Unimolecular reaction chemistry of a charge-tagged beta-hydroxyperoxyl radical

β-Hydroxyperoxyl radicals are formed during atmospheric oxidation of unsaturated volatile organic compounds such as isoprene. They are intermediates in the combustion of alcohols. In these environments the unimolecular isomerization and decomposition of β-hydroxyperoxyl radicals may be of importance, either through chemical or thermal activation. We have used ion-trap mass spectrometry to generate the distonic charge-tagged β-hydroxyalkyl radical anion, ˙CH2C(OH)(CH3)CH2C(O)O−, and investigated its subsequent reaction with O2 in the gas phase under conditions that are devoid of complicating radical–radical reactions. Quantum chemical calculations and master equation/RRKM theory modeling are used to rationalize the results and discern a reaction mechanism. Reaction is found to proceed via initial hydrogen abstraction from the γ-methylene group and from the β-hydroxyl group, with both reaction channels eventually forming isobaric product ions due to loss of either ˙OH + HCHO or ˙OH + CO2. Isotope labeling studies confirm that a 1,5-hydrogen shift from the β-hydroxyl functionality results in a hydroperoxyalkoxyl radical intermediate that can undergo further unimolecular dissociations. Furthermore, this study confirms that the facile decomposition of β-hydroxyperoxyl radicals can yield ˙OH in the gas phase.

Japanese Video Game Localization : A Case Study of Sony's Sairen Series

This thesis is a comparative case study in Japanese video game localization for the video games Sairen, Sairen 2 and Sairen Nyûtoransurêshon, and English-language localized versions of the same games as published in Scandinavia and Australia/New Zealand. All games are developed by Sony Computer Entertainment Inc. and published exclusively for Playstation2 and Playstation3 consoles. The fictional world of the Sairen games draws much influence from Japanese history, as well as from popular and contemporary culture, and in doing so caters mainly to a Japanese audience. For localization, i.e. the adaptation of a product to make it accessible to users outside the original market it was intended for in the first place, this is a challenging issue. Video games are media of entertainment, and therefore localization practice must preserve the games’ effects on the players’ emotions. Further, video games are digital products that are comprised of a multitude of distinct elements, some of which are part of the game world, while others regulate the connection between the player as part of the real world and the game as digital medium. As a result, video game localization is also a practice that has to cope with the technical restrictions that are inherent to the medium. The main theory used throughout the thesis is Anthony Pym’s framework for localization studies that considers the user of the localized product as a defining part of the localization process. This concept presupposes that localization is an adaptation that is performed to make a product better suited for use during a specific reception situation. Pym also addresses the factor that certain products may resist distribution into certain reception situations because of their content, and that certain aspects of localization aim to reduce this resistance through significant alterations of the original product. While Pym developed his ideas with mainly regular software in mind, they can also be adapted well to study video games from a localization angle. Since modern video games are highly complex entities that often switch between interactive and non-interactive modes, Pym’s ideas are adapted throughout the thesis to suit the particular elements being studied. Instances analyzed in this thesis include menu screens, video clips, in-game action and websites. The main research questions focus on how the games’ rules influence localization, and how the games’ fictional domain influences localization. Because there are so many peculiarities inherent to the medium of the video game, other theories are introduced as well to complement the research at hand. These include Lawrence Venuti’s discussions of foreiginizing and domesticating translation methods for literary translation, and Jesper Juul’s definition of games. Additionally, knowledge gathered from interviews with video game localization professionals in Japan during September and October 2009 is also utilized for this study. Apart from answering the aforementioned research questions, one of this thesis’ aims is to enrich the still rather small field of game localization studies, and the study of Japanese video games in particular, one of Japan’s most successful cultural exports.

Click Chemistry Inspired Synthesis of Novel Ferrocenyl-Substituted Amino Acids or Peptides

This work reports on the synthesis of a wide range of ferrocenyl-substituted amino acids and peptides in excellent yield. Conjugation is established via copper-catalyzed 1,3-dipolar cycloaddition. Two complementary strategies were employed for conjugation, one involving cycloaddition of amino acid derived azides with ethynyl ferrocene 1 and the other involves cycloaddition between amino acid derived alkynes with ferrocene-derived azides 2 and 3. Labeling of amino acids at multiple sites with ferrocene is discussed. A new route to 1,1'-unsymmetrically substituted ferrocene conjugates is reported. A novel ferrocenophane 19 is accessed via bimolecular condensation of amino acid derived bis-alkyne 9b with the azide 2. The electrochemical behavior of some selected ferrocene conjugates has been studied by cyclic voltammetry.

