Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C- and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and 15N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional [15N] 1H heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 microM concentration range, with a total of 30 nmol or approximately 40 micrograms of RNA in approximately 150 microliters, give strong NMR signals in a short accumulation time. The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems. This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure.

Definition of a metal-dependent/Li(+)-inhibited phosphomonoesterase protein family based upon a conserved three-dimensional core structure.

Inositol polyphosphate 1-phosphatase, inositol monophosphate phosphatase, and fructose 1,6-bisphosphatase share a sequence motif, Asp-Pro-(Ile or Leu)-Asp-(Gly or Ser)-(Thr or Ser), that has been shown by crystallographic and mutagenesis studies to bind metal ions and participate in catalysis. We compared the six alpha-carbon coordinates of this motif from the crystal structures of these three phosphatases and found that they are superimposable with rms deviations ranging from 0.27 to 0.60 A. Remarkably, when these proteins were aligned by this motif a common core structure emerged, defined by five alpha-helices and 11 beta-strands comprising 155 residues having rms deviations ranging from 1.48 to 2.66 A. We used the superimposed structures to align the sequences within the common core, and a distant relationship was observed suggesting a common ancestor. The common core was used to align the sequences of several other proteins that share significant similarity to inositol monophosphate phosphatase, including proteins encoded by fungal qa-X and qutG, bacterial suhB and cysQ (identical to amtA), and yeast met22 (identical to hal2). Evolutionary comparison of the core sequences indicate that five distinct branches exist within this family. These proteins share metal-dependent/Li(+)-sensitive phosphomonoesterase activity, and each predicted tree branch exhibits unique substrate specificity. Thus, these proteins define an ancient structurally conserved family involved in diverse metabolic pathways including inositol signaling, gluconeogenesis, sulfate assimilation, and possibly quinone metabolism. Furthermore, we suggest that this protein family identifies candidate enzymes to account for both the therapeutic and toxic actions of Li+ as it is used in patients treated for manic depressive disease.

Innovative analytical strategies for the development of sensor devices and mass spectrometry methods

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato.

Three-dimensional inverse modelling of magnetic anomaly sources based on a genetic algorithm.

We present a modelling method to estimate the 3-D geometry and location of homogeneously magnetized sources from magnetic anomaly data. As input information, the procedure needs the parameters defining the magnetization vector (intensity, inclination and declination) and the Earth's magnetic field direction. When these two vectors are expected to be different in direction, we propose to estimate the magnetization direction from the magnetic map. Then, using this information, we apply an inversion approach based on a genetic algorithm which finds the geometry of the sources by seeking the optimum solution from an initial population of models in successive iterations through an evolutionary process. The evolution consists of three genetic operators (selection, crossover and mutation), which act on each generation, and a smoothing operator, which looks for the best fit to the observed data and a solution consisting of plausible compact sources. The method allows the use of non-gridded, non-planar and inaccurate anomaly data and non-regular subsurface partitions. In addition, neither constraints for the depth to the top of the sources nor an initial model are necessary, although previous models can be incorporated into the process. We show the results of a test using two complex synthetic anomalies to demonstrate the efficiency of our inversion method. The application to real data is illustrated with aeromagnetic data of the volcanic island of Gran Canaria (Canary Islands).

Finite temperature dynamics of vortices in the two dimensional anisotropic Heisenberg model

We study the effects of finite temperature on the dynamics of non-planar vortices in the classical, two-dimensional anisotropic Heisenberg model with XY- or easy-plane symmetry. To this end, we analyze a generalized Landau-Lifshitz equation including additive white noise and Gilbert damping. Using a collective variable theory with no adjustable parameters we derive an equation of motion for the vortices with stochastic forces which are shown to represent white noise with an effective diffusion constant linearly dependent on temperature. We solve these stochastic equations of motion by means of a Green's function formalism and obtain the mean vortex trajectory and its variance. We find a non-standard time dependence for the variance of the components perpendicular to the driving force. We compare the analytical results with Langevin dynamics simulations and find a good agreement up to temperatures of the order of 25% of the Kosterlitz-Thouless transition temperature. Finally, we discuss the reasons why our approach is not appropriate for higher temperatures as well as the discreteness effects observed in the numerical simulations.

Development and Application of Improvements to the Tile-based Fisher Ratio Method and Fundamental Instrument Considerations for Non-targeted Analysis using Two-dimensional Gas Chromatography

Thesis (Ph.D.)--University of Washington, 2016-06

Optimization and Machine Learning Frameworks for Complex Network Analysis

Thesis (Ph.D.)--University of Washington, 2016-06

Solution structure of the cyclotide palicourein: Implications for the development of a pharmaceutical framework

The cyclotides are a family of disulfide-rich proteins from plants. They have the characteristic structural features of a circular protein backbone and a knotted arrangement of disulfide bonds. Structural and biochemical studies of the cyclotides suggest that their unique physiological stability can be loaned to bioactive peptide fragments for pharmaceutical and agricultural development. In particular, the cyclotides incorporate a number of solvent-exposed loops that are potentially suitable for epitope grafting applications. Here, we determine the structure of the largest known cyclotide, palicourein, which has an atypical size and composition within one of the surface-exposed loops. The structural data show that an increase in size of a palicourein loop does not perturb the core fold, to which the thermodynamic and chemical stability has been attributed. The cyclotide core fold, thus, can in principle be used as a framework for the development of useful pharmaceutical and agricultural bioactivities.

