Novel surface-tethered estrogen polymeric platforms in cardiovascular regenerative medicine

L’estradiol (E2) est une hormone femelle qui joue un rôle essentiel, à la fois dans la régulation et dans la détermination de certaines conditions physiologiques in vivo, telle que la différenciation et la prolifération cellulaire. Lorsque l’E2 est donné en supplément, par exemple dans le cas de thérapie hormonale, deux effets sont observés, un effet génomique et un effet non-génomique, de par son interaction avec les récepteurs à œstrogène du noyau ou de la membrane cellulaire, respectivement. L’effet non-génomique est plus difficile à étudier biologiquement parce que l’effet se produit sur une échelle de temps extrêmement courte et à cause de la nature hydrophobe de l’E2 qui réduit sa biodisponibilité et donc son accessibilité aux cellules cibles. C’est pourquoi il est nécessaire de développer des systèmes d’administration de l’E2 qui permettent de n’étudier que l’effet non-génomique de l’œstrogène. Une des stratégies employée consiste à greffer l’E2 à des macromolécules hydrophiles, comme de l’albumine de sérum bovin (BSA) ou des dendrimères de type poly(amido)amine, permettant de maintenir l’interaction de l’E2 avec les récepteurs d’œstrogène de la membrane cellulaire et d’éviter la pénétration de l’E2 dans le noyau des cellules. Toutefois, ces systèmes macromolécules-E2 sont critiquables car ils sont peu stables et l’E2 peut se retrouver sous forme libre, ce qui affecte sa localisation cellulaire. L’objectif de cette thèse est donc de développer de nouvelles plateformes fonctionnalisées avec de l’E2 en utilisant les approches de synthèses ascendantes et descendantes. Le but de ces plateformes est de permettre d’étudier le mécanisme de l’effet non-génomique de l’E2, ainsi que d’explorer des applications potentielles dans le domaine biomédical. L’approche ascendante est basée sur un ligand d’E2 activé, l’acide 17,α-éthinylestradiol-benzoïque, attaché de façon covalente à un polymère de chitosan avec des substitutions de phosphorylcholine (CH-PC-E2). L’estradiol est sous forme de pro-drogue attachée au polymère qui s’auto-assembler pour former un film. L’effet biologique de la composition chimique du film de chitosan-phosphorylcholine a été étudié sur des cellules endothéliales. Les films de compositions chimiques différentes ont préalablement été caractérisés de façon physicochimique. La topographie de la surface, la charge de surface, ainsi que la rhéologie des différents films contenant 15, 25, ou 40% molaires de phosphorylcholine, ont été étudiés par microscopie à force atomique (AFM), potentiel zêta, résonance plasmonique de surface et par microbalance à cristal de quartz avec dissipation (QCM-D). Les résultats de QCM-D ont montré que plus la part molaire en phosphorylcholine est grande moins il y a de fibrinogène qui s’adsorbe sur le film de CH-PC. Des cellules humaines de veine ombilicale (HUVECs) cultivées sur des films de CH-PC25 et de CH-PC40 forment des amas cellulaire appelés sphéroïdes au bout de 4 jours, alors que ce n’est pas le cas lorsque ces cellules sont cultivées sur des films de CH-PC15. L’attachement de l’estradiol au polymère a été caractérisé par plusieurs techniques, telles que la résonance magnétique nucléaire de proton (1H NMR), la spectroscopie infrarouge avec transformée de Fourier à réfraction totale atténuée (FTIR-ATR) et la spectroscopie UV-visible. La nature hydrogel des films (sa capacité à retenir l’eau) ainsi que l’interaction des films avec des récepteurs à E2, ont été étudiés par la QCM-D. Des études d’imagerie cellulaires utilisant du diacétate de diaminofluoresceine-FM ont révélé que les films hydrogels de CH-PC-E2 stimulent la production d’oxyde nitrique par les cellules endothéliales, qui joue un rôle protecteur pour le système cardiovasculaire. L’ensemble de ces études met en valeur les rôles différents et les applications potentielles qu’ont les films de type CH-PC-E2 et CH-PC dans le cadre de la médecine cardiovasculaire régénérative. L’approche descendante est basée sur l’attachement de façon covalente d’E2 sur des ilots d’or de 2 μm disposés en rangées et espacés par 12 μm sur un substrat en verre. Les ilots ont été préparés par photolithographie. La surface du verre a quant à elle été modifiée à l’aide d’un tripeptide cyclique, le cRGD, favorisant l’adhésion cellulaire. L’attachement d’E2 sur les surfaces d’or a été suivi et confirmé par les techniques de SPR et de QCM-D. Des études d’ELISA ont montré une augmentation significative du niveau de phosphorylation de la kinase ERK (marqueur important de l’effet non-génomique) après 1 heure d’exposition des cellules endothéliales aux motifs alternant l’E2 et le cRGD. Par contre lorsque des cellules cancéreuses sont déposées sur les surfaces présentant des motifs d’E2, ces cellules ne croissent pas, ce qui suggère que l’E2 n’exerce pas d’effet génomique. Les résultats de l’approche descendante montrent le potentiel des surfaces présentant des motifs d’E2 pour l’étude des effets non-génomiques de l’E2 dans un modèle in vitro.