Word Sense Discovery and Disambiguation

The work is based on the assumption that words with similar syntactic usage have similar meaning, which was proposed by Zellig S. Harris (1954,1968). We study his assumption from two aspects: Firstly, different meanings (word senses) of a word should manifest themselves in different usages (contexts), and secondly, similar usages (contexts) should lead to similar meanings (word senses). If we start with the different meanings of a word, we should be able to find distinct contexts for the meanings in text corpora. We separate the meanings by grouping and labeling contexts in an unsupervised or weakly supervised manner (Publication 1, 2 and 3). We are confronted with the question of how best to represent contexts in order to induce effective classifiers of contexts, because differences in context are the only means we have to separate word senses. If we start with words in similar contexts, we should be able to discover similarities in meaning. We can do this monolingually or multilingually. In the monolingual material, we find synonyms and other related words in an unsupervised way (Publication 4). In the multilingual material, we ?nd translations by supervised learning of transliterations (Publication 5). In both the monolingual and multilingual case, we first discover words with similar contexts, i.e., synonym or translation lists. In the monolingual case we also aim at finding structure in the lists by discovering groups of similar words, e.g., synonym sets. In this introduction to the publications of the thesis, we consider the larger background issues of how meaning arises, how it is quantized into word senses, and how it is modeled. We also consider how to define, collect and represent contexts. We discuss how to evaluate the trained context classi?ers and discovered word sense classifications, and ?nally we present the word sense discovery and disambiguation methods of the publications. This work supports Harris' hypothesis by implementing three new methods modeled on his hypothesis. The methods have practical consequences for creating thesauruses and translation dictionaries, e.g., for information retrieval and machine translation purposes. Keywords: Word senses, Context, Evaluation, Word sense disambiguation, Word sense discovery.

Aspects and Applications of Pulse Sequence Design for Solution-State Nuclear Magnetic Resonance Spectroscopy

NMR spectroscopy enables the study of biomolecules from peptides and carbohydrates to proteins at atomic resolution. The technique uniquely allows for structure determination of molecules in solution-state. It also gives insights into dynamics and intermolecular interactions important for determining biological function. Detailed molecular information is entangled in the nuclear spin states. The information can be extracted by pulse sequences designed to measure the desired molecular parameters. Advancement of pulse sequence methodology therefore plays a key role in the development of biomolecular NMR spectroscopy. A range of novel pulse sequences for solution-state NMR spectroscopy are presented in this thesis. The pulse sequences are described in relation to the molecular information they provide. The pulse sequence experiments represent several advances in NMR spectroscopy with particular emphasis on applications for proteins. Some of the novel methods are focusing on methyl-containing amino acids which are pivotal for structure determination. Methyl-specific assignment schemes are introduced for increasing the size range of 13C,15N labeled proteins amenable to structure determination without resolving to more elaborate labeling schemes. Furthermore, cost-effective means are presented for monitoring amide and methyl correlations simultaneously. Residual dipolar couplings can be applied for structure refinement as well as for studying dynamics. Accurate methods for measuring residual dipolar couplings in small proteins are devised along with special techniques applicable when proteins require high pH or high temperature solvent conditions. Finally, a new technique is demonstrated to diminish strong-coupling induced artifacts in HMBC, a routine experiment for establishing long-range correlations in unlabeled molecules. The presented experiments facilitate structural studies of biomolecules by NMR spectroscopy.

Role of heme in the synthesis of cytochrome c oxidase in Neurospora crassa

The role of heme in the synthesis of cytochrome c oxidase has been investigated in the mold Neurospora crassa. Iron-deficient cultures of the mold have low levels of cytochrome oxidase and delta-aminolevulinate dehydratase, the latter being the rate-limiting enzyme of the heme-biosynthetic pathway in this organism. Addition of iron to the iron-deficient cultures results in an immediate increase in the levels of delta-aminolevulinate dehydratase followed by an increase in the rate of heme synthesis and cytochrome oxidase levels. The rate of precursor labeling of the mitochondrial subunits of cytochrome oxidase is decreased preferentially under conditions of iron deficiency and addition of iron corrects this picture. Exogenous hemin addition which prevents iron-mediated induction of delta-aminolevulinate dehydratase also inhibits the increase in the activity of cytochrome oxidase and the enhanced precursor labeling of the mitochondrial subunits of cytochrome oxidase. Protein synthesis on mitoribosomes measured in vivo and in vitro is decreased under conditions of heme deficiency. Hemin addition in vitro to mitochondrial lysates prepared from heme-deficient mycelia restores a near normal rate of protein synthesis. It is concluded that heme is required for the optimal rate of translation on mitoribosomes.

