Transcription factor occupancy in differentiating skeletal muscle

With recent advances in high-throughput sequencing, mapping of genome-wide transcription factor occupancy has become feasible. To advance the understanding of skeletal muscle differentiation specifically and transcriptional regulation in general, I determined the genome-wide occupancy map for myogenin in differentiating C2C12 myocyte cells. I then analyzed the myogenin map for underlying sequence content and the association between occupied elements and expression trajectories of adjacent genes. Having determined that myogenin primarily associates with expressed genes, I performed a similar analysis on occupancy maps of other transcription factors active during skeletal muscle differentiation, including an extensive analysis of co-occupancy. This analysis provided strong motif evidence for protein-protein interactions as the primary driving force in the formation of Myogenin / Mef2 and MyoD / AP-1 complexes at jointly-occupied sites. Finally, factor occupancy analysis was extended to include bHLH transcription factors in tissues other than skeletal muscle. The cross-tissue analysis led to the emergence of a motif structure used by bHLH TFs to encode either tissue-specific or "general" (public) access in a variety of lineages.

Binding Site Structure and Stoichiometry in Serotonin Type 3 Receptors

This dissertation primarily describes studies of serotonin type 3 (5-HT₃) receptors of the Cys-loop super-family of ligand gated ion channels. The first chapter provides a general introduction to these important proteins and the methods used to interrogate their structure and function. The second chapter details the delineation of a structural unit of the ligand binding site of homomeric 5-HT₃A receptors on which the ligands serotonin (5-HT) and m-chlorophenyl biguanide (mCPBG) are reliant for effective receptor activation. Unnatural amino acid mutagenesis results show that the details of each ligand’s interaction with this organizing feature of the binding site differ, providing insights into general principles of receptor activation.

The third chapter describes a study in which florescent protein fusions of the A and B subunits of the heteromeric 5-HT₃AB receptor are employed to determine the subunit stoichiometry and order within functional receptors. Strong evidence is found for an A₃B₂ stoichiometry with A-A-B-A-B order. The fourth chapter investigates the potential for ligand binding across heteromeric binding sites in the 5-HT₃AB receptor. Unlike serotonin, mCPBG is found to bind the receptor at heteromeric binding sites. Further mCPBG is capable of allosterically modulating the response of serotonin on the 5-HT₃AB receptor from these heteromeric sites.

Finally, the fifth chapter describes progress towards the application of unnatural amino acid mutagenesis to an important new class of proteins, transcription factors. Experiments optimizing novel methods for the detection of function are described, using RARα of the nuclear receptor family of transcription factors.

DNA-mediated oxidation of transcription factor p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the π-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

To determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]³⁺, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.

The chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were ¹³C₂D₂-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward ¹³C₂D₂-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

Targeting DNA repeat sequences with Py-Im polyamides

Hairpin pyrrole-imdazole polyamides are cell-permeable, sequence-programmable oligomers that bind in the minor groove of DNA. This thesis describes studies of Py-Im polyamides targeted to biologically important DNA repeat sequences for the purpose of modulating disease states. Design of a hairpin polyamide that binds the CG dyad, a site of DNA methylation that can become dysregulated in cancer, is described. We report the synthesis of a DNA methylation antagonist, its sequence specificity and affinity informed by Bind-n-Seq and iteratively designed, which improves inhibitory activity in a cell-free assay by 1000-fold to low nanomolar IC50. Additionally, a hairpin polyamide targeted to the telomeric sequence is found to trigger a slow necrotic-type cell death with the release of inflammatory molecules in a model of B cell lymphoma. The effects of the polyamide are unique in this class of oligomers; its effects are characterized and a functional assay of phagocytosis by macrophages is described. Additionally, hairpin polyamides targeted to pathologically expanded CTG•CAG triplet repeat DNA sequences, the molecular cause of myotonic dystrophy type 1, are synthesized and assessed for toxicity. Lastly, ChIP-seq of Hypoxia-Inducible Factor is performed under hypoxia-induced conditions. The study results show that ChIP-seq can be employed to understand the genome-wide perturbation of Hypoxia-Inducible Factor occupancy by a Py-Im polyamide.

