Deciphering living networks : Perturbation strategies for functional genomics

Thesis: Deciphering living networks: Perturbation strategies for functional genomics Alberto de la Fuente [email protected] Molecular Cell Physiology Free University Amsterdam Advisors: Prof.Dr. H.V. Westerhoff Prof.Dr. J.L. Snoep Supervisor: Dr. P.J. Mendes Using modern experimental techniques it is possible to measure the concentrations of a great many, and ultimately all, cellular constituents such as mRNAs, proteins and metabolites. Given these experimental technologies, astronomical amounts of new data will appear. To enable us to see the forest for the trees, we need to find ways in which best to analyze the data so as to obtain better understanding of biochemical systems and predictive power. When those new ways of analyzing the data are found, this may even lead to a preference for a certain type of data or certain experimental methodologies. This may then help direct experimentation towards the highest possible impact for understanding of biochemical systems. Ideally, the three levels of biochemical organization, i.e. mRNAs, proteins and metabolites, are studied all together in an integrated fashion. However, due to the number of components and complexity of such integrated systems it is reasonable to try to decompose the system and to study the subsystems or to use simplified descriptions of the whole system. It will be important to decompose the system into subsystems that behave in isolation in much the same way as they do when they are embedded in the whole system. This is exactly what I deal with in my dissertation; on the one hand I show how and when it is possible to study the systems properties of metabolism in vivo, ignoring the effects of gene and protein expression, and on the other hand I develop a quantitative concept in terms of Metabolic Control Analysis to describe the properties of the whole system in a simplified form, i.e. as a gene network a description of only the dynamics of gene expression without explicit accounting for metabolites and proteins. This concept enables the inference of the topology of such gene networks from experimental data. The analysis guides the experimenter towards the specific experiments that need to be done in order to be able to infer the interactions between genes on a genome scale. After introducing the relevant preliminaries in Chapter 1, in Chapter 2 I introduce the concept of hierarchical biochemical systems and show how to express their properties in terms of properties of the individual flux-disconnected modules of which it is composed. In particular, I focus on the study of metabolic systems. I propose several methods with the goal of distinguishing regulation that takes place at the metabolic level only from regulation that involves transcription or translation, thus quantifying the relative importance of each of these processes to the global systems behavior. I verify the experimental applicability of these methods by analyzing data obtained by simulation of a biochemical system. In Chapter 3 I introduce the concept of the gene network. Gene networks are network models in which the nodes represent gene activities (mRNA levels) and the edges correspond to regulatory interactions between them. Such models are highly phenomenological because they do not represent explicitly the proteins and metabolites that mediate those interactions. I show the use of Regulatory Strengths to quantify gene-gene interactions and show how to express these coefficients in terms of the biochemical system underlying these interactions. This approach establishes a clear and formal link between the phenomenological gene network modeling and more detailed approaches considering the hierarchical structuring of biochemical networks as introduced in Chapter 2.Snoep, J.L. [Promotor]Westerhoff, H.V. [Promotor]Mendes, P. [Copromotor

Voorkomen van uitval bij Impatiens walleriana onder veldcondities

Abstract Wageningen UR Greenhouse Horticulture, together with Syngenta Seeds performed field trials with fungicides and fertilizing products to prolong the consumer value of Impatiens walleriana (busy lily). This project was funded by the Dutch Horticultural Board. The plants were situated on two field locations, both in the field and in pots to follow the natural disease development with and without interaction with soil pathogens (e.g. downy mildew). The results of this study show that Ridomil Gold is effective against early infestation from soil propagules of downy mildew, but there is no effectivity against leaf infections. Products based on phoshite (potassium phosphite, product X of Everris) slow down the infection process with a few weeks. Especially, later in the season when the conidia are most dominantly present. It is recommended to screen multiple products which are effective against resting spores of downy mildew. Sensitive Impatiens species should preferably be planted early in the season and not directly in soil with high risk of surviving propagules

Beheersing van valse meeldauw in Impatiens

De éénjarige zomerbloeier Impatiens walleriana vormt een belangrijk onderdeel in het assortiment van perkgoed. Ondermeer in 2007 waren er door de natte weersomstandigheden grote problemen met uitval door valse meeldauw in de consumentfase. Hierdoor kwam de vraag of er tijdens de opkweek niet meer preventieve maatregelen genomen kunnen worden om infectie door valse meeldauw onder vochtige omstandigheden te verminderen of te voorkomen. Daarnaast was het onbekend of de besmetting met valse meeldauw op Nederlandse locaties veroorzaakt wordt door één soort of meerdere soorten. In een literatuurstudie is alle beschikbare informatie over valse meeldauw en mogelijkheden voor beheersen/ bestrijding samengeva

Nieuwe middelen tegen valse meeldauw in Impatiens walleriana

Abstract Wageningen UR Greenhouse Horticulture, together with Syngenta Seeds tested various combinations of agents for their effects to increase plant defense against infection of downy mildew in the consumer phase. All means were dosed 2-3 times during the growers phase. There are three new products, which were found to strengthen Impatiens plants against infection by downy mildew. It is still unclear how these products can be combined with other agents that also control pathogenic soil fungi so that failure occurs less rapidly under field conditions at high disease pressure. In 2011, this research will be continued at two field locations