Identification and bioactivity evaluation of two novel temporins from the skin secretion of the European edible frog, Pelophylax kl. Esculentus

The amphibian temporins, amongst the smallest antimicrobial peptides (AMPs), are α-helical, amphipathic, hydrophobic and cationic and are active mainly against Gram-positive bacteria but inactive or weakly active against Gram-negative bacteria. Here, we report two novel members of the temporin family, named temporin-1Ee (FLPVIAGVLSKLFamide) and temporin-1Re (FLPGLLAGLLamide), whose biosynthetic precursor structures were deduced from clones obtained from skin secretion-derived cDNA libraries of the European edible frog, Pelophylax kl. esculentus, by ‘shotgun’ cloning. Deduction of the molecular masses of each mature processed peptide from respective cloned cDNAs was used to locate respective molecules in reverse-phase HPLC fractions of secretion. Temporin-1Ee (MIC = 10 μM) and temporin-1Re (MIC = 60 μM) were both found to be active against Gram-positive Staphylococcus aureus, but retaining a weak haemolytic activity. To our knowledge, Single-site substitutions can dramatically change the spectrum of activity of a given temporin. Compared with temporine-1Ec, just one chemically-conservative substitution (Val8 instead of Leu8), temporin-1Ee bearing a net charge of +2 displays broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC = 40 μM). These factors bode well for translating temporins to be potential drug candidates for the design of new and valuable anti-infective agents.

Evaluation of fly ash in water reduced paving mixtures, 1984

Fly ash was used to replace 15% of the cement in C3WR and C6WR concrete paving mixes containing ASTM C494 Type A water reducin9 admixtures. Two Class C ashes and one Class F ash from Iowa approved sources were examined in each mix. When Class C ashes were used they were substituted on the basis of 1 pound of ash added for each pound of cement deleted. When Class F was used it was substituted on the basis of 1.25 pounds of ash added for each pound of cement deleted. Compressive strengths of the water reduced mixes, with and without fly ash, were determined at 7, 28, and 56 days of age. In every case except one the mixes containing the fly ash exhibited higher strengths than the same concrete mix without the fly ash. An excellent correlation existed between the C3WR and C6WR mixes both with and without fly ash substitutions. The freeze-thaw durability of the concrete studied was not affected by presence or absence of fly ash. The data gathered suggests that the present Class C water reduced concrete paving mixes can be modified to allow the substitution of 15% of the cement with an approved fly ash.

Liver glucose-6-phosphatase proteins in suckling and weaned grey seal pups: structural similarities to other mammals and relationship to nutrition, insulin signalling and metabolite levels

Phocid seals have been proposed as models for diabetes because they exhibit limited insulin response to glucose, high blood glucose and increasing insulin resistance when fasting. Liver glucose-6-phosphatase (G6Pase) catalyses the final step in glucose production and is central to glucose regulation in other animals. G6Pase comprises a translocase (SLC37A4) and a catalytic subunit (G6PC). G6PC and SLC37A4 expression and activity are normally regulated by nutritional state and glucostatic hormones, particularly insulin, and are elevated in diabetes. We tested the hypotheses that (1) grey seal G6PC and SLC37A4 cDNA and predicted protein sequences differ from other species’ at functional sites, (2) relative G6Pase protein abundances are lower during feeding than fasting and (3) relative G6Pase protein abundances are related to insulin, insulin receptor phosphorylation and key metabolite levels. We show that G6PC and partial SLC37A4 cDNA sequences encode proteins sharing 82–95 % identity with other mammals. Seal G6PC contained no differences in sites responsible for activity, stability or subcellular location. Several substitutions in seal SLC37A4 were predicted to be tolerated with low probability, which could affect glucose production. Suckling pups had higher relative abundance of both subunits than healthy, postweaned fasting pups. Furthermore, relative G6PC abundance was negatively related to glucose levels. These findings contrast markedly with the response of relative hepatic G6Pase abundance to feeding, fasting, insulin, insulin sensitivity and key metabolites in other animals, and highlight the need to understand the regulation of enzymes involved in glucose control in phocids if these animals are to be informative models of diabetes.

