Guanosine nucleotides link cell wall metabolism and protein synthesis during entry into quiescence

Quiescence, a transitory period of non-growth, is a ubiquitous aspect that is present in all organisms. In addition to being present in all forms of life, quiescence is a feature that has been observed in cells that are important for human health, including stem cells in mammals and antibiotic tolerant cells in bacteria. In bacteria, quiescence per se has recently been suggested to underlie the transient tolerance to a wide range of antibiotics. Furthermore, most microbial life exists in a quiescent state. Despite their prevalence and importance, relatively little is known about the physiology of quiescent bacteria. One aspect of bacterial quiescence that has been repeatedly observed is their lowered metabolic activity compared to actively growing bacteria. How do cells that grow and divide enter into a temporary state of non-growth? In particular, how are the energy-intensive processes that are required for growing cells regulated during a non-growing state? The main subject of this thesis is to investigate how protein synthesis, the most energy-intensive process in growing bacterial cells, is regulated during entry into a quiescent phenotype (stationary phase). I first investigate how protein synthesis is regulated using a single cell method that fluorescently tags nascent polypeptide chains. In chapter 3, I show that during entry into stationary phase, protein synthesis is downregulated heterogeneously with one group of cells having comparatively low protein synthesis, resulting in a population that is approximately bimodal. I further show that this bimodality is dependent on a signaling system (PrkC and its partner phosphatase PrpC) that senses cell wall metabolism. I connect signaling from this system to the expression of an enzyme (SasA) that produces a group of nucleotides that are major regulators of growth in bacteria ((pp)pGpp). Lastly, I show that the bimodality is dependent on the three enzymes that synthesize (pp)pGpp. In chapter 4, I explore in detail how the bimodality in protein synthesis is generated. This heterogeneity requires the production of (pp)pGpp by three synthases: SasA, SasB, RelA. I first show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population exhibiting low protein synthesis, whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. The RelA product (pppGpp) allosterically activates SasB, and I find that the SasA product (pGpp) competitively inhibits this activation. I provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This chapter, therefore, identifies the mechanism underlying the generation of phenotypic heterogeneity in the central physiological process of protein synthesis. In chapter 5, I next turn to understand the biochemical mechanism by which cells with comparatively low levels of protein synthesis down-regulate this process. I first show that ppGpp is sufficient to inhibit protein synthesis in vivo. I then show that ppGpp inhibits protein synthesis by inhibiting translation initiation directly by binding to the essential GTPase, Initiation Factor 2 (IF2). In collaboration with Ruben Gonzalez’s lab, we also show that ppGpp prevents the allosteric activation of IF2. Finally, I demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (pp)pGpp and IF2

PIV data clustering of a buoyant jet in a stratified environment

Three spatial clustering approaches of a high-Reynolds number transient buoyant jet in a linearly stratified environment are applied along with proper orthogonal decomposition to identify similar/consistent regions in the domain of interest. The velocity fields analyzed are obtained from an experimental test with large scale, time-resolved, particle image velocimetry (PIV) measurements. Clustering is performed by the k-means method considering: (a) crosssection velocity profiles, (b) point-wise energy spectra, and (c) point-wise Reynolds stress tensor components. Three metrics are used for the assessment of clustering approaches, namely: (a) within-cluster sum of squares, (b) average silhouette, and (c) within-cluster number of POD modes required to resolve prescribed levels of total variance/energy. Results are promising and lay the foundation for an in depth analysis of local features of complex flows as well as the formulation of efficient reduced order models

Transcription factor mediated control of anthocyanin biosynthesis in vegetative tissues

Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato (Solanum lycopersicum) for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from Antirrhinum that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology

Space, time, desire, and the Atlantic in three Spanish films of the 1920s

This article looks at the relationship between Hollywood and Spanish cinema of the 1920s. The central issue is the impact on the representation of space and time, both of Hollywood film structures, and of the elision between 'Spanish' and 'North American' space. The problem of boundaries and elision between these two spaces hinges on the definition of sexual and gender roles

1,3-Bis(4-bromo­phen­yl)imidazolium chloride dihydrate

In the title hydrated salt, C15H11Br2N2 +·Cl−·2H2O, the complete imidazolium cation is generated by a crystallographic twofold axis, with one C atom lying on the axis. The chloride ion and both water mol­ecules of crystallization also lie on a crystallographic twofold axis of symmetry. The cation is non-planar, the dihedral angle formed between the central imidazolium and benzene rings being 12.9 (3)°; the dihedral angle between the symmetry-related benzene rings is 25.60 (13)°. In the crystal, O—H⋯Cl hydrogen bonds result in supra­molecular chains along c mediated by eight-membered {⋯HOH⋯Cl}2 synthons. These are consolidated by C—H⋯O and π–π [centroid–centroid distance = 3.687 (3) Å] inter­actions

