Understanding aspects of alginate biosynthesis and regulation by Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

Alginate is a medically and industrially important polymer produced by seaweeds and certain bacteria. The bacterium Pseudomonas aeruginosa over-produces alginate during cystic fibrosis lung infections, forming biofilms, making the infection difficult to treat. Bacteria make alginate using membrane spanning multi-protein complexes. Although alginate biosynthesis and regulation have been studied in detail, there are still major gaps in knowledge. In particular, the requirement of AlgL (a periplasmic alginate degrading enzyme) and role played by MucR (an inner membrane c-di-GMP modulator) are not well understood. Here I show that AlgL and MucR are not essential for alginate production during biofilm growth. My findings suggest that while catalytically active AlgL negatively affects alginate production, expressing catalytically inactive AlgL enhances alginate yields. Furthermore, preliminary data show AlgL is not required for the stability or functionality of the alginate biosynthesis complex, suggesting that it is a free periplasmic protein dispensable for alginate production. These findings support the prediction that the primary function of AlgL is to degrade misguided alginate from the periplasm. For MucR, I show for the first time that its sensor domain mediates nitrate-induced suppression of alginate biosynthesis. This appears to occur at multiple levels in a manner only partially dependent on c-di-GMP signaling. These results indicate that MucR is associated with the negative effect of nitrate (and possibly denitrification) on alginate production. On the basis of these results, I propose a combination of nitrate (or denitrification intermediates), exogenous lyases and antimicrobial agents could be used to eliminate established chronic biofilm infections. Furthermore, catalytically inactive AlgL and/or homologs of MucR with disabled sensor motifs could be harnessed in non-pathogenic bacteria for producing tailor-made alginates

The Time-Course of the Effects of Growth Hormone During Zebrafish (\u3ci\u3eDANIO RERIO\u3c/i\u3e) Auditory Hair Cell Regeneration

Growth hormone (GH) was upregulated in the zebrafish inner ear following sound exposure in a previous study. To identify the specific role of GH in hair cell regeneration and the possible cellular mechanisms of this regeneration, groups of zebrafish were divided into baseline (no sound exposure, no injection), buffer-injected and GH-injected groups. Buffer- and GH-injected fish were exposed to a 150 Hz tone at a source level of 179 dB re 1 μPa root mean squared (RMS) for 36 h. Phalloidin-staining was used to assess the effects of GH on hair cell bundle density; BrdU-labeling was used to assess the effects of GH on cellular proliferation; TUNEL-labeling was used to assess the effects of GH on apoptosis in the zebrafish inner ear following acoustic trauma. The time-course of hair cell bundle density, cell proliferation, and apoptosis was established by combining data for baseline fishes and sound-exposed fishes at post-sound exposure day 1 (psed1), psed2, and psed3. GH-injected fish exhibited greater densities of hair cells than bufferinjected controls. In addition, GH-injected fish had higher levels of cell proliferation and lower levels of apoptosis than buffer-injected controls. This suggests that GH may play an important role in zebrafish inner ear hair cell regeneration by stimulating cellular proliferation and inhibiting cellular apoptosis

Mechanisms and control of mine floor heave.

Floor heave has been one of the important ground control problems in the underground coal mining industry. However, the mechanisms of floor heave have not been fully understood although some efforts have been made by many researchers. In this research, two mines experiencing floor heave are selected to study the mechanisms of floor heave. By in situ observation, two types of floor heave are identified: Type I floor heave caused by buckling failure of floor, and the Type II floor heave caused by plastic failure and plastic flow of floor material. Based on the observation results, the mechanisms of floor heave are analyzed and the mechanical models for two types of floor heave are established. The model for type I floor heave is based on composite slab theory and the model for type II floor heave is based on foundation theory. Using the models established, the floor heaves in two mines were analyzed and one of them was redesigned. Results show that the floor heaves predicted by the models are quite accurate. The mine has eliminated the floor heave problems after it had been redesigned

