FeedOmics, an approach to evaluate the functional properties of protein containing feed ingredients
Kar, Soumya K. - \ 2017
Wageningen University. Promotor(en): M.A. Smits; J.M. Wells, co-promotor(en): A.J.M. Jansman; D. Schokker. - Wageningen : Wageningen University - ISBN 9789463434461 - 254
compound feeds - ingredients - protein sources - proteins - functional properties - metabolism - feed formulation - protein digestion - proteomics - digestive tract - nutrition physiology - animal nutrition - livestock feeding - mengvoer - ingrediënten - eiwitbronnen - eiwitten - functionele eigenschappen - metabolisme - voersamenstelling - eiwitvertering - eiwitexpressieanalyse - spijsverteringskanaal - voedingsfysiologie - diervoeding - veevoeding
This thesis presents FeedOmics approach as a toolkit, to evaluate (novel) protein containing feed ingredients of different origin considering both their nutritional and functional value in terms of their capacity to support or modify nutrient supply, the animal’s physiology, tissue development and functioning. Such knowledge may contribute to introduce novel and/or alternative protein containing feed ingredients in the diet of livestock, thus creating a sustainable food supply for growing human population.
Enrichment proteomics challenges and perspectives : analysis of the N-glycoproteome and plasma membrane proteome in glycosylation mutants and plant-pathogen interactions
Song, Wei - \ 2016
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Sander van der Krol; Twan America. - Wageningen : Wageningen University - ISBN 9789462578722 - 172
proteomics - glycoproteins - arabidopsis - plant-animal interactions - plant pathogens - plasma membranes - eiwitexpressieanalyse - glycoproteïnen - arabidopsis - plant-dier interacties - plantenziekteverwekkers - plasmamembranen
This thesis is based on two technology projects from the Centre for BioSystems Genomics (CBSG), entitled ‘Comparative proteomics on Plant Pathogen interactions through enrichment of the N-glycoproteome and tagged-glycoproteome’ (TD7) and ‘Plasma Membrane proteomics for Plant Pathogen interactions’ (TD5). In the former project we developed the protocol for isolation, identification and quantification of N-linked glycoproteins from plants and used it to obtain a comprehensive inventory of glycan-occupancy of Arabidopsis glycoproteins. In the second project, a protocol for the enrichment of plasma membrane (PM) fraction from plant material was developed and applied to study the role of the PM proteome in the interaction of plants with the plant pathogen Phytophthora infestans. Combined these activities have resulted in a thesis devoted to technical developments in label-free comparative enrichment proteomics, with validation in a number of different biological systems.
Biomarkers and mechanisms of natural disease resistance in dairy cows
Altena, S.E.C. van - \ 2016
Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Edwin Tijhaar. - Wageningen : Wageningen University - ISBN 9789462578005 - 158
dairy cows - biomarkers - disease resistance - immunity - antibodies - proteomics - immune response - dendritic cells - immunology - melkkoeien - biomarkers - ziekteresistentie - immuniteit - antilichamen - eiwitexpressieanalyse - immuniteitsreactie - dendritische cellen - immunologie
The aim of this thesis was to define and test biomarkers for disease resistance in dairy cows and to determine the underlying mechanism in natural disease resistance. The health status of the cows is an important issue in dairy farming. Due to the mandatory reduction in the use of antibiotics, alternatives are required to prevent the development and expression of illness in dairy cows. The identification of biomarkers associated with such disease offers the opportunity to adapt the management of cows at risk, and in this way, prevent them from developing overt disease. Previously, natural antibodies (NAbs) in serum and milk were used as candidate biomarkers for natural disease resistance in cows. In this thesis, we continue on the occurrence and mode of action of NAbs and also focus on their source: the B-1 cells. We performed a literature study on the identification and function of B-1 cells in different species and defined the limitations in the current identification of these cells in pigs, sheep and cows (Chapter 2). B-1 cells were described in cows by using widely accepted cell surface markers CD5 and CD11b. However, in literature several findings suggest that these cell surface markers are not unique markers for B-1 identification. The similarities between mice and veterinary animals in foetal B-cell development and antibody production, implies that B-1 cells are present in cows. In chapter 3, we carefully studied new markers to selectively identify B-1 cells in cows. The combination of B-1 cell markers IgM++ and pSYK++ (indicator constitutive intracellular signalling) identifies a distinct cell population with essential B-1 characteristics such as high CD80 expression. In addition, the development of these B-1 cells in calves before colostrum intake and 3 weeks afterwards shows the same kinetics as the development of NAbs represented by IgM antibodies binding to the well-accepted NAb-antigen phosphatidylcholine (PtC). In calves up to half a year of age, it is shown that the production of such NAbs increases from birth and stabilises from 6 weeks onwards. This implies an endogenous NAb production, which follows the same age-related kinetics as can be expected from B-1 cell development. In contrast, the development of total IgM antibody levels in calves shows a bimodal distribution, which is caused by the uptake and breakdown of maternally-derived IgM and simultaneous endogenous production of specific and natural IgM. Chapter 4 describes the role of such NAbs in bovine immunity. NAbs were represented by the binding of IgM to the naïve antigen keyhole limpet hemocyanin (KLH). Cows with high serum NAb levels were shown to have more IgM and IgG antibodies binding to common microbial structures LPS, LTA and PGN, than cows with low serum NAb levels. In addition, they also have more IgM antibodies binding to intact, fixed E. coli and S. Typhimurium bacteria. However, the killing of live E. coli and S. Typhimurium bacteria via antibody-mediated complement killing does not differ between cows with high and low NAb levels. The antibody-mediated complement killing was determined in a newly developed serum bactericidal test. Cows that performed less in the bactericidal test were more likely to develop mastitis in the future. This association was observed for the killing of E. coli and S. Typhimurium and the development of mastitis within the next one year. For S. Typhimurium the association was still present for the cases of mastitis occurring within four years after testing. Alternative biomarkers for disease resistance in cows were defined in chapter 5 by using a contemporary proteomics approach. Milk samples from high and low disease resistant cows were selected from the “Resilient Cattle” (Weerbaar Vee) biobank. Comparing the spectrum of milk proteins of high and low disease-resistant cows showed potential candidate biomarkers that were elevated in the milk of low-resistant cows. Two candidate marker proteins were validated with ELISA in a new and larger group of high- and low-resistant cows. Lactoferrin (LF) levels were significantly increased in milk of low-resistant cows. In addition, LF levels in milk were associated with clinical manifestations of lameness and had a predictive value for subsequent culling.
In conclusion, we found that also in cows NAbs are produced by B-1 cells that can be identified based on the combined expression of cell surface IgM and internal pSYK. In addition, the frequency of these B-1 cells after birth follows a similar kinetics as described before in mice. These NAbs can be more precisely identified based on their PtC binding ability and their functional activity in a bactericidal test. However, the true predictive value of B-1 cells and NAbs for the health status and immunocompetence of dairy cattle remains to be established. Proteomics turned out to be a useful approach for identifying potential new biomarkers for health and disease in milk of cows. Application and further development of their predictive capacity is dependent on the availability of robust, sensitive and quantitative assays. This project was part of the “Resilient Cattle” project providing biological samples and essential data on the health status during respective lactation periods of individual dairy cows. The impact of this research now requires translation into management tools and principles for the individual farmer impacting on the overall health status and economic performance of his herd of dairy cattle.