HIFI observations of warm gas in DR21: Shock versus radiative heating

The molecular gas in the DR21 massive star formation region is known to be affected by the strong UV field from the central star cluster and by a fast outflow creating a bright shock. The relative contribution of both heating mechanisms is the matter of a long debate. By better sampling the excitation ladder of various tracers we provide a quantitative distinction between the different heating mechanisms. HIFI observations of mid-J transitions of CO and HCO+ isotopes allow us to bridge the gap in excitation energies between observations from the ground, characterizing the cooler gas, and existing ISO LWS spectra, constraining the properties of the hot gas. Comparing the detailed line profiles allows to identify the physical structure of the different components. In spite of the known shock-excitation of H2 and the clearly visible strong outflow, we find that the emission of all lines up to > 2 THz can be explained by purely radiative heating of the material. However, the new Herschel/HIFI observations reveal two types of excitation conditions. We find hot and dense clumps close to the central cluster, probably dynamically affected by the outflow, and a more widespread distribution of cooler, but nevertheless dense, molecular clumps.Comment: Accepted for publication by A&

Beta interferon production is regulated by P38 mitogen-activated protein kinase in macrophages via both MSK1/2-and tristetraprolin-dependent pathways

Autocrine or paracrine signaling by beta interferon (IFN-β) is essential for many of the responses of macrophages to pathogen-associated molecular patterns. This feedback loop contributes to pathological responses to infectious agents and is therefore tightly regulated. We demonstrate here that macrophage expression of IFN-β is negatively regulated by mitogen- and stress-activated kinases 1 and 2 (MSK1/2). Lipopolysaccharide (LPS)-induced expression of IFN-β was elevated in both MSK1/2 knockout mice and macrophages. Although MSK1 and -2 promote the expression of the anti-inflammatory cytokine interleukin 10, it did not strongly contribute to the ability of MSKs to regulate IFN-β expression. Instead, MSK1 and -2 inhibit IFN-β expression via the induction of dual-specificity phosphatase 1 (DUSP1), which dephosphorylates and inactivates the mitogen-activated protein kinases p38 and Jun N-terminal protein kinase (JNK). Prolonged LPS-induced activation of p38 and JNK, phosphorylation of downstream transcription factors, and overexpression of IFN-β mRNA and protein were similar in MSK1/2 and DUSP1 knockout macrophages. Two distinct mechanisms were implicated in the overexpression of IFN-β: first, JNKmediated activation of c-jun, which binds to the IFN-β promoter, and second, p38-mediated inactivation of the mRNA-destabilizing factor tristetraprolin, which we show is able to target the IFN-β mRNA

Subclinical thyroid dysfunction and cognitive decline in old age

<p>Background: Subclinical thyroid dysfunction has been implicated as a risk factor for cognitive decline in old age, but results are inconsistent. We investigated the association between subclinical thyroid dysfunction and cognitive decline in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER).</p> <p>Methods: Prospective longitudinal study of men and women aged 70–82 years with pre-existing vascular disease or more than one risk factor to develop this condition (N = 5,154). Participants taking antithyroid medications, thyroid hormone supplementation and/or amiodarone were excluded. Thyroid function was measured at baseline: subclinical hyper- and hypothyroidism were defined as thyroid stimulating hormones (TSH) <0.45 mU/L or >4.50 mU/L respectively, with normal levels of free thyroxine (FT4). Cognitive performance was tested at baseline and at four subsequent time points during a mean follow-up of 3 years, using five neuropsychological performance tests.</p> <p>Results: Subclinical hyperthyroidism and hypothyroidism were found in 65 and 161 participants, respectively. We found no consistent association of subclinical hyper- or hypothyroidism with altered cognitive performance compared to euthyroid participants on the individual cognitive tests. Similarly, there was no association with rate of cognitive decline during follow-up.</p> <p>Conclusion: We found no consistent evidence that subclinical hyper- or hypothyroidism contribute to cognitive impairment or decline in old age. Although our data are not in support of treatment of subclinical thyroid dysfunction to prevent cognitive dysfunction in later life, only large randomized controlled trials can provide definitive evidence.</p&gt