PhosPhAt: a database of phosphorylation sites in Arabidopsis thaliana and a plant-specific phosphorylation site predictor

The PhosPhAt database provides a resource consolidating our current knowledge of mass spectrometry-based identified phosphorylation sites in Arabidopsis and combines it with phosphorylation site prediction specifically trained on experimentally identified Arabidopsis phosphorylation motifs. The database currently contains 1187 unique tryptic peptide sequences encompassing 1053 Arabidopsis proteins. Among the characterized phosphorylation sites, there are over 1000 with unambiguous site assignments, and nearly 500 for which the precise phosphorylation site could not be determined. The database is searchable by protein accession number, physical peptide characteristics, as well as by experimental conditions (tissue sampled, phosphopeptide enrichment method). For each protein, a phosphorylation site overview is presented in tabular form with detailed information on each identified phosphopeptide. We have utilized a set of 802 experimentally validated serine phosphorylation sites to develop a method for prediction of serine phosphorylation (pSer) in Arabidopsis. An analysis of the current annotated Arabidopsis proteome yielded in 27 782 predicted phosphoserine sites distributed across 17 035 proteins. These prediction results are summarized graphically in the database together with the experimental phosphorylation sites in a whole sequence context. The Arabidopsis Protein Phosphorylation Site Database (PhosPhAt) provides a valuable resource to the plant science community and can be accessed through the following link http://phosphat.mpimp-golm.mpg.d

The Ca2+-dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis

Plants use different signalling pathways to respond to external stimuli. Intracellular signalling via calcium-dependent protein kinases (CDPKs) or mitogen-activated protein kinases (MAPKs) present two major pathways that are widely used to react to a changing environment. Both CDPK and MAPK pathways are known to be involved in the signalling of abiotic and biotic stresses in animal, yeast and plant cells. Here, we show the essential function of the CDPK CPK3 (At4g23650) for salt stress acclimation in Arabidopsis thaliana, and test crosstalk between CPK3 and the major salt-stress activated MAPKs MPK4 and MPK6 in the salt stress response. CPK3 kinase activity was induced by salt and other stresses after transient overexpression in Arabidopsis protoplasts, but endogenous CPK3 appeared to be constitutively active in roots and leaves in a strictly Ca2+-dependent manner. cpk3 mutants show a salt-sensitive phenotype comparable with mutants in MAPK pathways. In contrast to animal cells, where crosstalk between Ca2+ and MAPK signalling is well established, CPK3 seems to act independently of those pathways. Salt-induced transcriptional induction of known salt stress-regulated and MAPK-dependent marker genes was not altered, whereas post-translational protein phosphorylation patterns from roots of wild type and cpk3 plants revealed clear differences. A significant portion of CPK3 was found to be associated with the plasma membrane and the vacuole, both depending on its N-terminal myristoylation. An initial proteomic study led to the identification of 28 potential CPK3 targets, predominantly membrane-associated proteins

Phospho.ELM: a database of phosphorylation sites—update 2011

The Phospho.ELM resource (http://phospho.elm.eu.org) is a relational database designed to store in vivo and in vitro phosphorylation data extracted from the scientific literature and phosphoproteomic analyses. The resource has been actively developed for more than 7 years and currently comprises 42 574 serine, threonine and tyrosine non-redundant phosphorylation sites. Several new features have been implemented, such as structural disorder/order and accessibility information and a conservation score. Additionally, the conservation of the phosphosites can now be visualized directly on the multiple sequence alignment used for the score calculation. Finally, special emphasis has been put on linking to external resources such as interaction networks and other databases

PhosPhAt: the Arabidopsis thaliana phosphorylation site database. An update

The PhosPhAt database of Arabidopsis phosphorylation sites was initially launched in August 2007. Since then, along with 10-fold increase in database entries, functionality of PhosPhAt (phosphat.mpimp-golm.mpg.de) has been considerably upgraded and re-designed. PhosPhAt is now more of a web application with the inclusion of advanced search functions allowing combinatorial searches by Boolean terms. The results output now includes interactive visualization of annotated fragmentation spectra and the ability to export spectra and peptide sequences as text files for use in other applications. We have also implemented dynamic links to other web resources thus augmenting PhosPhAt-specific information with external protein-related data. For experimental phosphorylation sites with information about dynamic behavior in response to external stimuli, we display simple time-resolved diagrams. We have included predictions for pT and pY sites and updated pS predictions. Access to prediction algorithm now allows ‘on-the-fly’ prediction of phosphorylation of any user-uploaded protein sequence. Protein Pfam domain structures are now mapped onto the protein sequence display next to experimental and predicted phosphorylation sites. Finally, we have implemented functional annotation of proteins using MAPMAN ontology. These new developments make the PhosPhAt resource a useful and powerful tool for the scientific community as a whole beyond the plant sciences