Proteomics as a tool to gain more insight into sub-lethal toxicological effects
Miller, Ingrid - \ 2016
Wageningen University. Promotor(en): Tinka Murk, co-promotor(en): A.C. Gutleb; T. Serchi. - Wageningen : Wageningen University - ISBN 9789462578210 - 182
proteomics - laboratory methods - sublethal effects - toxic substances - endocrine disruptors - food consumption - toxicology - animal experiments - eiwitexpressieanalyse - laboratoriummethoden - subletale effecten - toxische stoffen - hormoonverstoorders - voedselconsumptie - toxicologie - dierproeven
This thesis focuses on a modern analytical method, proteomics, to investigate its use in the field of toxicological research. Proteomics is a high resolution method which separates all proteins present in a sample at a clearly defined state and compares this pattern to another one, under slightly different conditions (e.g. after exposure to a chemical). Protein changes may give rise to or reflect disease/harm of the individual and can be attributed to alterations in body functions/regulation systems. Analysis conditions and different varieties of proteomic methods are explained, and a brief introduction given where proteomics is already applied in toxicology. A specific investigation has been performed with the flame retardant HBCD (i.e. hexabromocyclododecane). It is a compound that accumulates in lipid tissue from where it is only slowly removed. Its mechanism of action is not yet completely understood and sometimes seems to be contradictory. Rats were exposed to HBCD in very low doses for just one week and liver proteins were compared to those of unexposed animals. As HBCD is suggested to disturb the thyroid system, both healthy and hypothyroid rats were investigated, of both genders. In female rats, not in males, some specific liver protein changes were seen in glucose/carbohydrate and lipid metabolism, and also in some stress related proteins. Changes were not dependent on the thyroid function of the females. These results are in line with previous findings that female rats were more susceptible to HBCD than males. In a further step, protein patterns of unexposed animals of both genders were compared, revealing gender-dependent differences that exceeded the effects seen in any of the other comparisons, mainly in the pathways that were also affected by HBCD in females. A previous proteomic study on serum proteins has also shown clear gender-dependent concentration differences in rats. This underlines the importance of performing studies both in female and male individuals. The detection of considerable gender-dependent protein alterations confirms that proteomics is a biochemical tool with high sensitivity and large potential also in toxicological research.
Carboxypeptidase Z : an extracellular protein in zebrafish development
Kessels, M.Y. - \ 2015
Wageningen University. Promotor(en): Sacco de Vries; Johan van Leeuwen, co-promotor(en): Stefan Schulte-Merker; Sander Kranenbarg. - Wageningen : Wageningen University - ISBN 9789462575677 - 134
danio rerio - biologische ontwikkeling - carboxypeptidasen - skelet - eiwitten - eiwitexpressieanalyse - genexpressie - danio rerio - biological development - carboxypeptidases - skeleton - proteins - proteomics - gene expression
Historically the skeleton, in particular bone, was depicted as a rigid, inflexible, lifeless structure that readily breaks upon bending. We now know that bones in living organisms are complex, dynamic organs that combine toughness with flexibility. Astonishing is the fact that after their initial formation, bone structures are continuously remodeled (recycled and renewed), adapting to environmental demands at such a speed that in active healthy humans the distal part of the femur is completely replaced every six to twelve months. The zebrafish is a relatively new model organism in the field of skeletal development and primarily used as a powerful model for the identification of novel gene functions during skeletogenesis. At the molecular level, the biological similarity between zebrafish and humans is striking (Spoorendonk et al., 2010), but the actual skeletal composition of the zebrafish remained largely unknown.
The mechanical properties of the skeleton are largely dependent on the composition of proteins that are secreted into the extracellular matrix (ECM). In order to increase our understanding of normal skeletal development, it is necessary to identify and characterize changes in skeletal composition. The aim of this thesis was to analyze the composition of the extracellular matrix in zebrafish, in order to enable the identification of potential key regulatory proteins. By determination of the protein content in the zebrafish skeletal ECM with major changes in protein abundance during development we were able to identify various components of signaling pathways implicated in skeletogenesis. This first proteomic analysis of the zebrafish skeleton revealed the homology between the zebrafish and the skeleton of other vertebrate species including mammals. Our study provides a solid foundation for future studies on the composition and the regulation of the morphogenesis of the vertebrate skeleton.
After the identification of potential regulatory proteins in the developing zebrafish skeleton during the MS-based approach, the protein carboxypeptidase Z (Cpz) was selected for further analysis. This peptidase has previously been implicated in the Wnt signaling pathway, an elaborate pathway that regulates crucial aspects of development (Nusse and Varmus, 2012). Previous studies implicated a regulatory role for this peptidase through the processing of Wnt (Moeller et al., 2003; Wang et al., 2009). An analysis of the role of this peptidase in zebrafish was still lacking and therefore we mapped the spatio-temporal expression of cpz. We showed that expression of cpz is localized in and around juvenile zebrafish ossified structures. A more thorough analysis showed a complex expression pattern during early developmental stages that partially overlaps with that observed in other species. Furthermore, we provide a comparative view of cpz expression and the expression of its proposed ligand, Wnt4 (zebrafish Wnt4a and Wnt4b). Partial overlap with wnt4 expression provided the first evidence for a potential complementary function in the Wnt signaling pathway as observed in mammalian species.
In order to explore the role of cpz in zebrafish development, a loss-of-function mutant for zebrafish cpz was generated via TALEN-mediated mutagenesis. We show that mutant embryos display a variety of phenotypes during early development, most of which were similar to described defects in the Wnt/Calcium signaling pathway. These morphological phenotypes provide the first evidence for a connection between Cpz and regulation of the β-catenin independent Wnt signaling pathways which places the function of Cpz in a completely new perspective.
As a complement to these genetic studies an alternative approach was employed to examine the role of another pathway, the thyroid hormone system, in skeletogenesis. Thyroid hormones are required for skeletal development (Kim and Mohan, 2013), but to what extent thyroid hormone affects early bone development in zebrafish remained unclear. By exposing zebrafish embryos to the thyroid hormone triiodothyronine (T3) we showed that exposure accelerates ossification of craniofacial elements including the opercle and ceratohyal in a dose-dependent manner. This provides the first histological evidence of increased ossification in zebrafish due to thyroid hormone exposure which can be used as a starting point to explore the mechanism of thyroid hormone on skeletal development at a greater depth.
The zebrafish skeleton displays remarkable resemblance to that of other vertebrate species with regards to composition, regulatory components and hormonal response. The use of zebrafish as a model in future research will undoubtedly increase our understanding of vertebrate skeletal development and disease. In this thesis we provide a first insight in the extracellular protein content of the zebrafish skeleton, identify a role for the Cpz protein in β-catenin independent (non-canonical) Wnt signaling during development and shows the effect of thyroid hormone on ossification during early zebrafish development.
Identification and functional characterization of proteases and protease inhibitors involved in virulence of fungal tomato pathogens
Karimi Jashni, M. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jerome Collemare; Rahim Mehrabi. - Wageningen : Wageningen University - ISBN 9789462574571 - 183
passalora fulva - plantenziekteverwekkende schimmels - virulentie - proteïnasen - proteïnaseremmers - plant-microbe interacties - genomica - solanum lycopersicum - tomaten - eiwitexpressieanalyse - passalora fulva - plant pathogenic fungi - virulence - proteinases - proteinase inhibitors - plant-microbe interactions - genomics - solanum lycopersicum - tomatoes - proteomics
Pathogens cause disease on both animal and plant hosts. For successful infection and establishment of disease, pathogens need proper weaponry to protect themselves against host defenses and to promote host colonization to facilitate uptake of nutrients for growth and reproduction. Indeed, plant pathogens secrete various types of effector molecules (proteins and secondary metabolites) to manipulate host responses for their own needs. Secreted proteases and protease inhibitors (PIs) are such effector molecules. Proteases can hydrolyze plant defense proteins and PIs can inhibit plant proteases that are part of the host surveillance system. Despite the importance of proteases and PIs secreted by fungal pathogens, little information about their role in virulence is available. The recent advances in genomics, bioinformatics, transcriptomics and proteomics have facilitated identification and functional analysis of proteases and PIs relevant to plant-fungus interactions.