Molecular cloning and expression of leptin in gray and harbor seal blubber, bone marrow, and lung and its potential role in marine mammal respiratory physiology

Leptin is a multifunctional hormone, produced predominantly in adipocytes. It regulates energy balance through its impact on appetite and fat metabolism, and its concentration indicates the size of body fat reserves. Leptin also plays a vital role in stretch-induced surfactant production during alveolar development in the fetus. The structure, expression pattern, and role of leptin have not previously been explored in marine mammals. Phocid seals undergo cyclical changes in body composition as a result of prolonged fasting and intensive foraging bouts and experience rapid, dramatic, and repeated changes in lung volume during diving. Here, we report the tissue-specific expression pattern of leptin in these animals. This is the first demonstration of leptin expression in the lung tissue of a mature mammal, in addition to its expression in the blubber and bone marrow, in common with other animals. We propose a role for leptin in seal pulmonary surfactant production, in addition to its likely role in long-term energy balance. We identify substitutions in the phocine leptin sequence in regions normally highly conserved between widely distinct vertebrate groups, and, using a purified seal leptin antiserum, we confirm the presence of the leptin protein in gray seal lung and serum fractions. Finally, we report the substantial inadequacies of using heterologous antibodies to measure leptin in unextracted gray seal serum.

Development of a simple and fast “DNA extraction kit” for sea food identification and marine species

Seafood products fraud, the misrepresentation of them, have been discovered all around the world in different forms as false labeling, species substitution, short-weighting or over glazing in order to hide the correct identity, origin or weight of the seafood products. Due to the value of seafood products such as canned tuna, swordfish or grouper, these species are the subject of the commercial fraud is mainly there placement of valuable species with other little or no value species. A similar situation occurs with the shelled shrimp or shellfish that are reduced into pieces for the commercialization. Food fraud by species substitution is an emerging risk given the increasingly global food supply chain and the potential food safety issues. Economic food fraud is committed when food is deliberately placed on the market, for financial gain deceiving consumers (Woolfe, M. & Primrose, S. 2004). As a result of the increased demand and the globalization of the seafood supply, more fish species are encountered in the market. In this scenary, it becomes essential to unequivocally identify the species. The traditional taxonomy, based primarily on identification keys of species, has shown a number of limitations in the use of the distinctive features in many animal taxa, amplified when fish, crustacean or shellfish are commercially transformed. Many fish species show a similar texture, thus the certification of fish products is particularly important when fishes have undergone procedures which affect the overall anatomical structure, such as heading, slicing or filleting (Marko et al., 2004). The absence of morphological traits, a main characteristic usually used to identify animal species, represents a challenge and molecular identification methods are required. Among them, DNA-based methods are more frequently employed for food authentication (Lockley & Bardsley, 2000). In addition to food authentication and traceability, studies of taxonomy, population and conservation genetics as well as analysis of dietary habits and prey selection, also rely on genetic analyses including the DNA barcoding technology (Arroyave & Stiassny, 2014; Galimberti et al., 2013; Mafra, Ferreira, & Oliveira, 2008; Nicolé et al., 2012; Rasmussen & Morrissey, 2008), consisting in PCR amplification and sequencing of a COI mitochondrial gene specific region. The system proposed by P. Hebert et al. (2003) locates inside the mitochondrial COI gene (cytochrome oxidase subunit I) the bioidentification system useful in taxonomic identification of species (Lo Brutto et al., 2007). The COI region, used for genetic identification - DNA barcode - is short enough to allow, with the current technology, to decode sequence (the pairs of nucleotide bases) in a single step. Despite, this region only represents a tiny fraction of the mitochondrial DNA content in each cell, the COI region has sufficient variability to distinguish the majority of species among them (Biondo et al. 2016). This technique has been already employed to address the demand of assessing the actual identity and/or provenance of marketed products, as well as to unmask mislabelling and fraudulent substitutions, difficult to detect especially in manufactured seafood (Barbuto et al., 2010; Galimberti et al., 2013; Filonzi, Chiesa, Vaghi, & Nonnis Marzano, 2010). Nowadays,the research concerns the use of genetic markers to identify not only the species and/or varieties of fish, but also to identify molecular characters able to trace the origin and to provide an effective control tool forproducers and consumers as a supply chain in agreementwith local regulations.

