhancement methodology for both the light and electron microscopic detection of biotinated oligo-dT probes permitted a synoptic view of the intracellular distribution of poly(A) mRNA. At the light microscope, the distribution of poly(A) mRNA did not resemble the individual distribution patterns of microfilaments, intermediate filaments or microtubules. Ultrastructural examination revealed that poly(A) mRNA was not uniformly distributed along cytoskeletal filaments, but clustered at their intersections. The composition of these mRNA containing structures was investigated by both morphologic and in situ hybridization analysis using antibodies to cytoskeletal proteins. In thin sections, polysomes were observed attached to both microfilaments and intermediate filaments. To permit the simultaneous detection of oligo-dT hybridization and specific cytoskeletal proteins, a double labelling method using colloidal gold conjugated antibodies was developed. The majority of poly(A) mRNA was associated with the actin cytoskeleton, with 72% of the hybridization localized within 5nm of a labelled microfilament. Within the actin cytoskeleton, poly(A) mRNA was localized to intersections of orthogonal networks. Greater than 50% of poly(A) colocalized with the actin crosslinking proteins, filamin and α-actinin, but not vinculin.
A significant amount of poly(A) mRNA was found to be associated with intermediate filaments. The double label gold analysis demonstrated that 33% of the hybridization signal localized within 5nm of labelled vimentin filaments. Prior disorganization of the actin cytoskeleton using cytochalasin did not disrupt the association of mRNA with vimentin. These observations are consistent with our morphologic results of polysome-intermediate filament associations, and indicate that microfilaments are not the only filament system to which mRNA is bound. Furthermore, a small amount of hybridization signal (12%) consistently was observed along microtubules, providing an additional cytoskeletal network to distribute mRNA.
To further characterize the spatial organization of mRNA within the cytoskeleton, ultrastructural methods were developed to directly visualize individual mRNA molecules. First, oligonucleotide probes chemically modified with a single hapten and directly conjugated primary reagents were used to permit detection of an individual hybridized probe molecule by a single gold particle. Second, biotin and digoxigenin labelled oligonucleotide probes were used to simultaneously visualize the intermolecular and intramolecular relationships of two nucleic acid sequences. Third, reverse transcriptase was used to extend hybridized primers in situ which permitted visualization of the poly(A) sequence concomittant with the conformation of an mRNA molecule. These methods have permitted analysis of how single mRNA molecules may be positioned with respect to each other within the cytoskeleton.
The ultrastructural visualization of mRNA within its structural environment has demonstrated heterogeneous interactions with the cytoskeleton. Future work will be needed to further characterize the mechanism of mRNA attachment. The proteins which bridge nucleic acid sequences to specific intersections can be identified. It will be interesting to learn how the identified mRNA-cytoskeletal interactions might be involved in the regulation of both mRNA translation and intracellular location. Lastly, and perhaps the most challenging goal, is to investigate whether the identified mRNA-cytoskeletal interactions are used by the cell to influence its own shape, polarity and architecture.
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Pérez, Cano Laura. "Structural prediction and characterization of protein-RNA interactions / Predicción y caracterización estructural de interacciones proteína-ARN." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/120481.
Full textAbstract:
Computational methods are increasingly important to help to predict and characterize protein interactions. However, most of the efforts so far have focused on protein-protein and protein-ligand interactions, and few computer methods are available for modeling and characterizing protein-RNA interactions, in spite of their biological and biomedical importance. Given the difficulties and resource limitations of experimental procedures, developing computer methods for studying protein-RNA interactions is essential in order to get a better understanding of gene expression and cellular function. In this context, the main purpose of this thesis has been the development and application of computational methods for the structural prediction and characterization of protein-RNA complexes.
This Doctoral thesis has fulfilled all the expected objectives. A more detailed summary is given below.
In 2008 it was proposed the first protein-RNA docking case by the CAPRI (Critical Assessment of Prediction of Interactions) communitywide experiment. We devised a new protocol for this new challenge, based on our previous protein-protein docking programs, and obtained excellent results, generating the second best model among all participants. This experiment showed for the first time the potential of our new approach and the possibilities for further improvements.