Chapter 1 is an introduction to the thesis outlining the general concept of plant-microbe interactions. It briefly describes the current knowledge of pathogenicity mechanisms employed by fungal plant pathogens and defense mechanisms employed by their host plants. It further introduces proteases and PIs and their potential role in modifying pathogenesis-related (PR) proteins to facilitate fungal virulence. It completes with an outline of the PhD research project.
In chapter 2, we analyzed and compared the number of putatively secreted proteases present in the genomes of 30 fungi with different lifestyles. The analysis showed that fungi with a saprotrophic and hemibiotrophic lifestyle contain more secreted protease genes than biotrophs. Surprisingly, the number of protease genes present in the genome of Cladosporium fulvum, a biotrophic tomato pathogen, is comparable with that of hemibiotrophs and saprotrophs. We analyzed all C. fulvum protease genes both at the transcriptome and proteome level by means of RNA-Seq/RT-qrtPCR and mass spectrometry analyses, respectively. Results showed that many proteases of C. fulvum are not expressed during growth in planta, likely sustaining the biotrophic growth pattern of this fungus.
In chapter 3, using an alignment-based gene prediction tool, we identified pseudogenes containing disruptive mutations (DMs) that likely lead to the production of nonfunctional proteins, including a group of putatively secreted proteases from C. fulvum. Fewer DMs were observed in other fungi including Dothistroma septosporum, a hemibiotrophic pine needle pathogen and close relative of C. fulvum, and suggested that the difference in pseudogenization of proteases between these two pathogens might in part explain their different lifestyle.
In chapter 4, we analyzed the tomato genome and identified 30 candidate chitinases genes, of which six encoded chitin binding domain (CBD)-containing chitinases. Transcriptome and proteome data were collected after inoculation of tomato with several fungal pathogens and allowed the identification of two CBD-chitinases (SlChi2 and SlChi13) with a putative role in protecting tomato against C. fulvum and F. oxysporum f. sp. lycopersici (F. oxysporum), respectively. Purified CBD-chitinases SlChi1, SlChi2, SlChi4 and SlChi13 were incubated with secreted protein extracts (SPEs) from seven fungal tomato pathogens and we could show that SPEs from F. oxysporum, Verticillium dahliae, and Botrytis cinerea modified SlChi1 and SlChi13. LC-MS/MS analysis revealed that incubation with SPE from F. oxysporum removed the N-terminal 37 and 49 amino acids, comprising part and complete CBD domain from SlChi1 and SlChi13, respectively. Removal of the CBD of SlChi1 and SlChi13 by SPE of F. oxysporum reduced the antifungal activity of the two chitinases. We identified a fungal metalloprotease (FoMep1) and a subtilisin serine protease (FoSep1) that synergistically cleaved both SlChi1 and SlChi13. Transgenic F. oxysporum in which the genes encoding these two proteases were knocked out by homologous recombination lost the ability to cleave the two chitinases and were compromised in virulence on tomato compared to the parental wild type. These results suggest an important role of the two chitinases in defense of tomato against this pathogen.
In chapter 5, we searched for host target(s) of the apoplastic effector Avr9 secreted by C. fulvum during infection of tomato. Based on the structural homology of Avr9 with carboxy peptidase inhibitors, we hypothesized that the host target of Avr9 might be apoplastic proteases. To isolate and identify Avr9 targets in apoplastic fluids, we used synthetic biotinylated Avr9, and performed pull-down and far-western blotting assays with apoplastic fluids from tomato inoculated with a C. fulvum race lacking the Avr9 gene. However, we found no specific Avr9-interacting proteins from pull-down complexes analyzed by mass spectrometry or by far-western blotting. Then, we hypothesized that glycosylation of Avr9 might be required for its biological function. The results of mass spectrometry analysis revealed that Avr9 is N-glycosylated when secreted by C. fulvum, containing at least two GlcNac and six mannose residues. The necrosis-inducing activity of glycosylated and non-glycosylated Avr9 was assayed but appeared not significantly different; however, we could not produce sufficient amounts of (biotinylated)-glycosylated Avr9 to perform pull-down assays for identification of potential glycosylated Arv9-interacting proteins by mass spectrometry.
Previous studies as well as the results present in this PhD thesis showed that fungal pathogens secrete a plethora of effectors including proteases and PIs. Many of identified proteases and PIs mediate effector-triggered immunity in host plants. In chapter 6, we reviewed the recent advances on the various roles of proteases and PIs in compromising basal defense responses induced by microbe-associated molecular patterns.
Chapter 7 is a summarizing discussion of the PhD thesis. We showed determinative roles of proteases and PIs in shaping plant-pathogen interactions. The expression and pseudogenization studies on proteases of C. fulvum showed that the genome content does not necessarily reflect the lifestyle of this fungus. This is true for many classes of fungal genes, including proteases. Fungi contain many different types of proteases whose functions may partly overlap. This hampers the discovery of their biological functions. We could demonstrate that two different types of proteases (metalloprotease (FoMep1) and subtilisin serine protease (FoSep1)) of F. oxysporum act synergistically to modify and reduce antifungal activity of two plant CBD-chitinases. Identifying additional proteases is achievable by a targeted proteomics approach using known targets as we did in chapter 4. However, identification of biological functions of proteases is a technical challenge when targets are not known. Multi-gene targeting of protease and PI genes is required to reveal their function in plant-pathogen interactions, which can only be addressed by using advanced genetic tools in future research.
One-carbon metabolism in acetogenic and sulfate-reducing bacteria
Visser, M. - \ 2015
Wageningen University. Promotor(en): Fons Stams. - Wageningen : Wageningen University - ISBN 9789462571730 - 210
anaërobe microbiologie - metabolisme - koolmonoxide - methanol - alcohol dehydrogenase - sulfaat reducerende bacteriën - genetische analyse - eiwitexpressieanalyse - anaerobic microbiology - metabolism - carbon monoxide - methanol - alcohol dehydrogenase - sulfate reducing bacteria - genetic analysis - proteomics
One-carbon metabolism in acetogenic and sulfate-reducing bacteria
Life on earth is sustained by the constant cycling of six essential elements: oxygen, hydrogen, nitrogen, sulfur, phosphorous, and carbon. The continuous cycling of these elements is due to geo-chemical processes and the combined metabolism of all life on earth. Microorganisms like bacteria and archaea play a major role in this. This is also true for the carbon cycle. In this cycle carbon dioxide and methane are two important C-1 compounds present in the atmosphere. Carbon dioxide is the highest oxidative state of carbon while methane is the highest reduced form of carbon. The art to use light to produce organic compounds and conserve energy from the highest oxidative state of carbon is called photosynthesis and is performed by plants, algae and cyanobacteria. Photosynthesis is not the only system to fix carbon from carbon dioxide. Chemolithotrophs can fix carbon from carbon dioxide using inorganic electron donors, like hydrogen. Subsequently, fixed carbon can be used by other organisms, which also makes life possible for them. Microorganisms play a major role in the degradation of complex organic matter, producing smaller compounds including C-1 compounds. C-1 compounds other than carbon dioxide are e.g. carbon monoxide (CO), methanol and formate. Bacteria and archaea can utilize these relative simple compounds in the presence and absence of oxygen, alone and in cooperation with others (syntrophy). The complex and simple carbon compounds are finally oxidized to carbon dioxide, which closes the carbon cycle.
In addition to their importance to the carbon cycle, one carbon compounds like CO, methanol and formate are important for several applications. They are used as a building block for the production of chemicals. They are also used for bioremediation purposes and for wastewater treatment. Therefore, it is important to gain insight in the one carbon metabolism of microorganisms. The research described in this thesis focuses on the proteins and encoding genes involved in anaerobic degradation of C1 compounds by using genome and proteome analysis.