Top-K Query Processing in Edge-Labeled Graph Data

Edge-labeled graphs have proliferated rapidly over the last decade due to the increased popularity of social networks and the Semantic Web. In social networks, relationships between people are represented by edges and each edge is labeled with a semantic annotation. Hence, a huge single graph can express many different relationships between entities. The Semantic Web represents each single fragment of knowledge as a triple (subject, predicate, object), which is conceptually identical to an edge from subject to object labeled with predicates. A set of triples constitutes an edge-labeled graph on which knowledge inference is performed. Subgraph matching has been extensively used as a query language for patterns in the context of edge-labeled graphs. For example, in social networks, users can specify a subgraph matching query to find all people that have certain neighborhood relationships. Heavily used fragments of the SPARQL query language for the Semantic Web and graph queries of other graph DBMS can also be viewed as subgraph matching over large graphs. Though subgraph matching has been extensively studied as a query paradigm in the Semantic Web and in social networks, a user can get a large number of answers in response to a query. These answers can be shown to the user in accordance with an importance ranking. In this thesis proposal, we present four different scoring models along with scalable algorithms to find the top-k answers via a suite of intelligent pruning techniques. The suggested models consist of a practically important subset of the SPARQL query language augmented with some additional useful features. The first model called Substitution Importance Query (SIQ) identifies the top-k answers whose scores are calculated from matched vertices' properties in each answer in accordance with a user-specified notion of importance. The second model called Vertex Importance Query (VIQ) identifies important vertices in accordance with a user-defined scoring method that builds on top of various subgraphs articulated by the user. Approximate Importance Query (AIQ), our third model, allows partial and inexact matchings and returns top-k of them with a user-specified approximation terms and scoring functions. In the fourth model called Probabilistic Importance Query (PIQ), a query consists of several sub-blocks: one mandatory block that must be mapped and other blocks that can be opportunistically mapped. The probability is calculated from various aspects of answers such as the number of mapped blocks, vertices' properties in each block and so on and the most top-k probable answers are returned. An important distinguishing feature of our work is that we allow the user a huge amount of freedom in specifying: (i) what pattern and approximation he considers important, (ii) how to score answers - irrespective of whether they are vertices or substitution, and (iii) how to combine and aggregate scores generated by multiple patterns and/or multiple substitutions. Because so much power is given to the user, indexing is more challenging than in situations where additional restrictions are imposed on the queries the user can ask. The proposed algorithms for the first model can also be used for answering SPARQL queries with ORDER BY and LIMIT, and the method for the second model also works for SPARQL queries with GROUP BY, ORDER BY and LIMIT. We test our algorithms on multiple real-world graph databases, showing that our algorithms are far more efficient than popular triple stores.

Investigations into the mechanism of orotidine 5'-monophosphate decarboxylase: Sources of substrate destabilization and transition state stabilization

Orotidine 5′-monophosphate decarboxylase (OMPDC) achieves a rarely paralleled rate acceleration, yet the catalytic basis prompting this enhancement have yet to be fully elucidated. To accomplish decarboxylation, OMPDC must overcome the high energy barrier due to the localized anionic charge of the intermediate. Mechanistic studies employing enzyme mutagenesis and product or intermediate analogues were used to investigate possible transition state stabilization by a carbene resonance structure. Viability of the carbene structure depends upon a key hydrogen bond between O4 of the substrate and the amide backbone of a conserved serine or threonine. Substitution of the conserved residue with Pro resulted in a kcat/KM of 1 M-1s-1; deletion of the FUMP O4 resulted in a product analogue that does not undergo H6 exchange or inhibit decarboxylation. Hence, indirect evidence reveals the O4-backbone interaction plays an important role for binding and catalysis. OMPDC likely has honed multiple mechanisms to attain its remarkable catalysis. The successful crystallizations of OMPDC a decade ago sparked hypotheses that structure and sequence conserved residues induced productive strain on the substrate-enzyme complex. Here, we demonstrate a new source of stress: a hydrophobic pocket adjacent to the OMP carboxylate that exhibits kinetic parameters characteristic of substrate destabilization. Substitution of these residues with hydrophilic side-chains, by providing hydrogen-bonding partners, decreased kcat by 10 to 10^4–fold. The same substitutions display very little change in the rate of product H6 exchange, supporting that this hydrophobic pocket affects the substrate-enzyme complex before the transition state. We also provide evidence that hydrophilic residues can insert water molecules into the pocket with detrimental effects to catalysis.