Site-Specific Isotopes in Small Organic Molecules

Stable isotope geochemistry is a valuable toolkit for addressing a broad range of problems in the geosciences. Recent technical advances provide information that was previously unattainable or provide unprecedented precision and accuracy. Two such techniques are site-specific stable isotope mass spectrometry and clumped isotope thermometry. In this thesis, I use site-specific isotope and clumped isotope data to explore natural gas development and carbonate reaction kinetics. In the first chapter, I develop an equilibrium thermodynamics model to calculate equilibrium constants for isotope exchange reactions in small organic molecules. This equilibrium data provides a framework for interpreting the more complex data in the later chapters. In the second chapter, I demonstrate a method for measuring site-specific carbon isotopes in propane using high-resolution gas source mass spectrometry. This method relies on the characteristic fragments created during electron ionization, in which I measure the relative isotopic enrichment of separate parts of the molecule. My technique will be applied to a range of organic compounds in the future. For the third chapter, I use this technique to explore diffusion, mixing, and other natural processes in natural gas basins. As time progresses and the mixture matures, different components like kerogen and oil contribute to the propane in a natural gas sample. Each component imparts a distinct fingerprint on the site-specific isotope distribution within propane that I can observe to understand the source composition and maturation of the basin. Finally, in Chapter Four, I study the reaction kinetics of clumped isotopes in aragonite. Despite its frequent use as a clumped isotope thermometer, the aragonite blocking temperature is not known. Using laboratory heating experiments, I determine that the aragonite clumped isotope thermometer has a blocking temperature of 50-100°C. I compare this result to natural samples from the San Juan Islands that exhibit a maximum clumped isotope temperature that matches this blocking temperature. This thesis presents a framework for measuring site-specific carbon isotopes in organic molecules and new constraints on aragonite reaction kinetics. This study represents the foundation of a future generation of geochemical tools for the study of complex geologic systems.

Report on the Linda Vista bridge site

The Linda Vista Bridge spans the Arroyo Seco about a quarter of a mile above the Colorado Street Bridge, but serves an entirely different territory; as there is no road between there on the west bank. Los Angeles, Hollywood, and several of the beach cities can be reached by the way of the Colorado Street Bridge. The Linda Vista Bridge carries the traffic to the northwest of Pasadena, that is, Flintridge, Linda Vista, Montrose, Sunland. After leaving the bridge, the road follows the west bank of the Arroyo almost to the mouth of the canyon; then to the west along the foot of the mountains and into the San Fernando Valley.

Estimating Sighting Proportions of American Alligator Nests during Helicopter Survey

Proportions of American alligator (Alligator mississippiensis) nests sighted during aerial survey in Florida were estimated based upon multiple surveys by different observers. We compared sighting proportions across habitats, nesting seasons, and observer experience levels. The mean sighting proportion across all habitats and years was 0.736 (SE=0.024). Survey counts corrected by the mean sighting proportion reliably predicted total nest counts (R2=0.933). Sighting proportions did not differ by habitat type (P=0.668) or year P=0.328). Experienced observers detected a greater proportion of nests (P

I. Site effects and exciton structure in molecular crystals - Benzene. II. The first and second triplet states of solid benzene

The effect of intermolecular coupling in molecular energy levels (electronic and vibrational) has been investigated in neat and isotopic mixed crystals of benzene. In the isotopic mixed crystals of C₆H₆, C₆H₅D, m-C₆H₄D₂, p-C₆H₄D₂, sym-C₆H₃D₃, C₆D₅H, and C₆D₆ in either a C₆H₆ or C₆D₆ host, the following phenomena have been observed and interpreted in terms of a refined Frenkel exciton theory: a) Site shifts; b) site group splittings of the degenerate ground state vibrations of C₆H₆, C₆D₆, and sym-C₆H₃D₃; c) the orientational effect for the isotopes without a trigonal axis in both the ¹B_2u electronic state and the ground state vibrations; d) intrasite Fermi resonance between molecular fundamentals due to the reduced symmetry of the crystal site; and e) intermolecular or intersite Fermi resonance between nearly degenerate states of the host and guest molecules. In the neat crystal experiments on the ground state vibrations it was possible to observe many of these phenomena in conjunction with and in addition to the exciton structure.

To theoretically interpret these diverse experimental data, the concepts of interchange symmetry, the ideal mixed crystal, and site wave functions have been developed and are presented in detail. In the interpretation of the exciton data the relative signs of the intermolecular coupling constants have been emphasized, and in the limit of the ideal mixed crystal a technique is discussed for locating the exciton band center or unobserved exciton components. A differentiation between static and dynamic interactions is made in the Frenkel limit which enables the concepts of site effects and exciton coupling to be sharpened. It is thus possible to treat the crystal induced effects in such a fashion as to make their similarities and differences quite apparent.