In Chapter 2 the genomes of two closely related sulfate-reducing bacteria, Desulfotomaculum nigrificans and D. nigrificans strain CO-1-SRB, are compared including their CO metabolism. Both the D. nigrificans type strain and strain CO-1-SRB can grow with CO. However, there are differences. The type strain can grow with 20% CO coupled to sulfate reduction in the presence of yeast extract, while strain CO-1-SRB can grow with 100% CO in the presence of yeast extract. Moreover, strain CO-1-SRB can grow with CO in the presence and absence of sulfate. It couples the oxidation of CO to carbon dioxide to hydrogen production. This conversion, the protein complex involved, and the genes coding for these proteins have been described before in other microorganisms. The genome of strain CO-1-SRB contains the genes coding for this protein complex while the genome of the D. nigrificans type strain does not. However, the genome of the type strain contains genes encoding two other CO dehydrogenases. This indicates that one or both are necessary for the type strain to grow with 20% CO. Additional research on the different CO dehydrogenases and their regulation is essential to assess if all different CO dehydrogenases can facilitate growth and how they are linked to for example creating a proton motive force for ATP production.
The methanol metabolism of anaerobic bacteria seems to differ more from that of methanogens than initially described. Methanogens use a methanol methyltransferase system that consists of two methyltranferases, methyltransferase 1 (subunits MtaB and MtaC) and methyltransferase 2 (MtaA). The methyl group from methanol is transferred to the MtaC subunit by MtaB. Subsequently, MtaA transports the methyl group from MtaC to coenzyme M. A genome and proteome analysis of the acetogenic bacterium Sporomusa strain An4 suggests that instead of MtaA a methyl-tetrahydrofolate methyltransferase is involved in the transport of the methyl bound to MtaC to tetrahydrofolate (Chapter 3).
Research done on the methanol metabolism of the sulfate-reducing bacterium Desulfotomaculum kuznetsovii also shows differences with that of methanogens (Chapter 5). The methanol methyltransferase system is vitamin B12 and cobalt dependent. D. kuznetsovii grows with methanol and sulfate, but can do this in presence and absence of vitamin B12 and cobalt. In the absence of vitamin B12 and cobalt D. kuznetsovii grows slower and reaches a lower optical density compared to growth in the presence of vitamin B12 and cobalt. This suggests that D. kuznetsovii can use both a methyltransferase system and a vitamin B12 and cobalt independent system for the degradation of methanol. Proteome results confirm this and suggest that the vitamin B12 and cobalt independent system consists of an alcohol dehydrogenase and an aldehyde ferredoxin oxidoreductase. Moreover, the alcohol dehydrogenase seems to be involved in the oxidation of both methanol and ethanol (Chapter 5). The presence of two methanol degradation pathways give an ecological advantage to D. kuznetsovii in environments containing methanol and sulfate but limiting cobalt and vitamin B12 concentrations. Future research should elucidate if more sulfate-reducing bacteria, or perhaps even acetogenic bacteria, have two methanol degrading pathways.
Additional to the genome analysis of D. kuznetsovii to assess the genes coding for the proteins involved in the two methanol degradation pathways, the genome was also analyzed to assess genes encoding other degradation pathways (Chapter 4). This analysis shows many genes present in D. kuznetsovii are also present in Pelotomaculum thermopropionicum. P. thermopropionicum is known to degrade propionate in syntrophic interaction with a methanogen. D. kuznetsovii can also degrade propionate, but only coupled to sulfate reduction and not in syntrophy with methanogens. Moreover, P. thermopropionicum is not able to reduce sulfate. D. kuznetsovii is the only close related, non-syntrophic, propionate degrader of which the genome is available. Therefore, a genome comparison was performed between D. kuznetsovii and P. thermopropionicum to define the differences between a non-syntrophic and a syntrophic lifestyle. D. kuznetsovii misses membrane bound protein complexes like hydrogenases and an extra-cytoplasmic formate dehydrogenase. In order to expand the analysis between non-syntrophs and syntrophs, more genomes of propionate- and butyrate-degrading bacteria were included (Chapter 6). This extended analysis shows that the genomes of non-syntrophs do not contain genes coding for an extra-cytoplasmic formate dehydrogenase, in contrast to all syntrophs included in the analysis. This indicates the importance of this protein complex and the importance of formate as an interspecies electron carrier in syntrophic degradation of propionate and butyrate. Thanks to the extra cytoplasmic formate dehydrogenase the syntrophic bacteria can couple the degradation of propionate and butyrate to formate production. Subsequently, the formate is utilized by methanogens to produce methane. This keeps the formate concentration low, which is necessary for the entire process to be energetically favorable.
On the involvement of host proteins in Cowpea mosaic virus intercellular spread
Hollander, P.W. den - \ 2014
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Jan van Lent. - Wageningen : Wageningen University - ISBN 9789462570986 - 146
plantenvirussen - koebonenmozaïekvirus - plasmodesmata - planteiwitten - pathogenesis-gerelateerde eiwitten - viruseiwitten - virale transporteiwitten - chaperoninen - uitschakelen van genexpressie - genexpressie - eiwittransport - eiwitexpressieanalyse - plant viruses - cowpea mosaic virus - plasmodesmata - plant proteins - pathogenesis-related proteins - viral proteins - movement proteins - chaperonins - gene silencing - gene expression - protein transport - proteomics
Abstract of thesis Paulus den Hollander entitled “On the involvement of host proteins in Cowpea mosaic virus intercellular spread”.
Defence: 18th of November 13.30 h
Intercellular spread of Cowpea mosaic virus (CPMV) occurs via movement tubules inserted into the cell wall spanning plasmodesmata (PD) channels. These tubules are composed solely of viral movement proteins (MPs) and carry virus particles to neighbouring cells. The insertion of movement tubules into PD requires severe structural modification of these channels. These modifications are thought to rely on the concerted action of host and viral proteins. The aim of this thesis research has been to identify those host factors involved in the intercellular transport of CPMV.
Movement tubules collected from protoplasts, isolated plants cells without cell walls, were analysed by tandem mass spectrometry. This analysis revealed that seven host proteins associated specifically with these movement structures. Among these tubule-associated host proteins were CDC48-type AAA-proteins, heat shock protein (HSP) 60 and HSP70 isoforms. These three proteins were selected for further analysis in order to test their potential role in CPMV infection and spread. Targeted gene silencing of either CDC48 or HSP70 proteins caused a reduction in viral titres and also reduced the spread of CPMV in infected leaves. These results show that altered expression of these two tubule-associated host proteins influences the viral infection cycle.
Furthermore, interactions between the MP of CPMV and both plasma membrane intrinsic proteins (PIPs) and plasmodesmata-located proteins (PDLPs) were investigated using fluorescently labelled fusion proteins. An interaction between the MP and PIP-isoforms could not be established, however, MPs were found to co-localise with PDLP1 in PD. Using fluorescence lifetime imaging (FRET-FLIM) the molecular interaction between PDLPs and viral MPs was established in PD. This interaction was, however, only found in plant tissues and was not observed in protoplasts. This suggests that the interaction is restricted to structural context of the PD. Further experiments in protoplasts showed that co-localisation of MPs with PDLP1 was not required for the formation of movement tubules in these cells, which supports the hypothesis that the role of PDLP in plant virus intercellular transport is restricted to the PD.