"Mutações no gene OCT4 em células eritroleucêmicas humanas: implicações sobre o fenótipo de resistência a múltiplas drogas (MDR)”

O fator de transcrição OCT4 é um importante marcador de células tronco e tem sido relacionado com o conceito de células tronco tumorais (CTTs). Recentemente, ele tem sido também relacionado ao fenótipo de resistência a múltiplas drogas (MDR). O objetivo deste trabalho foi testar se o OCT4 está ligado ao fenótipo MDR em células eritroleucêmicas derivadas da linhagem LMC-K562. Para isso, foi realizada a análise de expressão de genes associados à superfamília de transportadores ABC (ATP Binding Cassette) e, também, de fatores de transcrição relacionados a células tronco. Os primeiros resultados apontaram uma relação direta entre OCT4 e transportadores ABC na linhagem MDR derivada de K562 (Lucena). O sequenciamento de promotores ABC não revelou qualquer mutação que pudesse explicar a expressão diferenciada do OCT4 na linhagem MDR. Posteriormente, o sequenciamento da região contendo o domínio homeobox do gene OCT4 de ambas as linhagens celulares evidenciou, pela primeira vez, que este fator de transcrição é alvo de mutações que podem estar relacionados com o fenótipo MDR. As mutações encontradas implicam substituições de vários aminoácidos em ambas as linhagens celulares. K562 teve sete substituições (três exclusivas), enquanto Lucena teve 13 (nove exclusivas). Além disso, um busca in silico por motivos de fosforilação dentro da sequência de aminoácidos comparada, revelou que o OCT4 humano normal possui sete motivos potenciais de fosforilação. Entretanto, K562 perdeu um motivo e Lucena dois deles. Moléculas com diferentes padrões de fosforilação podem ter sua função modificada. Estes resultados indicam o OCT4 com uma alvo potencial para o tratamento do câncer, especialmente aqueles resistentes à quimioterapia.

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

Trapping phosphorus (III) into polyoxometalates: synthesis, characterisation and electronic behaviour study