A calculation of the ground state vibrational phenomena (site shifts and splittings, orientational effects, and exciton structure) and of the crystal lattice modes has been carried out for these systems. This calculation serves as a test of the approximations of first order Frenkel theory and the atom-atom, pair wise interaction model for the intermolecular potentials. The general form of the potential employed was V(r) = Be^-Cr - A/r⁶ ; the force constants were obtained from the potential by assuming the atoms were undergoing simple harmonic motion.

In part II the location and identification of the benzene first and second triplet states (³B_1u and ³E_1u) is given.

Estimating the underwater shape of tuna longlines with micro-bathythermographs

An estimation method for the three-dimensional underwater shape of tuna longlines is developed, using measurements of depth obtained from micro-bathythermographs (BTs) attached to the main line at equally spaced intervals. The shape of the main line is approximated by a model which consists of a chain of unit length lines (folding-rule model), where the junction points are placed at the observed depths. Among the infinite number of possible shapes, the most likely shape is considered to be the smoothest one that can be obtained with a numerical optimization algorithm. To validate the method, a series of experimental longline operations were conducted in the equatorial region of the eastern Pacific Ocean, using 13 or 14 micro-BTs per basket of main line. Concurrent observations of oceanographic conditions (currents and temperature structure) were obtained. The shape of the main line can be calculated at arbitrary times during operations. Shapes were consistent with the current structure. On the equator, the line was elevated significantly by the Equatorial Undercurrent. It is shown that the shape of main line depends primarily upon the vertical shear and direction of the current relative to the gear. Time sequences of calculated shapes reveals that observed periodic (1-2 hours) oscillations in depth of the gear was caused by swinging movements of the main line. The shortening rate of the main line is an important parameter for formulating the shape of the longline, and its precise measurement is desirable.

Análise das estimativas de emissão de metano por aterros sanitários em projetos de MDL no Brasil.

Para a maioria dos municípios brasileiros, a instalação de um aterro sanitário é um desafio, sendo uma das dificuldades o custo elevado. Existem algumas formas de mitigar estes custos e uma delas é através do mercado de emissões. Com planejamento prévio suficiente, é possível queimar o metano gerado através da degradação do resíduo, podendo resultar em benefícios para o aterro tanto através do aproveitamento (geração de energia ou venda direta) quanto recebimento de algum tipo de certificado de emissões negociável. Incluído neste planejamento prévio suficiente está a realização da estimativa ex-ante de emissão de metano para saber previamente qual será o aproveitamento mais indicado e a eventual receita oriunda da queima. Quando analisados os projetos de MDL feitos em aterros sanitários, pode ser notado que estas estimativas são muitas vezes mal feitas, gerando valores estimados muito acima do realmente observado durante a operação. Este erro acarreta uma perda de credibilidade deste tipo de projeto, já que o número esperado é raramente alcançado. Existem alguns fatores que contribuem para esta discrepância de valores, sendo problemas operacionais (como exemplo podem ser citados deficiência no sistema de captura do biogás e problemas na captação e recirculação de lixiviado) e de modelagem (utilização de valores de entrada experimentais obtidos sob situações muito diferentes das encontradas nos aterros brasileiros, por exemplo) os possíveis principais vilões. Este trabalho visa apresentar e discutir os principais problemas na realização de estimativas prévias de emissão de metano em aterros sanitários utilizando projetos brasileiros de MDL registrados e que estejam atualmente emitindo créditos de carbono como base para analisar a qualidade das estimativas feitas atualmente. Além disto, busca-se também entrevistar profissionais da área para tentar obter diferentes pontos de vista sobre esta questão. Fica claro que os valores estimados, de um modo geral, são entre 40 e 50% superiores aos observados. Metade dos especialistas aponta problemas operacionais diversos como os principais contribuintes desta diferença, mas problemas na modelagem parecem influenciar decisivamente na realização das estimativas. A utilização de valores de entrada no modelo precisa ser criteriosamente analisada e devem ser utilizados números obtidos através de pesquisas que representem a realidade do aterro em questão.

Estimating reflectivity values from wind turbines for analyzing the potential impact on weather radar services

Locate full-text(opens in a new window)|View at Publisher| Export | Download | More... Atmospheric Measurement Techniques Volume 8, Issue 5, 27 May 2015, Pages 2183-2193 Estimating reflectivity values from wind turbines for analyzing the potential impact on weather radar services (Article) Angulo, I.a, Grande, O.a, Jenn, D.b, Guerra, D.a, De La Vega, D.a a University of the Basque Country (UPV/EHU), Bilbao, Spain b Naval Postgraduate School, Monterey, United States View references (28) Abstract The World Meteorological Organization (WMO) has repeatedly expressed concern over the increasing number of impact cases of wind turbine farms on weather radars. Current signal processing techniques to mitigate wind turbine clutter (WTC) are scarce, so the most practical approach to this issue is the assessment of the potential interference from a wind farm before it is installed. To do so, and in order to obtain a WTC reflectivity model, it is crucial to estimate the radar cross section (RCS) of the wind turbines to be built, which represents the power percentage of the radar signal that is backscattered to the radar receiver.