Flame-like Ellerman Bombs and Their Connection to Solar UV Bursts

Ellerman bombs (EBs) are small-scale intense brightenings in H$\alpha$ wing images, which are generally believed to be signatures of magnetic reconnection events around the temperature minimum region of the solar atmosphere. They have a flame-like morphology when observed near the solar limb. Recent observations from the Interface Region Imaging Spectrograph (IRIS) reveal another type of small-scale reconnection events, termed UV bursts, in the lower atmosphere. Though previous observations have shown a clear coincidence of some UV bursts and EBs, the exact relationship between these two phenomena is still under debate. We investigate the spatial and temporal relationship between flame-like EBs and UV bursts using joint near-limb observations between the 1.6--meter Goode Solar Telescope (GST) and IRIS. In total 161 EBs have been identified from the GST observations, and 20 of them reveal signatures of UV bursts in the IRIS images. Interestingly, we find that these UV bursts have a tendency to appear at the upper parts of their associated flame-like EBs. The intensity variations of most EB-related UV bursts and their corresponding EBs match well. Our results suggest that these UV bursts and EBs are likely formed at different heights during a common reconnection process.Comment: 5 figures; accepted by ApJ

Design and engineering of multi-step (bio)catalytic systems

Nature has been a perpetual source of inspiration for biochemists. It is not only the vast diversity of compounds that living beings can create, but also the extraordinary strategies of synthesis deployed. Evidently, the catalysts used by living beings -enzymes- are key to nature’s synthesis strategies. Biocatalysis is undoubtfully one of the most invaluable gifts given by nature to flourish the development of green chemical and pharmaceutical industries. With the development of protein and metabolic engineering tools and strategies, more and more enzymes have been used in the industries to improve the chemical processing; and microbes such as Escherichia coli and Saccharomyces cerevisiae have been engineered to produce a wide variety of value-added and bulk chemicals to replace traditional chemical synthesis. However, researchers have just explored the tip of the iceberg in the biocatalysis area. Proteins with new catalytic functionality should be discovered or engineered to broaden current biotransformation boundaries. New in vitro enzymatic or chemoenzymatic cascade reactions need to be designed and optimized to realize stronger synthetic power and more stable systems. Metabolic networks of traditional or new microorganisms should be largely rewired to meet the manufacturing standards. In this work, I aimed at designing and engineering multi-step (bio) catalytic systems for selective synthesis of value-added chemicals. Microorganisms synthesize complex molecules from simple substrates by a series of enzymes working cooperatively. Inspired by how aromatic polyketides are synthesized by the teamwork between enzymes, I sought to couple biocatalysis with organometallic catalysis, two distinct catalytic disciplines, in one pot to realize synthetic power that cannot be achieved by either of them. I first developed a modular, one-pot, sequential chemoenzymatic system for the formal enantioselective construction of C-C bond in 2-aryl 1,4-dicarbonyl compounds. This sequence comprises a rhodium-catalyzed diazocoupling that provides >9:1 selectivity for heterocoupling of two diazoesters and a reduction mediated by an ene-reductase (ER), which occurs in up to 99% enantiomeric excess (ee). The high yield and enantioselectivity of this system were resulted from the preferential generation of an (E)-alkene from the diazo coupling reaction and selective reduction of the (E)-alkene in a mixture of (E) and (Z) isomers by the ER. This work demonstrates the benefit of combining organometallic and enzymatic catalysis to create unusual overall transformations that do not require the isolation and purification of intermediates. To make the system works better on a broader range of substrates, I later developed a new class of cooperative chemoenzymatic reactions that combine photocatalysts that isomerize alkenes with ene-reductases that reduce carbon-carbon double bonds to generate valuable enantioenriched products. I demonstrated that this method enables the stereoconvergent reduction of E/Z mixtures of alkenes or reduction of the unreactive stereoisomer of an alkene in yields and ee’s that match those obtained from the reduction of the pure, more reactive isomer. This new cooperative system overcomes the limitations of both individual catalysts and affords a range of synthetically valuable and biologically active enantioenriched compounds. More generally, these results illustrate the value of driving a chemical reaction with light to ensure compatibility between the chemical and enzymatic catalysts. In vitro biocatalytic reaction normally has poor tolerance to harsh conditions such as low pH or high substrate concentrations. Cells membrane is natural compartmentalization and protects the enzymes from extracellular inhibitors. In addition, cell factories-based production provides an attractive alternative to chemical synthesis of value-added chemicals. I also worked on engineering a S. cerevisiae strain as a whole-cell catalyst for L-lactic acid overproduction in industrially preferred low pH environment (pH 3) by metabolic engineering and genome-wide engineering methods. In addition, to establish an automated cellular engineering platform, I developed a growth-based L-lactic acid biosensor and an automated quantification assay by BioProfile Analyzer