The introduction of electronically-active heteroanions into polyoxometalates (POMs) is one of the emerging topics in this field. The novel clusters have shown unprecedented intramolecular electron-transfer features that can be directly mediated by the incorporated heteroanions. In this thesis, we will focus on the study of phosphite (HPO32-) as new non-traditional heteroanions, discover HPO32- templated nanostructures, investigate their electronic behaviours as well as understand the self-assembly process of HPO32--templated species. The thesis starts with incorporating HPO32- into POM cages. The feasibility of this work was illustrated by the successful trapping of HPO32- into a “Trojan Horse” type {W18O56} nanocage. The reactivity of embedded {HPO3} was fully studied, showing the cluster undergoes a structural rearrangement in solution whereby the {HPO3} moieties dimerise to form a weakly interacting (O3PH···HPO3) moiety. In the crystalline state a temperature-dependent intramolecular redox reaction and structural rearrangement occurs. This rearrangement appears to proceed via an intermediate containing two different templates, a pyramidal {HPO3} and a tetrahedral {PO4} moiety. {HPO3} templated POM cages were then vigorously expanded and led to the isolation of five either fully oxidised or mixed-valence clusters trapped with mono-, di-, or tri- {HPO3}. Interestingly, an intriguing 3D honeycomb-like host-guest structure was also synthesised. The porous framework was self-aggregated by a tri-phopshite anion templated {W21} cluster with a {VO4} templated Wells-Dawson type {W18} acting as a guest species within the hexagonal channels. Based on this work, we further extended the templating anions to two different redox-active heteroanions, and discovered a unique mixed-heteroatom templated system built by pairing redox-active {HPIIIO3} with {TeO3}, {SeO3} or {AsO3}. Two molecular systems were developed, ie. “Trojan Horse” type [W18O56(HPO3)0.8(SeO3)1.2(H2O)2]8- and cross-shaped [H4P4X4W64O224]32-/36-, where X=TeIV, SeIV, AsIII. In the case of {W18(HPO3)0.8(SeO3)1.2}, the compound is found to be a mixture of heteroleptic {W18(HPO3)(SeO3)} and homoleptic {W18(SeO3)2} and {W18(HPO3)2}, identified by single crystal x-ray diffraction, NMR as well as high resolution mass spectrometry. The cluster exhibited similar temperature-dependent electronic features to “Trojan Horse” type {W18(HPO3)2O56}. However, due to the intrinsic reactivity difference between {HPO3} and {SeO3}, the thermal treatment leads to the formation of an unusual species [W18O55(PO4)(SeO3)]5-, in which {HPO3} was fully oxidised to {PO4} within the cage, whereas and lone-pair-containing {SeO3} heteroanions were kept intact inside the shell. This finding is extremely interesting, as it demonstrated that multiple and independent intramolecular electronic performance can be achieved by the coexistence of distinct heteroatoms within a single molecule. On the other hand, the cross-shaped [H4P4X4W64O224]32-/36- were constructed by four {W15(HPO3)(XO3)} building units linked by four {WO6} octahedra. Each building unit traps two different heteroatoms. It is interesting to note that the mixed heteroatom species show self-sorting, with a highly selective positional preference. Smaller ionic sized {HPO3} are self-organised into the uncapped side of {W15} cavity, whereas closed side are occupied by larger heteroatoms, which is surprisingly opposed to steric hindrance. Density functional theory (DFT) calculations are currently underway to have a full understanding of the preference of heteroatom substitutions. This series of clusters is of great interest in terms of achieving single molecule-based heteroatom-dependent multiple levels of electron transfer. It has opened a new way to design and synthesise POMs with higher diversity of electrical states, which may lead to a new type of Q-bits for quantum computing. The third chapter is focused on developing polyoxotungstate building blocks templated by {HPO3}. A series of building blocks, {W15O48(HPO3)2}, {W9O30(HPO3)} {W12O40(HPO3)2} and hexagonal {W6O18(HPO3)} have been obtained. The first four building blocks have been reported with {SeO3} and/or {TeO3} heteroanions. This result demonstrates {HPO3} has a similar reactivity as {SeO3} and {TeO3}, therefore studying the self-assembly of {HPO3}-based building blocks would be helpful to have a general understanding of pyramidal heteroatom-based molecular systems. The hexagonal {W6O18(HPO3)} is observed for the first time in polyoxotungstates, showing some of reactivity difference between {HPO3} and {SeO3} and {TeO3}. Furthermore, inorganic salts and pH values have some directing influence on the formation and transformation of various building blocks, resulting in the discovery of a family of {HPO3}-based clusters with nuclearity ranging from {W29} to {W106}. High resolution mass spectrometry was also carried out to investigate the cluster solution behaviour and also gain information of building block speciation. It is found that some clusters experienced decomposition, which gives rise to potential building blocks accountable for the self-assembly.

AnnoTALE: Bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present 'AnnoTALE', a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities. © 2016, Nature Publishing Group. All rights reserved.