For the proposed model, a representative scenario has been chosen in which both the weather radar and the wind farm are placed on clear areas; i.e., wind turbines are supposed to be illuminated only by the lowest elevation angles of the radar beam.

This paper first characterizes the RCS of wind turbines in the weather radar frequency bands by means of computer simulations based on the physical optics theory and then proposes a simplified model to estimate wind turbine RCS values. This model is of great help in the evaluation of the potential impact of a certain wind farm on the weather radar operation.

Estimating relative abundance from catch and effort data, using neural networks

We develop and test a method to estimate relative abundance from catch and effort data using neural networks. Most stock assessment models use time series of relative abundance as their major source of information on abundance levels. These time series of relative abundance are frequently derived from catch-per-unit-of-effort (CPUE) data, using general linearized models (GLMs). GLMs are used to attempt to remove variation in CPUE that is not related to the abundance of the population. However, GLMs are restricted in the types of relationships between the CPUE and the explanatory variables. An alternative approach is to use structural models based on scientific understanding to develop complex non-linear relationships between CPUE and the explanatory variables. Unfortunately, the scientific understanding required to develop these models may not be available. In contrast to structural models, neural networks uses the data to estimate the structure of the non-linear relationship between CPUE and the explanatory variables. Therefore neural networks may provide a better alternative when the structure of the relationship is uncertain. We use simulated data based on a habitat based-method to test the neural network approach and to compare it to the GLM approach. Cross validation and simulation tests show that the neural network performed better than nominal effort and the GLM approach. However, the improvement over GLMs is not substantial. We applied the neural network model to CPUE data for bigeye tuna (Thunnus obesus) in the Pacific Ocean.

An Evaluation of the area stratification used for sampling tunas in the eastern Pacific Ocean and implications for estimating total annual catches

The Inter-American Tropical Tuna Commission (IATTC) staff has been sampling the size distributions of tunas in the eastern Pacific Ocean (EPO) since 1954, and the species composition of the catches since 2000. The IATTC staff use the data from the species composition samples, in conjunction with observer and/or logbook data, and unloading data from the canneries to estimate the total annual catches of yellowfin (Thunnus albacares), skipjack (Katsuwonus pelamis), and bigeye (Thunnus obesus) tunas. These sample data are collected based on a stratified sampling design. I propose an update of the stratification of the EPO into more homogenous areas in order to reduce the variance in the estimates of the total annual catches and incorporate the geographical shifts resulting from the expansion of the floating-object fishery during the 1990s. The sampling model used by the IATTC is a stratified two-stage (cluster) random sampling design with first stage units varying (unequal) in size. The strata are month, area, and set type. Wells, the first cluster stage, are selected to be sampled only if all of the fish were caught in the same month, same area, and same set type. Fish, the second cluster stage, are sampled for lengths, and independently, for species composition of the catch. The EPO is divided into 13 sampling areas, which were defined in 1968, based on the catch distributions of yellowfin and skipjack tunas. This area stratification does not reflect the multi-species, multi-set-type fishery of today. In order to define more homogenous areas, I used agglomerative cluster analysis to look for groupings of the size data and the catch and effort data for 2000–2006. I plotted the results from both datasets against the IATTC Sampling Areas, and then created new areas. I also used the results of the cluster analysis to update the substitution scheme for strata with catch, but no sample. I then calculated the total annual catch (and variance) by species by stratifying the data into new Proposed Sampling Areas and compared the results to those reported by the IATTC. Results showed that re-stratifying the areas produced smaller variances of the catch estimates for some species in some years, but the results were not significant.

Variations saisonnières des facteurs hydrologiques et du biovolume microalgal benthique au sein d'un habitat artificiel (acadja-enclos) dans la lagune Ebrié: site d'Adjapoté

This paper presents the results of a comparative study of chemical and physical characteristics (temperature, oxygen, salinity, turbidity, pH, nutrient: phosphates, nitrates, nitrites, ammoniac) within the extensive aquaculture structure (acadja-enclosure or artificial habitats for fishes) and in the surrounding water. Biological characteristics of the periphyton growing on bamboos from acadja-enclosure were also described.