The next step was the extraction of statistical potentials to be applied for protein-RNA docking and interface prediction. For that purpose, we compiled the largest structural set of non-redundant protein-RNA complexes reported so far in order to derive individual and pairwise propensities of ribonucleotides and amino acid residues to be located at the binding interfaces. We found that the most significantly populated residues at protein-RNA interfaces were Arg, Lys and His, while the less favoured were Asp, Glu, Cys, Val, Leu and Ile. On the other hand, we did not observe a significant preference among the four types of ribonucleotides to be at protein-RNA interfaces. In the same line, pairwise propensities showed similar propensity values for the different types of ribonucleotides.
We then developed the OPRA method to identify regions on protein surface with global preference to bind RNA. This method was tested with an independent set of known protein-RNA structures and showed to have a high positive predictive value for the prediction of residues involved in RNA binding. In addition, we found that this method was able to identify RNA-binding proteins.
The next objective was the application of pairwise statistical potentials to the scoring of protein-RNA docking solutions. Unexpectedly, the statistical potentials showed worse predictive success rates than the FTDock scoring function (highly related to structural complementarity), although the results improved when both scoring terms were combined. However, we still needed more test cases in order to extract more reliable and general conclusions.
Therefore, the next objective was to build a benchmark that could be used for the optimization and development of protein-RNA docking methods. For this, we collected as many non-redundant protein-RNA cases as possible with known complex structure and known or modeled structure for at least one of the subunits. This was the first publicly available protein-RNA docking benchmark and was composed of 106 cases, with 71 cases with at least the available unbound coordinates for one of the molecules, and 35 cases in which at least one of the molecules was built by homology modelling. One of the conclusions that emerged from the analysis of this set of structures is that protein-RNA complexes are much more flexible than protein-protein and even protein-DNA complexes.
We then performed a docking study over the full protein-RNA docking benchmark which showed that the use of pairwise statistical potentials for identifying protein-RNA near-native solutions is noisy. The results confirmed that the best docking success determinant is structural interface complementarity as defined by parameters such as the FTDock score or the van der Waals energy. The combination of these efficient terms with electrostatics yields a scoring function that is able to identify high quality models in most of the cases when the bound coordinates of the interacting molecules are used. However, its efficiency in a more realistic scenario (using the unbound coordinates of the molecules) is highly dependent on the capability of sampling methods to generate high quality docking solutions. Results also underlined important differences with protein-protein interactions.
The experience acquired during these more methodological parts of this PhD thesis has facilitated the application of computational methods to the study of translin, a highly conserved nucleic acid-binding protein of significant biomedical interest. By combining computational tools with experimental techniques, we contributed to the elucidation of the translin multimerization interfaces and nucleic acids binding sites and provided a first structural and dynamic picture of the functions carried out by the protein.La caracterización estructural de complejos proteína-ARN es esencial para lograr una mayor comprensión en el campo de la biología molecular y la regulación celular. Los métodos computacionales de predicción estructural representan una alternativa rápida y poco costosa para la detección y caracterización de complejos biológicos. No obstante, en contraste con la gran variedad de métodos computacionales orientados a la predicción estructural de las interacciones proteína-proteína, existen muy pocos métodos enfocados al estudio de complejos proteína-ARN. En este contexto, el propósito principal de este proyecto de tesis ha sido el desarrollo y aplicación de métodos computacionales para el análisis, caracterización y predicción estructural de complejos proteína-ARN. Con este objetivo, en la primera fase de esta tesis doctoral, se han desarrollado nuevos protocolos para la predicción estructural de este tipo de complejos a partir de métodos de docking entre proteínas previamente descritos, y se han generado potenciales estadísticos por residuo, nucleótido y por pares residuo-nucleótido a partir de estructuras conocidas de complejos proteína-ARN. Dichos potenciales estadísticos por residuo se han aplicado al desarrollo de un método para la predicción de sitios de unión a ARN en proteínas y la identificación de proteínas que unen ARN. Por otro lado, se ha construido un conjunto de pruebas de complejos proteína-ARN para la evaluación de métodos de docking. Usando dicho conjunto de pruebas, se ha estudiado el poder predictivo de los potenciales estadísticos de pares residuo-nucleótido, así como otros términos energéticos, para la evaluación de soluciones de docking de complejos proteína-ARN y se ha desarrollado una nueva función de evaluación de posibles orientaciones de docking en complejos proteína-ARN, integrando aquellos términos energéticos más efectivos a nivel individual. La experiencia acumulada durante las fases iniciales de la tesis permitió la aplicación de técnicas de modelado computacional, en combinación con técnicas experimentales llevadas a cabo por colaboradores, al estudio de translin, una proteína de unión a ácidos nucleicos de gran interés biológico. Así pues, durante la fase final de este proyecto de tesis doctoral se contribuyó a la identificación de los sitios de multimerización y de unión a ácidos nucleicos en translin, y se propuso una primera aproximación estructural y dinámica de las funciones llevadas a cabo por la proteína, contribuyendo a resolver aspectos tan fundamentales como los determinantes estructurales de la unión a ARN.