Vroege detectie van dracht bij koeien door Proteomics Biomerkers in melk = Early pregnancy detection using proteomics biomarkers in milk
Pas, M.F.W. te; Kruijt, L. ; Wit, A.A.C. de; Hulsegge, B. ; Riel, J.W. van; Heeres-van der Tol, J.J. ; Sulkers, H. ; Woelders, H. - \ 2014
Lelystad : Wageningen UR Livestock Research (Rapport / Wageningen UR Livestock Research nr. 747) - 10
melkkoeien - zwangerschap - eiwitexpressieanalyse - merkers - melkproductie - melkveehouderij - rundveehouderij - productiebeperkingen - optimalisatie - voortplantingsefficiëntie - dairy cows - pregnancy - proteomics - markers - milk production - dairy farming - cattle husbandry - production restrictions - optimization - reproductive efficiency
The aim of this study is to develop an accurate, fast, cheap, and reliable test to detect pregnancy before day 35 on the basis of markers in milk. The ultimate goal is to have a method that can be implemented in a practical setting.
Glossina hytrosavirus control strategies in tsetse fly factories: application of infectomics in virus management
Kariithi, H.M. - \ 2013
Wageningen University. Promotor(en): Just Vlak; Monique van Oers, co-promotor(en): A.M.M. Abd-Alla; G.A. Murilla. - Wageningen : Wageningen UR - ISBN 9789461737533 - 207
glossina pallidipes - dierenvirussen - speekselklierziekten - massakweek - virusziekten - ziektebestrijding - eiwitexpressieanalyse - genomica - glossina pallidipes - animal viruses - salivary gland diseases - mass rearing - viral diseases - disease control - proteomics - genomics
African trypanosomosis is a fatal zoonotic disease transmitted by tsetse flies (Diptera; Glossinidae); blood-sucking insects found only in sub-Saharan Africa. Two forms of trypanosomoses occur: the animal African trypanosomosis (AAT; nagana), and the human African trypanosomosis (HAT; sleeping sickness). Since there are no effective vaccines against trypanosomosis, tsetse fly eradication is the most effective disease control method. Tsetse flies can be effectively eradicated by the sterile insect technique (SIT), which is applied in an area-wide integrated pest management approach. SIT is an environmentally benign method with a long and solid record of accomplishments. SIT requires large-scale production of sexually sterilized male flies (by exposure to a precise and specific dose of ionizing radiation, usually from a 60Co or 137Ce source), which are sequentially released into a target wild insect population to out-compete wild type males in inseminating wild virgin females. Once inseminated by sterile males, the virgin females do not produce viable progeny flies. Importantly, these females do not typically re-mate. Ultimately, the target wild insect population can decrease to extinction. However, tsetse SIT programs are faced with a unique problem: laboratory colonies of many tsetse species are infected by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae). GpSGHV-infected flies have male aspermia or oligospermia, underdeveloped female ovarioles, sterility, salivary gland hypertrophy syndrome (SGH), distorted sex ratios, and reduced insemination rates. Without proper management, symptomatic GpSGHV infections (characterized by SGH symptoms) can cause collapse of Glossina colonies. To ensure colony productivity and survival, GpSGHV management strategies are required. This will ensure a sustained supply of sterile males for SIT programs. The aim of this PhD research was to investigate the functional and structural genomics and proteomics (infectomics) of GpSGHV as a prerequisite to development of rationally designed viral control strategies. A series of experiments were designed to: (i) investigate epidemiology and diversity of GpSGHV; (ii) identify GpSGHV proteome and how viral and host proteins contribute to the pathobiology of the virus; and (iii) investigate the interplay between GpSGHV, the microbiome and the host, and how these interactions influence the outcomes of viral infections. By relating GpSGHV and host infectomics data, cost-effective viral management strategies were developed. This resulted in significant reduction of GpSGHV loads and elimination of SGH from laboratory colonies of G. pallidipes.
|Control of Pig Reproduction IX
Rodriguez-Martinez, H. ; Soede, N.M. ; Flowers, W.L. - \ 2013
Leicestershire, United Kingdom : Context Products Ltd (Society of Reproduction and Fertility volume 68) - ISBN 9781899043484 - 345
varkens - geslachtelijke voortplanting - gameten - embryo's - kunstmatige inseminatie - embryotransplantatie - zwangerschap - partus - pasgeborenen - biggen - overleving - biotechnologie - metabolomica - eiwitexpressieanalyse - kunstmatige selectie - pigs - sexual reproduction - gametes - embryos - artificial insemination - embryo transfer - pregnancy - parturition - neonates - piglets - survival - biotechnology - metabolomics - proteomics - artificial selection
Proteomic and mechanistic analysis of Auxin Response Factors in the Arabidopsis embryo
Llavata Peris, C.I. - \ 2013
Wageningen University. Promotor(en): Dolf Weijers. - S.l. : s.n. - ISBN 9789461736734 - 143
arabidopsis - auxinen - plantengroeiregulatoren - reacties - eiwitexpressieanalyse - genexpressie - embryonale ontwikkeling - embryogenese - arabidopsis - auxins - plant growth regulators - responses - proteomics - gene expression - embryonic development - embryogenesis
Auxin is a phytohormone that is crucial for many aspects of plant development. The processes in which this hormone has been implicated span from embryo development to flower transition, defense, tropic responses, and many other processes during plant life. A key question in auxin biology is how this molecule is able to elicit such diverse responses. Auxin regulates the transcriptional activation or repression of genes through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. In my studies I focus in the ARF transcription factors as a likely source of variation in output specificity. We consider three levels at which ARFs differ. First, ARFs differ in their ability to interact with different Aux/IAA (antagonistic family of transcription factors), or to form homo- or heterodimers. Second, ARFs assemble into different protein complexes, transcription factors interact with other transcriptional regulators or other proteins to form transcription complexes. These, when different, may contribute to different functions of ARF complexes. Thirdly, ARFs bind to and regulate different target genes. My work offers a plausible explanation how specific auxin responses are generated and through which genes the developmental responses to auxin are generated.
Systems biology and statistical data integration of ~omics data sets
Acharjee, A. - \ 2013
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Chris Maliepaard. - S.l. : s.n. - ISBN 9789461735843 - 177
systeembiologie - statistische gegevens - gegevensanalyse - gegevens verzamelen - metabolomica - loci voor kwantitatief kenmerk - genomica - eiwitexpressieanalyse - solanum tuberosum - aardappelen - databanken - systems biology - statistical data - data analysis - data collection - metabolomics - quantitative trait loci - genomics - proteomics - solanum tuberosum - potatoes - databases
In this thesis quality traits of potato were related to different highly multivariate ~omics datasets containing information on proteins, primary and secondary metabolites and gene expression. The objectives were to explore and compare different statistical techniques that are able to quantify these relationships, and to identify components responsible for prediction of quality. We propose a strategy to integrate two or more of such datasets and to select subsets of predictive components. We used potato flesh colour as an example trait and identified metabolites and expressed genes that are associated with flesh colour. We identified two putative novel non-volatile glycosides of carotenoid-derived metabolites and a novel putative connection with the flavonoid pathway. From a gas chromatography data set we identified genetic factors underlying variation in primary metabolism and found the amino acid beta-alanine associated with starch content. Finally we performed an integrated analysis with gene expression, metabolites and proteomics data and present an approach to select a limited set of predictive genes, metabolites and proteins.
The biology of milk synthesis from a proteomics perspective
Lu, J. - \ 2013
Wageningen University. Promotor(en): Toon van Hooijdonk, co-promotor(en): Kasper Hettinga; Jacques Vervoort. - S.l. : s.n. - ISBN 9789461734600 - 177
melksynthese - melksecretie - melksamenstelling - oppervlakte-eiwitten - eiwitexpressieanalyse - variatie - milk synthesis - milk secretion - milk composition - surface proteins - proteomics - variation
Large variation in bovine milk composition of Dutch Holstein cows has been observed. The factors influencing the milk synthesis and secretion process in the mammary gland and the variations in this process lead to variation in milk composition. The understanding of milk synthesis was improved during the last decades, however, much is still unknown, especially with regard to lipid synthesis and secretion. In this research, a proteomics technique (FASP-Dimethyl labeling-NanoLC-Orbitrap-MS/MS) was used to identify and quantify low abundant proteins in milk. By using this proteomics technique combined with a metabolomics technique (1H-NMR), the variation in milk synthesis and secretion processes were shown as affected by several factors influencing milk composition: DGAT1 polymorphism, fat globule size and energy balance of the cow. Stomatin, cholesterol and cholesterol synthesis enzymes were shown to be important for milk synthesis and secretion in lactating cows in all the different studies. Cytoplasmic vesicle and cytoskeleton proteins were shown to be involved in milk fat globule secretion. These results led to new insights in milk synthesis and secretion.