Three-dimensional solitons in coupled atomic-molecular Bose-Einstein condensates

We present a theoretical analysis of three-dimensional (3D) matter-wave solitons and their stability properties in coupled atomic and molecular Bose-Einstein condensates (BECs). The soliton solutions to the mean-field equations are obtained in an approximate analytical form by means of a variational approach. We investigate soliton stability within the parameter space described by the atom-molecule conversion coupling, the atom-atom s-wave scattering, and the bare formation energy of the molecular species. In terms of ordinary optics, this is analogous to the process of sub- or second-harmonic generation in a quadratic nonlinear medium modified by a cubic nonlinearity, together with a phase mismatch term between the fields. While the possibility of formation of multidimensional spatiotemporal solitons in pure quadratic media has been theoretically demonstrated previously, here we extend this prediction to matter-wave interactions in BEC systems where higher-order nonlinear processes due to interparticle collisions are unavoidable and may not be neglected. The stability of the solitons predicted for repulsive atom-atom interactions is investigated by direct numerical simulations of the equations of motion in a full 3D lattice. Our analysis also leads to a possible technique for demonstrating the ground state of the Schrodinger-Newton and related equations that describe Bose-Einstein condensates with nonlocal interparticle forces.

Similarity solution of axisymmetric flow in porous media

Applications of the axisymmetric Boussinesq equation to groundwater hydrology and reservoir engineering have long been recognised. An archetypal example is invasion by drilling fluid into a permeable bed where there is initially no such fluid present, a circumstance of some importance in the oil industry. It is well known that the governing Boussinesq model can be reduced to a nonlinear ordinary differential equation using a similarity variable, a transformation that is valid for a certain time-dependent flux at the origin. Here, a new analytical approximation is obtained for this case. The new solution,, which has a simple form, is demonstrated to be highly accurate. (c) 2005 Elsevier Ltd. All rights reserved.

Isolation and characterization of novel cyclotides from Viola hederaceae - Solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide

Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 ( vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional H-1 NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded β- sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and ∼ 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 μ m.

Wave-generated flow on coral reefs - an analysis for two-dimensional horizontal reef-tops with steep faces

Waves breaking on the seaward rim of a coral reef generate a flow of water from the exposed side of the reef to the sheltered side and/or to either channels through the reef-rim or lower sections of the latter. This wave-generated flow is driven by the water surface gradient resulting from the wave set-up created by the breaking waves. This paper reviews previous approaches to modelling wave-generated flows across coral reefs and discusses the influence of reef morphology and roughness upon these flows. Laboratory measurements upon a two-dimensional horizontal reef platform with a steep reef face provide the basis for extending a previous theoretical analysis for wave set-up on a reef in the absence of a flow [Gourlay, M.R., 1996b. Wave set-up on coral reefs. 2. Set-up on reefs with various profiles. Coastal Engineering 28, 1755] to include the interaction between a unidirectional flow and the wave set-up. The laboratory model results are then used to demonstrate that there are two basic reef-top flow regimes-reef-top control and reef-rim control. Using open channel flow theory, analytical relationships are derived for the reef-top current velocity in terms of the offreef wave conditions, the reef-top water depth and the physical characteristics of the reef-top topography. The wave set-up and wave-generated flow relationships are found to predict experimental values with reasonable accuracy in most cases. The analytical relationships are used to investigate wave-generated flows into a boat harbour channel on Heron Reef in the southern Great Barrier Reef. (c) 2005 Elsevier B.V. All rights reserved.

Isolation, solution structure, and insecticidal activity of Kalata B2, a circular protein with a twist: Do Mobius strips exist in nature?

A large number of macrocyclic miniproteins with diverse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using H-1 NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.

An adaptive and dynamic dimensionality reduction method for high-dimensional indexing

The notorious "dimensionality curse" is a well-known phenomenon for any multi-dimensional indexes attempting to scale up to high dimensions. One well-known approach to overcome degradation in performance with respect to increasing dimensions is to reduce the dimensionality of the original dataset before constructing the index. However, identifying the correlation among the dimensions and effectively reducing them are challenging tasks. In this paper, we present an adaptive Multi-level Mahalanobis-based Dimensionality Reduction (MMDR) technique for high-dimensional indexing. Our MMDR technique has four notable features compared to existing methods. First, it discovers elliptical clusters for more effective dimensionality reduction by using only the low-dimensional subspaces. Second, data points in the different axis systems are indexed using a single B+-tree. Third, our technique is highly scalable in terms of data size and dimension. Finally, it is also dynamic and adaptive to insertions. An extensive performance study was conducted using both real and synthetic datasets, and the results show that our technique not only achieves higher precision, but also enables queries to be processed efficiently. Copyright Springer-Verlag 2005

A method for screening the temperature dependence of three-dimensional crystal formation

Temperature is an important parameter controlling protein crystal growth. A new temperature-screening system (Thermo-screen) is described consisting of a gradient thermocycler fitted with a special crystallization-plate adapter onto which a 192-well sitting-drop crystallization plate can be mounted (temperature range 277-372 K; maximum temperature gradient 20 K; interval precision 0.3 K). The system allows 16 different conditions to be monitored simultaneously over a range of 12 temperatures and is well suited to conduct wide (similar to 20 K) and fine (similar to 3 K) temperature-optimization screens. It can potentially aid in the determination of temperature phase diagrams and run more complex temperature-cycling experiments for seeding and crystal growth.