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Sigova, Alla A. "RNA Silencing Pathways in Schizosaccharomyces pombe and Drosophila melanogaster: A Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/225.
Full textAbstract:
RNA silencing is an evolutionary conserved sequence-specific mechanism of regulation of gene expression. RNA interference (RNAi), a type of RNA silencing in animals, is based on recognition and endonucleolytic cleavage of target mRNA complimentary in sequence to 21-nucleotide (nt) small RNA guides, called small interfering RNAs (siRNAs). Another class of 21-nt small RNAs, called micro RNAs (miRNAs), is endogenously encoded in eukaryotic genomes. Both production of siRNAs from long double-stranded RNA (dsRNA) and biogenesis of miRNAs from hairpin structures are governed by the ribonuclease III enzyme Dicer. Although produced as duplex molecules, siRNAs and miRNAs are assembled into effector complex, called the RNA-induced silencing complex (RISC), as single-strands. A member of the Argonaute family of small RNA-binding proteins lies at the core of all known RNA silencing effector complexes. Plants and animals contain multiple Argonaute paralogs. In addition to endonucleolytic cleavage, Argonaute proteins can direct translational repression/destabilization of mRNA or transcriptional silencing of DNA sequences by the siRNAdirected production of silent heterochromatin.
The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. That only one Dicer, RdRP and Argonaute is expressed in S. pombe might reflect the extreme specialization of RNA silencing pathways regulating targets only at the transcriptional level in this organism. We decided to test if classical RNAi can be induced in S. pombe. We introduced a dsRNA hairpin corresponding to a GFP transgene. GFP silencing triggered by dsRNA reflected a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe required dcr1, rdp1, and ago1, but did not require chp1, tas3, or swi6, genes required for transcriptional silencing. We concluded that the RNAi machinery in S. pombecould direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. Our findings suggest that, in spite of specialization in distinct siRNA-directed silencing pathways, these three proteins fulfill a common biochemical function.
In Drosophila, miRNA and RNAi pathways are both genetically and biochemically distinct. Dicer-2 (Dcr-2) generates siRNAs, whereas the Dicer-1 (Dcr-1)/Loquacious complex produces miRNAs. Argonaute proteins can be divided by sequence similarity into two classes: in flies, the Ago subfamily includes Argonaute1 (Ago1) and Argonaute2 (Ago2), whereas the Piwi subfamily includes Aubergine, Piwi and Argonaute 3. siRNAs and miRNAs direct posttranscriptional gene silencing through effector complexes containing Ago1 or Ago2. The third class of small RNAs, called repeat-associated small interfering RNAs (rasiRNAs), is produced endogenously in the Drosophilagerm line. rasiRNAs mediate silencing of endogenous selfish genetic elements such as retrotransposons and repetitive sequences to ensure genomic stability.
We examined the genetic requirements for biogenesis of rasiRNAs in both male and female germ line of Drosophilaand silencing of 8 different selfish elements, including tree LTR retrotransposons, two non-LTR retrotransposons, and three repetitive sequences. We find that biogenesis of rasiRNAs is different from that of miRNAs and siRNAs. rasiRNA production appears not to require Dicer-1 or Dicer-2. rasiRNAs lack the 2´,3´ hydroxy termini characteristic of animal siRNA and miRNA. While siRNAs derive from both the sense and antisense strands of their dsRNA precursors, rasiRNAs accumulate in antisense polarity to their corresponding target mRNAs. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. We find that rasiRNAs silence their target RNAs posttranscriptionally: mutations that abrogate rasiRNA function dramatically increase the steady-state mRNA level of rasiRNA targets, but do not alter their rate of transcription, measured by nuclear run-on assay.
Our data suggest that rasiRNAs protect the fly germ line through a silencing mechanism distinct from both the miRNA and RNAi pathways.
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Doherty, Johnna E. "Cellular and Molecular Mechanisms Driving Glial Engulfment of Degenerating Axons: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/577.