From existing data to novel hypotheses : design and application of structure-based Molecular Class Specific Information Systems
Kuipers, R.K.P. - \ 2012
Wageningen University. Promotor(en): Vitor Martins dos Santos; G. Vriend, co-promotor(en): Peter Schaap. - S.l. : s.n. - ISBN 9789461733504 - 231
systeembiologie - bio-informatica - genomica - informatiesystemen - computerwetenschappen - databanken - datamining - eiwitten - eiwitexpressieanalyse - systems biology - bioinformatics - genomics - information systems - computer sciences - databases - data mining - proteins - proteomics
As the active component of many biological systems, proteins are of great interest to life scientists. Proteins are used in a large number of different applications such as the production of precursors and compounds, for bioremediation, as drug targets, to diagnose patients suffering from genetic disorders, etc. Many research projects have therefore focused on the characterization of proteins and on improving the understanding of the functional and mechanistic properties of proteins. Studies have examined folding mechanisms, reaction mechanisms, stability under stress, effects of mutations, etc. All these research projects have resulted in an enormous amount of available data in lots of different formats that are difficult to retrieve, combine, and use efficiently.
The main topic of this thesis is the 3DM platform that was developed to generate Molecular Class Specific Information Systems (3DM systems) for protein superfamilies. These superfamily systems can be used to collect and interlink heterogeneous data sets based on structure based multiple sequence alignments. 3DM systems can be used to integrate protein, structure, mutation, reaction, conservation, correlation, contact, and many other types of data. Data is visualized using websites, directly in protein structures using YASARA, and in literature using Utopia Documents. 3DM systems contain a number of modules that can be used to analyze superfamily characteristics namely Comulator for correlated mutation analyses, Mutator for mutation retrieval, and Validator for mutant pathogenicity prediction. To be able to determine the characteristics of subsets of proteins and to be able to compare the characteristics of different subsets a powerful filtering mechanism is available. 3DM systems can be used as a central knowledge base for projects in protein engineering, DNA diagnostics, and drug design.
The scientific and technical background of the 3DM platform is described in the first two chapters. Chapter 1 describes the scientific background, starting with an overview of the foundations of the 3DM platform. Alignment methods and tools for both structure and sequence alignments, and the techniques used in the 3DM modules are described in detail. Alternative methods are also described with the advantages and disadvantages of the various strategies. Chapter 2 contains a technical description of the implementation of the 3DM platform and the 3DM modules. A schematic overview of the database used to store the data is provided together with a description of the various tables and the steps required to create new 3DM systems. The techniques used in the Comulator, Mutator and Validator modules of the 3DM platforms are discussed in more detail.
Chapter 3 contains a concise overview of the 3DM platform, its capabilities, and the results of protein engineering projects using 3DM systems. Thirteen 3DM systems were generated for superfamilies such as the PEPM/ICL and Nuclear Receptors. These systems are available online for further examination. Protein engineering studies aimed at optimizing substrate specificity, enzyme activity, or thermostability were designed targeting proteins from these superfamilies. Preliminary results of drug design and DNA diagnostics projects are also included to highlight the diversity of projects 3DM systems can be applied to.
Project HOPE: a biomedical tool to predict the effect of a mutation on the structure of a protein is described in chapter 4. Project HOPE is developed at the Radboud University Nijmegen Medical Center under supervision of H. Venselaar. Project HOPE employs webservices to optimally reuse existing databases and computing facilities. After selection of a mutant in a protein, data is collected from various sources such as UniProt and PISA. A homology model is created to determine features such as contacts and side-chain accessibility directly in the structure. Using a decision tree, the available data is evaluated to predict the effects of the mutation on the protein.
Chapter 5 describes Comulator: the 3DM module for correlated mutation analyses. Two positions in an alignment correlate when they co-evolve, that is they mutate simultaneously or not at all. Comulator uses a statistical coupling algorithm to calculate correlated mutation analyses. Correlated mutations are visualized using heatmaps, or directly in protein structures using YASARA. Analyses of correlated mutations in various superfamilies showed that positions that correlate are often found in networks and that the positions in these networks often share a common function. Using these networks, mutants were predicted to increase the specificity or activity of proteins. Mutational studies confirmed that correlated mutation analyses are a valuable tool for rational design of proteins.
Mutator, the text mining tool used to incorporate mutations into 3DM systems is described in chapter 6. Mutator was designed to automatically retrieve mutations from literature and store these mutations in a 3DM system. A PubMed search using keywords from the 3DM system is used to preselect articles of interest. These articles are retrieved from the internet, converted to text, and parsed for mutations. Mutations are then grounded to proteins and stored in a 3DM database. Mutation retrieval was tested on the alpha-amylase superfamily as this superfamily contains the enzyme involved in Fabry’s disease: an x linked lysosomal storage disease. Compared to existing mutant databases, such as the HGMD and SwissProt, Mutator retrieved 30% more mutations from literature. A major problem in DNA diagnostics is the differentiation between natural variants and pathogenic mutations. To distinguish between pathogenic mutations and natural variation in proteins the Validator modules was added to 3DM. Validator uses the data available in a 3DM system to predict the pathogenicity of a mutant using, for example, the residue conservation of the mutants alignment position, side-chain accessibility of the mutant in the structure, and the number of mutations found in literature for the alignment position. Mutator and Validator can be used to study mutants found in disorder related genes. Although these tools are not the definitive solution for DNA diagnostics they can hopefully be used to increase our understanding of the molecular basis of disorders.
Chapter 7 and 8 describe applied research projects using 3DM systems containg proteins of potential commercial interest. A 3DM system for the a/b-beta hydrolases superfamily is described in chapter 7. This superfamily consists of almost 20,000 proteins with a diverse range of functions. Superfamily alignments were generated for the common beta-barrel fold shared by all superfamily members, and for five distinct subtypes within the superfamily. Due to the size and functional diversity of the superfamily, there is a lot of potential for industrial application of superfamily members. Chapter 8 describes a study focusing on a sucrose phosphorylase enzyme from the a-amylase superfamily. This enzyme can be potentially used in an industrial setting for the transfer of glucose to a wide variety of molecules. The aim of the study was to increase the stability of the protein at higher temperatures. A combination of rational design using a 3DM system, and in-depth study of the protein structure, led to a series of mutations that resulted in more than doubling the half-life of the protein at 60°C.
3DM systems have been successfully applied in a wide range of protein engineering and DNA diagnostics studies. Currently, 3DM systems are applied most successfully in project studying a single protein family or monogenetic disorder. In the future, we hope to be able to apply 3DM to more complex scenarios such as enzyme factories and polygenetic disorders by combining multiple 3DM systems for interacting proteins.