Functional characterization of breast cancer using pathway profiles

BACKGROUND: The molecular characteristics of human diseases are often represented by a list of genes termed “signature genes”. A significant challenge facing this approach is that of reproducibility: signatures developed on a set of patients may fail to perform well on different sets of patients. As diseases are resulted from perturbed cellular functions, irrespective of the particular genes that contribute to the function, it may be more appropriate to characterize diseases based on these perturbed cellular functions. METHODS: We proposed a profile-based approach to characterize a disease using a binary vector whose elements indicate whether a given function is perturbed based on the enrichment analysis of expression data between normal and tumor tissues. Using breast cancer and its four primary clinically relevant subtypes as examples, this approach is evaluated based on the reproducibility, accuracy and resolution of the resulting pathway profiles. RESULTS: Pathway profiles for breast cancer and its subtypes are constructed based on data obtained from microarray and RNA-Seq data sets provided by The Cancer Genome Atlas (TCGA), and an additional microarray data set provided by The European Genome-phenome Archive (EGA). An average reproducibility of 68% is achieved between different data sets (TCGA microarray vs. EGA microarray data) and 67% average reproducibility is achieved between different technologies (TCGA microarray vs. TCGA RNA-Seq data). Among the enriched pathways, 74% of them are known to be associated with breast cancer or other cancers. About 40% of the identified pathways are enriched in all four subtypes, with 4, 2, 4, and 7 pathways enriched only in luminal A, luminal B, triple-negative, and HER2+ subtypes, respectively. Comparison of profiles between subtypes, as well as other diseases, shows that luminal A and luminal B subtypes are more similar to the HER2+ subtype than to the triple-negative subtype, and subtypes of breast cancer are more likely to be closer to each other than to other diseases. CONCLUSIONS: Our results demonstrate that pathway profiles can successfully characterize both common and distinct functional characteristics of four subtypes of breast cancer and other related diseases, with acceptable reproducibility, high accuracy and reasonable resolution

Comercio electrónico conectando China y América Latina a través de la ruta de la seda digital

Latin America is the natural extension of the “Maritime Silk Road” and its exchanges and cooperation with China have become increasingly frequent in recent years. Under the current globale conomic backdrop, e-commerce as an emerging industry is gaining momentum and playing a bigger role in international  cooperation.  The  Belt  and  Road  top-level  design  features  policy  coordination, unimpededtrade, facilities connectivity and people-to-people bond respectively, which play a guiding role in China-LAC cooperation. Coupled with the status quo, the paper will mainly focus on these four sections so as to better analyze the China-LAC cooperation in Silk-Road E-commerce. This paper intends to analyze China-LAC e-commerce cooperation in fields of policy, trade, facilities, and people-to-people contact and study in which areas still remain room for bilateral cooperation and how can both sides better achieve win-win results under the framework of China’s “Belt and Road Initiative”.América Latina es la extensión natural de la “Ruta Marítima de la Seda” y sus intercambios y cooperación con China se han vuelto cada vez más frecuentes en los últimos años. En el contexto económico mundial actual, el comercio electrónico como industria emergente está cobrando impulso y desempeñando un papel más importante en la cooperación internacional. El diseño de alto nivel de la Franja y la Ruta presenta coordinación de políticas, comercio sin trabas, conectividad de instalaciones y vínculo entre personas, respectivamente, que desempeñan un papel rector en la cooperación entre China y ALC. Junto con el statu quo, el artículo se centrará principalmente en estas cuatro secciones para analizar mejor la cooperación entre China y ALC en el comercio electrónico de la Ruta de la Seda. Este documento tiene la intención de analizar la cooperación en comercio electrónico entre China y ALC en los campos de política, comercio, instalaciones y contacto entre personas y estudiar en qué áreas aún quedan espacios para la cooperación bilateral y cómo ambas partes pueden lograr mejores resultados en los que todos ganan en el marco de la “Iniciativa de la Franja y la Ruta” de China