Full textAbstract:
The nervous system is made up of two major cell types, neurons and glia. The major distinguishing feature between neuronal cells and glial cells is that neurons are capable of transmitting action potentials while glial cells are electrically incompetent. For over a century glial cells were neglected and it was thought they existed merely to provide trophic and structural support to neurons. However, in the past few decades it has become increasingly clear that glial cell functions underlie almost all aspects of nervous system development, maintenance, and health. During development, glia act as permissive substrates for axons, provide guidance cues, regulate axon bundling, facilitate synapse formation, refine synaptic connections, and promote neuronal survival. In the mature nervous system glial cells regulate adult neurogenesis through phagocytosis, act as the primary immune cell, and contribute to complex processes such as learning and memory. In recent years, glial cells have also become a primary focus in the study of neurodegenerative diseases. Mounting evidence shows that glial cells exert both beneficial as well as detrimental effects in the pathology of several nervous system disorders, and modulation of glial activity is emerging as a viable therapeutic strategy for many diseases. Although glial cells are critical to the proper development and functioning of the nervous system, there is still relatively little known about the molecular mechanisms used by glial cells, how they exert their effects on neurons, and how glia and neurons communicate.
Despite the relative simplicity and small size of the Drosophila nervous system, glial cell organization and function in flies shows a remarkable complexity similar to vertebrate glial cells. In this study I use Drosophila as a model organism to study cellular and molecular mechanisms of glial clearance of axonal debris after acute axotomy. In chapter two of this thesis, I characterize three distinct subtypes of glial cells in the adult brain; cell body glia which ensheath neuronal cell bodies in the cortex region of the brain, astrocyte like glial cells which bear striking morphological similarity to mammalian astrocytes and share common molecular components, and ensheathing glial cells which I show act as the primary phagocytic cell type in the neuropil region of the brain. In addition, I identify dCed-6, the ortholog of mammalian GULP, as a necessary component of the glial phagocytic machinery.
In chapter three of this thesis, I perform a candidate based, in vivo, RNAi screen to identify novel genes involved in the glial engulfment of degenerating axon material. The Gal4/UAS system was used to drive UAS-RNAi for approximately 300 candidate genes with the glial specific repo-Gal4 driver. Two assays were used as a readout in this screen, clearance of axon material five days after injury, and Draper upregulation one day after maxillary palp or antennal injury. Overall, I identified 20 genes which, when knocked down specifically in glial cells, result in axon clearance defects after injury. Finally, in chapter four I identify Stat92E as a novel glial gene required for glial phagocytic function. I show that Stat92E regulates both basal and injury induced Draper expression. Injury-induced Draper expression is transcriptionally regulated through a Stat92E dependent non-canonical signaling mechanism whereby signaling through the Draper receptor activates Stat92E which in turn transcriptionally activates draper through a binding site located in the first intron of Draper. Draper represents only the second receptor known to positively regulate Stat92E transcriptional activity under normal physiological conditions.
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Ferron, André. "An appraisal of condition measures for marine fish larvae with particular emphasis on maternal contribution, circadian periodicity, and the time response of nucleic acids and proteins /." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36540.
Full textAbstract:
The physical and physiological condition of larval fishes throughout their development is believed to influence their survival, and ultimately to contribute to recruitment to the adult population. The study of larval condition is therefore central to larval fish ecology.In this thesis I sought to (1) carry out an appraisal of the characteristics and the reliability of condition measures now being used, (2) assess the importance of maternal contribution to the nutritional status of larval fish, (3) evaluate the possibility that diel variability in metabolism could lead to serious biasing of the interpretation of condition measures obtained over time, and (4) assess the time course of the condition of larval fishes subjected to periods of intermittent feeding.
The experiments described in chapter 2 were designed (1) to assess the impact of female nutritional status on the quality of the eggs and larvae they produced, (2) to determine which of a series of nucleic acid and protein measurements were most responsive to post-hatching starvation, and (3) to determine whether the starvation dynamics of those measures was affected by female source. No significant correlation could be found between any of the maternal traits studied and eggs and larval measures. The results did show, however, that egg size was more variable between-clutches than within-clutch, was independent of embryonic developmental rate, but was positively related to larval size.
The existence and ontogeny of circadian (24 hrs.) and ultradian (<24 hrs.) oscillations in the nucleic acids and protein content of larval capelin was investigated in the laboratory experiments outlined in chapter 3. The most obvious long-terms trends occurred during the embryonic period when DNA and RNA content increased constantly, and during the post-yolk-sac period when RNA and protein decreased following sub-optimal feeding.