Functional genomics of chilo iridescent virus: a transcriptoproteomic approach
Ince, I.A. - \ 2012
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Monique van Oers. - S.l. : s.n. - ISBN 9789461731449 - 114
invertebrate iridescent virus 6 - iridovirus - insectenvirussen - functionele genomica - transcriptomica - eiwitexpressieanalyse - dierenvirussen - biologische bestrijding - virale insecticiden - invertebrate iridescent virus 6 - iridovirus - insect viruses - functional genomics - transcriptomics - proteomics - animal viruses - biological control - viral insecticides
Iridoviruses are disease causing agents in (pest) insects, fishes and amphibians with serious ecological and economic impacts. Insight in the composition of the virions and the transcriptional regulation of the virion protein genes is crucial to unravel the biology of this lesser known family of viruses. In this thesis, the virions of Chilo iridescent virus (CIV) (genus Iridovirus) were analyzed by mass spectrometry, revealing 54 virion proteins. A novel transcriptomic approach for non-polyadenylated RNA transcripts, called LACE, was developed and applied to unravel the temporal class of the virion protein genes. This showed that many virion protein genes were expressed as early genes. Another intriguing finding is that an infected cell-specific 100 kDa protein interacted with a crucial delayed-early promoter motif in the DNA polymerase gene and it turned out that this motif was conserved in other (putative) delayed early genes in CIV and other iridoviruses. The hypothesis is that this 100 kDa protein is responsible for transcriptional activation of delayed-early genes. CIV is an example of an invertebrate iridoviruses that deals with induction and inhibition of apoptosis during infection. In this study, a gene for a functional inhibitor of apoptosis (193R), unique for an iridovirus, was identified. In addition, several candidates for pro-apoptotic proteins were found in the virion. In this dissertation fundamental knowledge was obtained on the proteome of CIV virions and the regulation of CIV gene expression. Due to the development and application of novel technics, this thesis provides new venues to answer remaining questions concerning the infection cycle of this interesting iridovirus.
Iridovirus, transcriptomics, proteomics, virus-host interaction
Genome structure and pathogenicity of the fungal wheat pathogen Mycosphaerella graminicola
M'Barek, S. Ben - \ 2011
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Gert Kema. - [S.l.] : S.n. - ISBN 9789085859970 - 229
triticum aestivum - triticum turgidum - tarwe - mycosphaerella graminicola - plantenziekteverwekkende schimmels - genoomstructuur - eiwitexpressieanalyse - pathogeniteit - pathogenesis-gerelateerde eiwitten - genomen - plasticiteit - plant-microbe interacties - triticum aestivum - triticum turgidum - wheat - mycosphaerella graminicola - plant pathogenic fungi - genomic structure - proteomics - pathogenicity - pathogenesis-related proteins - genomes - plasticity - plant-microbe interactions
The phytopathogenic fungus Mycosphaerella graminicola (Fuckel) J. Schröt. in Cohn (asexual stage: Zymoseptoria tritici (Desm.) Quaedvlieg & Crous) causes septoria tritici leaf blotch (STB) in wheat and is one of the most important diseases of this crop worldwide. However, STB control, mainly based on the use of resistant cultivars and fungicides, is significantly hampered by the limited understanding of the genetic and biochemical bases of pathogenicity, and mechanisms of infection and resistance in the host. M. graminicola has a very active sexual cycle under field conditions, which is an important driver of STB epidemics. Moreover, it results in high genetic diversity of field populations that causes a major challenge for the development and sustainable management of resistant cultivars and the discovery of new antifungal compounds. Understanding the role of the sexual and asexual life cycles on genome composition of this versatile pathogen and its infection strategy is crucial in order to develop novel control methods.
Chapter 1 is an introduction to the biology and pathogenicity of M. graminicola. In addition, it shortly describes the impact of improved and novel technologies on the speed, scope and scale of comparative genomics research.
Chapter 2 provides detailed genetic analyses of two M. graminicola mapping populations, using mainly DArT markers, and the analysis of the meiotic transmission of unequal chromosome numbers. Polymorphisms in chromosome length and number were frequently observed in progeny isolates, of which 15–20% lacked one or more chromosomes despite their presence in one or both parents, but these had no apparent effect on sexual and pathogenic fitness. M. graminicola has up to eight so called dispensable chromosomes that can be easily lost - collectively called the dispensome - which is, so far, the highest number of dispensable chromosomes reported in filamentous fungi. They represent small-sized chromosomes and make up 38% of the chromosome complement of this pathogen. Much of the observed genome plasticity is generated during meiosis and could explain the high adaptability of M. graminicola in the field. The generated linkage map was crucial for finishing the M. graminicola genome sequence.
Chapter 3 describes the M. graminicola genome sequence with highlights on genome structure and organization including the eight dispensable chromosomes. The genome comprises a core set of 13 chromosomes and a dispensome, consisting of eight chromosomes that are distinct from the core chromosomes in structure, gene and repeat content. The dispensome contains a higher frequency of transposons and the genes have a different codon use. Most of the genes present one the dispensome are also present on the core chromosomes but little synteny is observed neither between the M. graminicola dispensome and the core chromosomes nor with the chromosomes of other related Dothideomycetes. The dispensome likely originates from ancient horizontal transfer(s) from (an) unknown donor(s).
Chapter 4 shows a global analysis of proteins secreted by M. graminicola in apoplastic fluids during infection. It focuses mainly on fungal proteins secreted in a compatible interaction. The study showed that many of the annotated secreted proteins have putative functions in fungal pathogenicity, such as cell wall degrading enzymes and proteases, but the function of a substantial number of the identified proteins is unknown. During compatible interactions proteins are primarily secreted during the later stages. However, many pathogenesis-related host proteins, such as PR-2, PR-3 and PR-9, accumulated earlier and at higher concentrations during incompatible interactions, indicating that fungal effectors are recognized by resistant plants and trigger resistant gene-mediated defence responses, though without a visible hypersensitive response.
Chapter 5 further details the initial identification and characterization of necrosis-inducing proteins that are produced in culture filtrates (CFs) of M. graminicola. The necrosis-inducing activity of CFs is light dependent and inactivated by proteinase K and heat treatment (100C). This is reminiscent of the necrosis-inducing properties of host selective toxins of other Dothideomycete pathogens such as Stagonospora nodorum and Pyrenophora tritici-repentis. Subsequent purifications of CFs and mass spectrometry identified several candidate proteins with necrosis-inducing activity. Heterologous expression of the two most prominent proteins in Pichia pastoris produced sufficient quantities for infiltration assays in a panel of wheat cultivars that showed differential responses, suggesting specific recognition.
Chapter 6 provides a general discussion of the thesis and puts the results obtained in a broader perspective with a focus on the genome structure of M. graminicola and its function. In addition, aspects of the hemi-biotrophic lifestyle, the relevance of secreted proteins for the wheat-M. graminicola pathosystem in relation to gene-for-gene models and the potential implications for resistance breeding strategies are discussed.