Graphical simulation of Petri Nets

An interactive graphics package for modeling with Petri Nets has been implemented. It uses the VT-11 graphics terminal supported on the PDP-11/35 computer to draw, execute, analyze, edit and redraw a Petri Net. Each of the above mentioned tasks can be performed by selecting appropriate items from a menu displayed on the screen. Petri Nets with a reasonably large number of nodes can be created and analyzed using this package. The number of nodes supported may be increased by making simple changes in the program. Being interactive, the program seeks information from the user after displaying appropriate messages on the terminal. After completing the Petri Net, it may be executed step by step and the changes in the number of tokens may be observed on the screen, at each place. Some properties of Petri Nets like safety, boundedness, conservation and redundancy can be checked using this package. This package can be used very effectively for modeling asynchronous (concurrent) systems with Petri Nets and simulating the model by “graphical execution.”

Mechanism of RNA Translocation by a Viral Packaging Motor

Molecular motors are proteins that convert chemical energy into mechanical work. The viral packaging ATPase P4 is a hexameric molecular motor that translocates RNA into preformed viral capsids. P4 belongs to the ubiquitous class of hexameric helicases. Although its structure is known, the mechanism of RNA translocation remains elusive. Here we present a detailed kinetic study of nucleotide binding, hydrolysis, and product release by P4. We propose a stochastic-sequential cooperative model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive. Further, we use hydrogen/deuterium exchange detected by high resolution mass spectrometry to visualize P4 conformational dynamics during the catalytic cycle. Concerted changes of exchange kinetics reveal a cooperative unit that dynamically links ATP binding sites and the central RNA binding channel. The cooperative unit is compatible with the structure-based model in which translocation is effected by conformational changes of a limited protein region. Deuterium labeling also discloses the transition state associated with RNA loading which proceeds via opening of the hexameric ring. Hydrogen/deuterium exchange is further used to delineate the interactions of the P4 hexamer with the viral procapsid. P4 associates with the procapsid via its C-terminal face. The interactions stabilize subunit interfaces within the hexamer. The conformation of the virus-bound hexamer is more stable than the hexamer in solution, which is prone to spontaneous ring openings. We propose that the stabilization within the viral capsid increases the packaging processivity and confers selectivity during RNA loading. Finally, we use single molecule techniques to characterize P4 translocation along RNA. While the P4 hexamer encloses RNA topologically within the central channel, it diffuses randomly along the RNA. In the presence of ATP, unidirectional net movement is discernible in addition to the stochastic motion. The diffusion is hindered by activation energy barriers that depend on the nucleotide binding state. The results suggest that P4 employs an electrostatic clutch instead of cycling through stable, discrete, RNA binding states during translocation. Conformational changes coupled to ATP hydrolysis modify the electrostatic potential inside the central channel, which in turn biases RNA motion in one direction. Implications of the P4 model for other hexameric molecular motors are discussed.

Project Energise: Using participatory approaches and real time computer prompts to reduce occupational sitting and increase work time physical activity in office workers

Objectives This efficacy study assessed the added impact real time computer prompts had on a participatory approach to reduce occupational sedentary exposure and increase physical activity. Design Quasi-experimental. Methods 57 Australian office workers (mean [SD]; age = 47 [11] years; BMI = 28 [5] kg/m2; 46 men) generated a menu of 20 occupational ‘sit less and move more’ strategies through participatory workshops, and were then tasked with implementing strategies for five months (July–November 2014). During implementation, a sub-sample of workers (n = 24) used a chair sensor/software package (Sitting Pad) that gave real time prompts to interrupt desk sitting. Baseline and intervention sedentary behaviour and physical activity (GENEActiv accelerometer; mean work time percentages), and minutes spent sitting at desks (Sitting Pad; mean total time and longest bout) were compared between non-prompt and prompt workers using a two-way ANOVA. Results Workers spent close to three quarters of their work time sedentary, mostly sitting at desks (mean [SD]; total desk sitting time = 371 [71] min/day; longest bout spent desk sitting = 104 [43] min/day). Intervention effects were four times greater in workers who used real time computer prompts (8% decrease in work time sedentary behaviour and increase in light intensity physical activity; p < 0.01). Respective mean differences between baseline and intervention total time spent sitting at desks, and the longest bout spent desk sitting, were 23 and 32 min/day lower in prompt than in non-prompt workers (p < 0.01). Conclusions In this sample of office workers, real time computer prompts facilitated the impact of a participatory approach on reductions in occupational sedentary exposure, and increases in physical activity.