The objectives of the study described in chapter 4 were threefold, (1) to determine which of a series of nucleic acid and protein measurements were affected by intermittent (delayed-fed and delayed-starved) feeding conditions in capelin larvae, (2) to determine the dynamics and shape of the time response, and (3) to determine whether the empirical data gathered were consistent with models developed as a consequence of the review of the literature (chapter 1). Only the dry weight, and the quantity of DNA, RNA, and protein per dry weight differed significantly between starvation and ad libitum feeding controls. Starvation dynamics were less consistent with predictions. Of the indices investigated, the dynamics of the quantity of DNA and RNA per dry weight were the most consistent dynamics with model predictions. (Abstract shortened by UMI.)
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Pagano, John M. Jr. "RNA Recognition by the Caenorhabditis elegans Embryonic Determinants MEX-5 and MEX-3: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/486.
Full textAbstract:
Post-transcriptional regulation of gene expression is a mechanism that governs developmental and cellular events in metazoans. In early embryogenesis, transcriptionally quiescent cells depend upon maternally supplied factors such as RNA binding proteins and RNA that control key decisions. Morphogen gradients form and in turn pattern the early embryo generating different cell types and spatial order. In the nematode Caenorhabditis elegans, the early embryo relies upon several RNA binding proteins that control mRNA stability, translation efficiency, and/or mRNA localization of cell fate determinants essential for proper development.
MEX-5 and MEX-3 are two conserved RNA-binding proteins required to pattern the anterior/posterior axis and early embryo. Mutation of either gene results in a maternal effect lethal phenotype with proliferating posterior muscle into the anterior blastomeres (Muscle EXcess). Several cell-fate determinants are aberrantly expressed in mex-5 and mex-3 embryos. Both proteins are thought to interact with cis-regulatory elements present in 3’-UTRs of target RNAs controlling their metabolism. However, previous studies failed to demonstrate that these proteins regulate maternal transcripts directly.
This dissertation presents a thorough assessment of the RNA binding properties of MEX-5 and MEX-3. Quantitative biochemical approaches were used to determine the RNA binding specificity of both proteins. MEX-5 has a relaxed specificity, binding with high affinity to linear RNA containing a tract of six or more uridines within an eight-nucleotide window. This is very different from its mammalian homologs Tristetraprolin (TTP) and ERF-2. I was able to identify two amino acids present within the MEX-5 RNA binding domain that are required for the differential RNA recognition observed between MEX-5 and TTP. MEX-3 on the other hand is a specific RNA binding protein, recognizing a bipartite element with flexible spacing between two four-nucleotide half-sites. I demonstrate that this element is required for MEX-3 dependent regulation in vivo. Previous studies only identify a small number of candidate regulatory targets of MEX-5 and MEX-3. The defined sequence specificity of both proteins is used to predict new putative targets that may be regulated by either protein. Collectively, this study examines the RNA binding properties of MEX-5 and MEX-3 to clarify their role as post-transcriptional regulators in nematode development.
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Lin, Chien-Ling. "Studies on the Regulation of Cytoplasmic Polyadenylation Element-Binding Protein: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/583.
Full textAbstract:
Post-transcriptional regulation of gene expression sits at the core of proteomic complexity; trans-acting factors that regulate RNA localization and translation capacity are thus indispensible. In this thesis, I present studies of the cytoplasmic polyadenylation element binding protein (CPEB), a sequence specific RNA-binding protein important for cell cycle progression and neural synaptic plasticity. I focus on CPEB because the activity of RNA-binding proteins affects the destiny of their mRNA substrates. As presented in Chapter II, CPEB, though mostly cytoplasmic at steady state, shuttles between the nucleus and the cytoplasm. Surprisingly, the RNA recognition motifs are essential for the nuclear localization. CPEB associates with the polyadenylation machinery in both compartments, suggesting it is involved in both nuclear mRNA processing and cytoplasmic translational regulation. Moreover, the nuclear translocalization is critical to relay a tight translation repression on CPE-containing mRNAs. Chapter III focuses on the regulation of CPEB dimerization. CPEB dimerizes through the RNA-binding domains to inhibit its own RNA binding ability in a cell cycle-dependent manner. By dimerizing, CPEB has enhanced binding to protein destruction factors so that robust active degradation occurs in the later cell cycle. The degradation of CPEB is required for translation activation of a subset of mRNAs and cell cycle progression. In addition, dimerization protects cells from being overloaded with excess CPEB. In sum, the localization and dimerization status of CPEB is dynamic and highly regulated; they in turn regulate the activity of CPEB, which results in responsive translation control. These studies provide a strong foundation to decipher CPEB-mediated gene expression.