Functional aspects of baculovirus DNA photolyases
Xu, F. - \ 2010
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Monique van Oers. - [S.l. : S.n. - ISBN 9789085857730 - 112
baculoviridae - organismen ingezet bij biologische bestrijding - insectenplagen - fotolyse - chrysodeixis chalcites - kernpolyedervirussen - lyasen - genen - genexpressie - fylogenetica - lokalisatie - ultraviolette straling - eiwitexpressieanalyse - gevoeligheid - baculoviridae - biological control agents - insect pests - photolysis - chrysodeixis chalcites - nuclear polyhedrosis viruses - lyases - genes - gene expression - phylogenetics - localization - ultraviolet radiation - proteomics - sensitivity
Keywords: baculovirus, ChchNPV, CPD photolyase, phylogeny, UV resistance, DNA binding, localization, proteomics
Baculoviruses are insect viruses that are applied as biological control agents due to adequate virulence, host specificity and safety for the environment. Solar light negatively affects field performance of baculoviruses by reducing their infectivity, most likely as a consequence of the formation of cyclobutane pyrimidine dimers (CPDs) in the viral DNA upon ultraviolet (UV) irradiation. CPDs can be repaired by CPD photolyases when exposed to blue light photons, a process called photoreactivation. From previous work it was known that the Cc-phr2 gene of the baculovirus Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) encodes a biochemically active photolyase. The research in this thesis focuses on (i) the degree of conservation of CPD photolyase (phr) genes in a subgroup of baculoviruses, (ii) the localization of baculovirus photolyase proteins in insect cells and occlusion derived virus (ODV), and (iii) the in vivo effect of phr genes on the UV sensitivity of baculoviruses. Homologues of the Cc-phr genes were found in all studied group II NPVs in the genus Alphabaculovirus that infect insects in the subfamily Plusiinae insects. Phylogenetic analysis suggested that these phr-like genes have a common ancestor. Intracellular localization of the two ChchNPV encoded PHR proteins in insect cells was studied using enhanced GFP fusion. Both PHR1 and PHR2 localized in the nucleus and associated with chromosomes, spindle, aster and midbody structures during host cell mitosis. Moreover, Cc-PHR2 co-localized with virogenic stroma, when PHR2-EGFP-transfected cells were infected with Autographa californica (Ac) MNPV. Neither of the two Cc-PHR proteins was identified by LC/MS-MS in the ODVs of ChchNPV. To evaluate the potential of the Cc-PHR2 protein to reduce the UV sensitivity of a baculovirus, the Cc-phr2 gene was incorporated in the genome of Helicoverpa armigera (Hear) NPV, which does not have a UV damage repair system. This resulted in a decreased sensitivity to UV-light compared to wild type HearNPV. A cell line was established from embryos of the insect C. chalcites. This cell line was shown to be permissive for both ChchNPV and the related Trichoplusia ni NPV (TnSNPV). This novel cell line will be a useful tool for making ChchNPV phr mutant viruses to study the impact of DNA repair mediated by photolyases on baculovirus ecology. The collected data support the hypothesis that the Cc- phr2 gene provides a baculovirus with an ecological benefit by increasing the resistance to UV.
Functional genomics analysis of the secretory pathway in Aspergillus niger
Oliveira, J.M. - \ 2010
Wageningen University. Promotor(en): John van der Oost, co-promotor(en): Leo de Graaff. - [S.l. : S.n. - ISBN 9789085857693 - 191
aspergillus niger - eiwitsecretie - genexpressie - eiwitten - transcriptie - microsomen - genexpressieanalyse - functionele genomica - eiwitexpressieanalyse - gene silencing - aspergillus niger - protein secretion - gene expression - proteins - transcription - microsomes - genomics - functional genomics - proteomics - gene silencing
Filamentous fungi can be found in the majority of habitats of our planet. The wide-spread presence of filamentous fungi is related to their versatile metabolism, which allows them to grow on simple substrates, such as nitrate, acetate, ethanol, ammonia, or on complex matter such as biopolymers from plant or animal tissues. In order to grow on complex biopolymers such as plant cell wall polysaccharides, fungi must secret hydrolytic and modifying enzymes. These enzymes allow polysaccharide degradation and subsequent internalization of simpler molecules, such as sugar monomers.
The filamentous fungus Aspergillus niger has been the subject of intense research in the past decades. This organism is responsible for the largest production of citric acid worldwide. In addition to this, A. niger produces high amounts of enzymes with important applications in the bioindustry, such as enzymes for food and feed processing, or enzymes used for the simultaneous saccharification and fermentation of cellulose for bioethanol production. The secretion of extracellular enzymes in A. niger has been mostly focused on the prediction of gene function based on genome annotation and on the analysis of gene expression. However, there is a gap in the knowledge of all the proteins present in cell, given by proteomics.
The aim of the work presented in this thesis was to use a functional genomics approach to identify genes and proteins involved in protein secretion in A. niger and to investigate the dynamic changes of the secretory proteome under high-secretion conditions. For this purpose, we used a combination of gene expression profiling with shotgun proteomics of secretory organelles.
Chapter 2 describes a method for gene silencing in filamentous fungi via RNA interference. This method makes use of vectors which express long hairpin RNAs. In A. niger, gene knock-out strategies have been the main method for the determination of gene function. These strategies have proven to be particularly useful when carried out in strains with defective pathways for non-homologous integration, such as the kusA mutant. Nevertheless, a gene knock-down strategy such as the one described in chapter 2 could be relevant for the study of gene function, for two reasons: a) essential genes could be studied as RNAi does not necessarily lead to loss-of-function, and b) multiple gene copies of a gene or paralogous genes could be targeted with a single construct. In our work, the gene coding the transcriptional activator of hemicellulases XlnR was silenced. Gene silencing resulted in various degrees of hemicellulase production depending on the different transformed fungal strains.
In chapter 3, the effect of D-xylose on gene expression in A. niger was investigated. The inducer of (hemi)cellulases D-xylose was added to cultures of A. niger growing on the non-inducer sorbitol. Genes differentially expressed on D-xylose were identified as candidate genes involved in the response to this sugar. This study confirmed that D-xylose activates enzymes involved in xylan degradation and D-xylose utilisation, but also enzymes responsible for the removal of other monomers that occurr on arabinoxylan and cellulases. Statistical analysis of variance components was used to assess the contribution of each external factors affecting the measured gene expression. Such analysis of variance components is important for reproducible sample processing for microarray analysis.
Chapter 4 describes the A. niger secretory pathway proteins that are involved in the production of (hemi)cellulases, via induction by D-xylose. For this, A. niger was grown under the same conditions as the ones described in chapter 3. After the isolation of microsomes, the corresponding proteins were analysed by shotgun proteomics. Induction by D-xylose was correlated with an increase in proteins related to protein secretion, namely small GTPases for vesicle transport and polarised growth. Most importantly, under induction by D-xylose, the complex for protein degradation 20S proteasome was associated with microsomes. These results indicate a novel mode of regulation in which the proteasome is recruited to secretory organelles upon the induction of extracellular enzymes.
In chapter 5, the analysis of secretory proteins described in chapter 4 is now applied to a system in which D-maltose is an inducer of starch-degrading enzymes. This chapter also includes the study of the proteins secreted after D-maltose or D-xylose. After D-maltose addition, three starch-degrading enzymes were found more abundant and after D-xylose addition, several enzymes were more abundant and these enzymes were mostly related to arabinoxylan and cellulose degradation. The effects of D-maltose on the microsomal proteome are similar to the effects of D-xylose. Both the induction by D-maltose and by D-xylose resulted in increased amounts of mitochondrial proteins. Moreover, the 20S proteasome assembly is an ATP-dependent process. For this reason, it is hypothesised that the assembly and association of 20S proteasome upon induction is related to an increased ATP production in the vicinity of secretory organelles.
Unravelling the mechanism of differential biological responses induced by food-borne xeno- and phyto-estrogenic compounds
Sotoca Covaleda, A.M. - \ 2010
Wageningen University. Promotor(en): Ivonne Rietjens; Tinka Murk, co-promotor(en): Jacques Vervoort. - [S.l. : S.n. - ISBN 9789085857075 - 186
oestrogenen - plantenoestrogenen - synthetische oestrogenen - oestrogeenreceptoren - genexpressie - eiwitexpressieanalyse - transcriptomics - oestrogens - plant oestrogens - synthetic oestrogens - oestrogen receptors - gene expression - proteomics - transcriptomics
The multiple actions of estradiol and other estrogenic compounds in mammalian physiology are brought about, on a molecular level, as a result of complex signalling pathways, and mediated by at least two receptors namely estrogen receptor (ER) α and ERβ.
The aim of the work presented in this thesis was to obtain insight in the role of ERα, ERβ and the ratio of ERα/ERβ present within a cell, in the cellular response to estrogen-like compounds. To this end, this thesis addressed the transcriptional activity at both the gene and protein level and effects on cell proliferation under the influence of specifically-acting estrogen-like molecules when varying the ratio of ERα/ERβ present in the cells under study. The ultimate aim was to link the data on cell proliferation as the biological end-point to the transcriptomics and proteomics data.