Identification and Characterization of a Novel Deoxyhypusine Synthase in Leishmania donovani

Deoxyhypusine synthase, an NAD(+)-dependent enzyme, catalyzes the first step in the post-translational synthesis of an unusual amino acid, hypusine (N-epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic initiation factor 5A precursor protein. Two putative deoxyhypusine synthase (DHS) sequences have been identified in the Leishmania donovani genome, which are present on chromosomes 20: DHSL20 (DHS-like gene from chromosome 20) and DHS34 (DHS from chromosome 34). Although both sequences exhibit an overall conservation of key residues, DHSL20 protein lacks a critical lysine residue, and the recombinant protein showed no DHS activity in vitro. However, DHS34 contains the critical lysine residue, and the recombinant DHS34 effectively catalyzed deoxyhypusine synthesis. Furthermore, in vivo labeling confirmed that hypusination of eukaryotic initiation factor 5A occurs in intact Leishmania parasites. Interestingly, the DHS34 is much longer, with 601 amino acids, compared with the human DHS enzyme (369 amino acids) and contains several unique insertions. To study the physiological role of DHS34 in Leishmania, gene deletion mutations were attempted via targeted gene replacement. However, chromosomal null mutants of DHS34 could only be obtained in the presence of a DHS34-containing episome. The present data provide evidence that DHS34 is essential for L. donovani and that structural differences in the human and leishmanial DHS enzyme may be exploited for designing selective inhibitors against the parasite.

X-ray and neutron scattering studies on some nanoscale structures in molecular biology

Scattering of X-rays and neutrons has been applied to the study of nanostructures with interesting biological functions. The systems studied were the protein calmodulin and its complexes, bacterial virus bacteriophage phi6, and the photosynthetic antenna complex from green sulfur bacteria, chlorosome. Information gathered using various structure determination methods has been combined to the low resolution information obtained from solution scattering. Conformational changes in calmodulin-ligand complex were studied by combining the directional information obtained from residual dipole couplings in nuclear magnetic resonance to the size information obtained from small-angle X-ray scattering from solution. The locations of non-structural protein components in a model of bacteriophage phi6, based mainly on electron microscopy, were determined by neutron scattering, deuterium labeling and contrast variation. New data are presented on the structure of the photosynthetic antenna complex of green sulfur bacteria and filamentous anoxygenic phototrophs, also known as the chlorosome. The X-ray scattering and electron cryomicroscopy results from this system are interpreted in the context of a new structural model detailed in the third paper of this dissertation. The model is found to be consistent with the results obtained from various chlorosome containing bacteria. The effect of carotenoid synthesis on the chlorosome structure and self-assembly are studied by carotenoid extraction, biosynthesis inhibition and genetic manipulation of the enzymes involved in carotenoid biosynthesis. Carotenoid composition and content are found to have a marked effect on the structural parameters and morphology of chlorosomes.

Assessment of crop insect damage using unmanned aerial systems: A machine learning approach

Agricultural pests are responsible for millions of dollars in crop losses and management costs every year. In order to implement optimal site-specific treatments and reduce control costs, new methods to accurately monitor and assess pest damage need to be investigated. In this paper we explore the combination of unmanned aerial vehicles (UAV), remote sensing and machine learning techniques as a promising technology to address this challenge. The deployment of UAVs as a sensor platform is a rapidly growing field of study for biosecurity and precision agriculture applications. In this experiment, a data collection campaign is performed over a sorghum crop severely damaged by white grubs (Coleoptera: Scarabaeidae). The larvae of these scarab beetles feed on the roots of plants, which in turn impairs root exploration of the soil profile. In the field, crop health status could be classified according to three levels: bare soil where plants were decimated, transition zones of reduced plant density and healthy canopy areas. In this study, we describe the UAV platform deployed to collect high-resolution RGB imagery as well as the image processing pipeline implemented to create an orthoimage. An unsupervised machine learning approach is formulated in order to create a meaningful partition of the image into each of the crop levels. The aim of the approach is to simplify the image analysis step by minimizing user input requirements and avoiding the manual data labeling necessary in supervised learning approaches. The implemented algorithm is based on the K-means clustering algorithm. In order to control high-frequency components present in the feature space, a neighbourhood-oriented parameter is introduced by applying Gaussian convolution kernels prior to K-means. The outcome of this approach is a soft K-means algorithm similar to the EM algorithm for Gaussian mixture models. The results show the algorithm delivers decision boundaries that consistently classify the field into three clusters, one for each crop health level. The methodology presented in this paper represents a venue for further research towards automated crop damage assessments and biosecurity surveillance.

Performance of RT-PCR-ELISA for the detection of peste des petits ruminants virus

A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR-ELISA) was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the amplification/probing of the 'N' gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer binding sites of the 'N' gene sequences available and the ability to amplify PPR viral genome from different sources of samples. RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR followed by agarose gel based detection of PCR product. Based on the Mean (mean +/- 3S.D.) optical density (OD) values of 47 RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54% positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6 days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node, lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of RT-PCR products are discussed. (c) 2006 Elsevier B.V. All rights reserved.