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Amer, Ayman Salah-el-deen. "Cytoanalysis of pancreatic B-cells using an avian model, mammalian tissue culture and implications of antisense oligonucleotides transfection /." Huntington, WV : [Marshall University Libraries], 2004. http://www.marshall.edu/etd/descript.asp?ref=474.
Full textAbstract:
Theses (Ph. D.)--Marshall University, 2004.Title from document title page. Includes abstract. Document formatted into pages: contains xiv, 192 p. including illustrations. Bibliography: p. 157-192.
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Du, Ling. "CIS/SOCS Proteins in Growth Hormone Action: A Dissertation." eScholarship@UMMS, 2000. https://escholarship.umassmed.edu/gsbs_diss/92.
Full textAbstract:
CIS/SOCS (cytokine-inducible SH2 protein/suppressor of cytokine signaling) are a family of proteins that are thought to act as negative regulators of signaling by erythropoetin, interleukin-6 and other cytokines whose receptors are related to the growth hormone receptor (GHR), and like growth hormone (GH), signal through the JAK/STAT pathway. We examined the possibility that CIS/SOCS proteins may also be involved in GH signaling, in particular, in termination of the transient insulin-like effects of GH. mRNAs for CIS, SOCS3, and to a lesser extent SOCS1 were detectable by Northern blot analysis of rat adipocyte total RNA, and the expression of CIS and SOCS3 was markedly increased 30 min after incubation with 500 ng/ml hGH. Both CIS and SOCS3 were detected in adipocyte extracts by immunoprecipitation and immunoblotting with their corresponding antisera. GH stimulated the tyrosine phosphorylation of a 120 kDa protein (p120) that was co-precipitated from adipocyte extracts along with αCIS and detected in Western blots with phospho-tyrosine antibodies. However, no tyrosine phosphorylated proteins in these cell extracts were immunoprecipitated with antibodies to CIS3/SOCS3. p120 was later identified as the GHR based on the observations that two GHR antibodies recognized p120 in scale-up experiments and that p120 and the GHR share several characteristics, including their molecular weights, tyrosine phosphorylation upon GH stimulation, interaction with CIS, similar extent of glycosylation as judged by electrophoretic mobility shift after Endo F digestion, comparable mobility shifts upon thrombin digestion, and N-terminal histidine-tagging. The findings, however, do not rule out the possibility that there might be other tyrosine phosphorylated 120 kDa protein(s) that interact with CIS and contribute to the p120 signal, as well as the GHR.
Further studies of the association of CIS with the GHR revealed that CIS might selectively interact with multiply tyrosine phosphorylated forms of the GHR, and these tyrosines are likely located near the carboxyl end of the GHR. Overexpression of CIS partially inhibited GH-induced STAT5 phosphorylation in CHO cells. Studies in freshly isolated and GH-deprived (sensitive) adipocytes revealed that the abundance of CIS does not correlate with the termination of the insulin-like effects of GH or the emergence of refractoriness. Neither the association of CIS with the GHR nor the tyrosine phosphorylation status of the GHR, JAK2 and STAT5 appear responsible for refractoriness in adipocytes. These data imply that some negative regulators other than CIS might contribute to the termination of GH-induced insulin-like effects in adipocytes.
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Laine, Jennifer M. "Protein Ligand Interactions Probed by NMR: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/617.
Full textAbstract:
Molecular recognition, defined as the specific interactions between two or more molecules, is at the center of many biological processes including catalysis, signal transduction, gene regulation and allostery. Allosteric regulation is the modification of function caused by an intermolecular interaction. Allosteric proteins modify their activity in response to a biological signal that is often transmitted through the interaction with a small effector molecule. Therefore, determination of the origins of intermolecular interactions involved in molecular recognition and allostery are essential for understanding biological processes. Classically, molecular recognition and allosteric regulation have been associated to structural changes of the system. NMR spectroscopic methods have indicated that changes in protein dynamics may also contribute to molecular recognition and allostery. This thesis is an investigation of the contributions of both structure and dynamics in molecular binding phenomena.