STRUCTURE AND FUNCTION OF THE GROUP III CHAPERONINS, A UNIQUE CLADE OF PROTEIN FOLDING NANOMACHINES

The survival and descent of cells is universally dependent on maintaining their proteins in a properly folded condition. It is widely accepted that the information for the folding of the nascent polypeptide chain into a native protein is encrypted in the amino acid sequence, and the Nobel Laureate Christian Anfinsen was the first to demonstrate that a protein could spontaneously refold after complete unfolding. However, it became clear that the observed folding rates for many proteins were much slower than rates estimated in vivo. This led to the recognition of required protein-protein interactions that promote proper folding. A unique group of proteins, the molecular chaperones, are responsible for maintaining protein homeostasis during normal growth as well as stress conditions. Chaperonins (CPNs) are ubiquitous and essential chaperones. They form ATP-dependent, hollow complexes that encapsulate polypeptides in two back-to-back stacked multisubunit rings, facilitating protein folding through highly cooperative allosteric articulation. CPNs are usually classified into Group I and Group II. Here, I report the characterization of a novel CPN belonging to a third Group, recently discovered in bacteria. Group III CPNs have close phylogenetic association to the Group II CPNs found in Archaea and Eukarya, and may be a relic of the Last Common Ancestor of the CPN family. The gene encoding the Group III CPN from Carboxydothermus hydrogenoformans and Candidatus Desulforudis audaxviator was cloned in E. coli and overexpressed in order to both characterize the protein and to demonstrate its ability to function as an ATPase chaperone. The opening and closing cycle of the Chy chaperonin was examined via site-directed mutations affecting the ATP binding site at R155. To relate the mutational analysis to the structure of the CPN, the crystal structure of both the AMP-PNP (an ATP analogue) and ADP bound forms were obtained in collaboration with Sun-Shin Cha in Seoul, South Korea. The ADP and ATP binding site substitutions resulted in frozen forms of the structures in open and closed conformations. From this, mutants were designed to validate hypotheses regarding key ATP interacting sites as well as important stabilizing interactions, and to observe the physical properties of the resulting complexes by calorimetry.

JOINT ENERGY BEAMFORMING AND TIME ASSIGNMENT FOR COOPERATIVE WIRELESS POWERED NETWORKS

Wireless power transfer (WPT) and radio frequency (RF)-based energy har- vesting arouses a new wireless network paradigm termed as wireless powered com- munication network (WPCN), where some energy-constrained nodes are enabled to harvest energy from the RF signals transferred by other energy-sufficient nodes to support the communication operations in the network, which brings a promising approach for future energy-constrained wireless network design. In this paper, we focus on the optimal WPCN design. We consider a net- work composed of two communication groups, where the first group has sufficient power supply but no available bandwidth, and the second group has licensed band- width but very limited power to perform required information transmission. For such a system, we introduce the power and bandwidth cooperation between the two groups so that both group can accomplish their expected information delivering tasks. Multiple antennas are employed at the hybrid access point (H-AP) to en- hance both energy and information transfer efficiency and the cooperative relaying is employed to help the power-limited group to enhance its information transmission throughput. Compared with existing works, cooperative relaying, time assignment, power allocation, and energy beamforming are jointly designed in a single system. Firstly, we propose a cooperative transmission protocol for the considered system, where group 1 transmits some power to group 2 to help group 2 with information transmission and then group 2 gives some bandwidth to group 1 in return. Sec- ondly, to explore the information transmission performance limit of the system, we formulate two optimization problems to maximize the system weighted sum rate by jointly optimizing the time assignment, power allocation, and energy beamforming under two different power constraints, i.e., the fixed power constraint and the aver- age power constraint, respectively. In order to make the cooperation between the two groups meaningful and guarantee the quality of service (QoS) requirements of both groups, the minimal required data rates of the two groups are considered as constraints for the optimal system design. As both problems are non-convex and have no known solutions, we solve it by using proper variable substitutions and the semi-definite relaxation (SDR). We theoretically prove that our proposed solution method can guarantee to find the global optimal solution. Thirdly, consider that the WPCN has promising application potentials in future energy-constrained net- works, e.g., wireless sensor network (WSN), wireless body area network (WBAN) and Internet of Things (IoT), where the power consumption is very critical. We investigate the minimal power consumption optimal design for the considered co- operation WPCN. For this, we formulate an optimization problem to minimize the total consumed power by jointly optimizing the time assignment, power allocation, and energy beamforming under required data rate constraints. As the problem is also non-convex and has no known solutions, we solve it by using some variable substitutions and the SDR method. We also theoretically prove that our proposed solution method for the minimal power consumption design guarantees the global optimal solution. Extensive experimental results are provided to discuss the system performance behaviors, which provide some useful insights for future WPCN design. It shows that the average power constrained system achieves higher weighted sum rate than the fixed power constrained system. Besides, it also shows that in such a WPCN, relay should be placed closer to the multi-antenna H-AP to achieve higher weighted sum rate and consume lower total power.

Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae

Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower Km for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK(a) of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK(a) values of the yGrx1 and yGix2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 angstrom, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity. (C) 2008 Elsevier Ltd. All rights reserved.