In chapter I, I describe molecular recognition, allostery and examples of allostery and cooperativity. Then I discuss the contribution of protein dynamics to function with a special focus on allosteric regulation. Lastly I introduce the hemoglobin homodimer, HbI of Scapharca inaequivalvis and the mRNA binding protein TIS11d.
Chapter II is the primary focus of this thesis on the contribution of protein dynamics to allostery in the dimeric hemoglobin of scapharca inaequivalvis, HbI. Thereafter I concentrate on the mechanism of adenine recognition of the Tristetraprolin-like (TTP) protein TIS11d; this study is detailed in Chapter III. In Chapter IV I discuss broader impacts and future directions of my research.
This thesis presents an example of the use of protein NMR spectroscopy to probe ligand binding. The studies presented in this thesis emphasize the importance of dynamics in understanding protein function. Measurements of protein motions will be an element of future studies to understand protein function in health and disease.
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Yang, Shun-Jen. "The Molecular Mechanisms Underlying the Polarized Distribution of Drosophila Dscam in Neurons: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/390.
Full textAbstract:
Neurons exhibit highly polarized structures, including two morphologically and functionally distinct domains, axons and dendrites. Dendrites and axons receive versus send information, and proper execution of each requires different sets of molecules. Differential distribution of membrane proteins in distinct neuronal compartments plays essential roles in neuronal functions. The major goal of my doctoral thesis was to study the molecular mechanisms that govern the differential distribution of membrane proteins in neurons, using the Drosophilalarval mushroom body (MB) as a model system.
My work was initiated by an observation of differential distribution of distinct Dscam isoforms in neurons. Dscam stands for Down Syndrome Cell Adhesion Molecule, which is a Drosophila homolog of human DSCAM. According to genomic analysis, DrosophilaDscam gene can generate more than 38,000 isoforms through alternative splicing in its exons 4, 6, 9 and 17. All Dscam isoforms share similar domain structures, with 10 immunoglobulin domains and 6 fibronectin type III repeats in the ectodomain, a single transmembrane domain and a cytoplasmic endodomain. There are two alternative exons in exon 17 (17.1 and 17.2), which encodes Dscam’s transmembrane domain. Interestingly, in ectopic expression, Dscam isoforms carrying exon 17.1 (Dscam[TM1]) can be preferentially localized to dendrites and cell bodies, while Dscam isoforms carrying exon 17.2 (Dscam[TM2]) are distributed throughout the entire neuron including axons and dendrites.
To unravel the mechanisms involved in the differential distribution of Dscam[TM1] versus Dscam[TM2], I conducted a mosaic genetic screening to identify the possible factors affecting dendritic distribution of Dscam[TM1], established an in vivoTARGET system to better distinguish the differential distribution of Dscam, identified the axonal and dendritic targeting motifs of Dscam molecules and further showed that Dscam’s differential roles in dendrites versus axons are correlated with its localization.
Several mutants affecting dendritic distribution of Dscam[TM1] have been identified using a MARCM genetic screen. Three of these mutants (Dlis1, Dmn and p24) are components of the dynein/dynactin complex. Silencing of other dynein/dynactin subunits and blocking dynein function with a dominant-negative Glued mutant also resulted in mislocalization of Dscam[TM1] from dendrites to axons. However, microtubule polarity in the mutant axons was maintained. Taken together, this was the first demonstration that the dynein/dynactin complex is involved in the polarized distribution of membrane proteins in neurons. To further examine how dynein/dynactin is involved in the dendritic distribution of Dscam[TM1], I compromised dynenin/dynactin function with dominant-negative Glued and transiently induced Dscam[TM1] expression. The results suggested that dynein/dynactin may not be directly involved in the targeting of newly synthesized Dscam[TM1] to dendrites. Instead, it plays a role in maintaining dendritic restriction of Dscam[TM1]. Notably, dynein/dynactin dysfunction did not alter distribution of another dendritic transmembrane protein Rdl (Resistant to Dieldrin), supporting involvement of diverse mechanisms in distributing distinct molecules to the dendritic membrane.
To identify the targeting motifs of Dscam, I incorporated the TARGET (Temporal and regional gene expression targeting) system into mushroom body (MB) neurons, and this allowed the demonstration of the differential distribution of Dscam[TM1] and Dscam[TM2] with more clarity than conventional overexpression techniques. Using the TARGET system, I identified an axonal targeting motif located in the cytoplasmic juxtamemebrane domain of Dscam[TM2]. This axonal targeting motif is dominant over the dendritic targeting motif located in Dscam’s ectodomain. Scanning alanine mutagenesis demonstrated that two amino acids in the axonal targeting motif were essential for Dscam’s axonal distribution. Interestingly, swapping the cytoplasmic juxtamembrane portions between TM1 and TM2 not only reversed TM1’s and TM2’s differential distribution patterns but also their functional properties in dendrites versus axons.
My thesis research also involved studying endodomain diversity of Dscam isoforms. Besides the diversity originally found in the ectodomain and transmembrane domain of Dscam, my colleagues and I further demonstrated the existence of four additional endodomain variants. These four variants are generated by skipping or retaining exon 19 or exon 23 through independent alternative splicing. Interestingly, different Dscam endodomain isoforms are expressed at different developmental stages and in different areas of the nervous system. Through isoform-specific RNA interference, we showed the differential involvement of distinct Dscam endodomains in specific neuronal morphogenetic processes. Analysis of the primary sequence of the Dscam endodomain indicated that endodomain variants may confer activation of different signaling pathways and functional roles in neuronal morphogenesis.
In Summary, my thesis work identified and characterized several previously unknown mechanisms related to the differential distribution of membrane proteins in neurons. I showed that there may be a dynein/dynactin-independent mechanism for selective transport of dendritic membrane proteins to dendrites. Second, dynein/dynactin plays a maintenance role in dendritic restriction of Dscam[TM1]. Third, different membrane proteins may require distinct combinations of mechanisms to be properly targeted and maintained in certain neuronal compartments. Further analysis of the mutants indentified from my genetic screen will definitely help to resolve the missing pieces of the puzzle. These findings provide novel mechanistic insight into the differential distribution of membrane proteins in polarized neurons.
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Straza, Michael W. "A Tale of Two ARFs: Tumor Suppressor and Anti-viral Functions of p14ARF: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/472.
Full textAbstract:
Animals have evolved complicated and overlapping mechanisms to guard against the development of cancer and infection by pathogenic organisms. ARF, a potent tumor suppressor, positively regulates p53 by antagonizing p53’s negative regulator, MDM2, which in turn results in either apoptosis or cell cycle arrest. ARF also has p53-independent tumor suppressor activity. The CtBP transcriptional co-repressors promote cancer cell survival and migration/invasion. CtBP senses cellular metabolism via a regulatory dehydrogenase domain, and is a target for negative regulation by ARF. ARF targets CtBP to the proteasome for degradation, which results in the up regulation of proapoptotic BH3-only proteins, and p53-independent apoptosis. CtBP inhibition by ARF also up regulates PTEN, reducing cancer cell motility, making CtBP a potential therapeutic target in human cancer.
The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and is cytotoxic to cancer cells from a wide variety of tissues. MTOB induced apoptosis was independent of p53, and correlated with the de-repression of the pro-apoptotic CtBP repression target Bik. CtBP over-expression, or Bik silencing, rescued MTOB-induced cell death. MTOB did not induce apoptosis in mouse embryonic fibroblasts (MEFs), but was increasingly cytotoxic to immortalized and transformed MEFs, suggesting that CtBP inhibition may provide a suitable therapeutic index for cancer therapy.
In human colon cancer cell peritoneal xenografts, MTOB treatment decreased tumor burden, and induced tumor cell apoptosis. To verify the potential utility of CtBP as a therapeutic target in human cancer the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Targeting CtBP with a small molecule like MTOB may thus represent a useful and widely applicable therapeutic strategy in human malignancies.
ARF has long been known to respond to virally encoded oncogenes. Recently, p14ARF was linked to the innate immune response to non-transforming viruses in mice. Therefore a wider role for the ARF pathway in viral infection was considered. Previous studies linking p53 to multiple points of the Human Immunodeficiency Virus-1 (HIV-1) life cycle suggested that ARF may also play a role in the HIV life cycle. In this study the interdependency of ARF and HIV infection was investigated. ARF expression was determined for a variety of cell types upon HIV infection. In every case, ARF levels exhibited dynamic changes upon HIV infection-in most cases ARF levels were reduced in infected cells. The impact of ARF over-expression or silencing by RNAi on HIV infection was also examined. Consistently, p24 levels were increased with ARF overexpression, and decreased when ARF was silenced. Thus ARF and HIV modulate each other, and ARF may paradoxically play a positive role in the HIV life cycle.