Functional genomics analysis of the secretory pathway in Aspergillus niger

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. <br/

Efficient cloning system for construction of gene silencing vectors in Aspergillus niger

An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two semi-quantitative plate assays testing different activities for xylan degradation. Upon induction on D-xylose, transcript levels of xlnR were decreased in the xlnR-silenced strains, compared to a wild-type background. Under these conditions, the transcript levels of xyrA and xynB (two genes regulated by XlnR) were also decreased for these xlnR-silenced strains. These results indicate that the newly developed system for rapid generation of silencing vectors is an effective tool for A. niger, and this can be used to generate strains with a tailored spectrum of enzyme activities or product formation by silencing specific genes encoding, e.g., regulators such as Xln

Disruption Management in Airline Operations Control – An Intelligent Agent-Based Approach

Operations control is one of the most important areas for an airline company. Through operations control mechanisms an airline company monitors all the flights checking if they follow the schedule that was previously defined by other areas of the company. Unfortunately, some problems may arise during this stage (Clausen et al., 2005). Those problems can be related with crewmembers, aircrafts and passengers. The Airline Operations Control Centre (AOCC) includes teams of experts specialized in solving the above problems under the supervision of an operation control manager. Each team has a specific goal contributing to the common and general goal of having the airline operation running under as few problems as possible. The process of solving these kinds of problems is known as Disruption Management (Kohl et al., 2004) or Operations Recovery

Market segmentation in behavioral perspective

A segmentation approach is presented using both traditional demographic segmentation bases (age, social class/occupation, and working status) and a segmentation by benefits sought. The benefits sought in this case are utilitarian and informational reinforcement, variables developed from the Behavioral Perspective Model (BPM). Using data from 1,847 consumers and from a total of 76,682 individual purchases, brand choice and price and reinforcement responsiveness were assessed for each segment across the UK cookie (biscuits) market. Building on previous work, the results suggest that the segmentation of brand choice using benefits sought is useful. This is especially the case alongside demographic variables. This article provides a theoretical and practical segmentation approach to both the behavioral psychology literature and the wider marketing segmentation literature

Consumer brand choice: Money allocation as a function of brand reinforcing attributes

Previous applications of the matching law to the analysis of consumer brand choice have shown that the amount of money spent purchasing a favorite brand tends to match the quantity bought of the favorite brand divided by the quantity bought of all other brands. Although these results suggest matching between spending and purchased quantity, branded goods differ qualitatively among themselves, rendering previous matching analyses incomplete. Consumer panel data containing information about more than 1,500 British consumers purchasing four grocery product categories (baked beans, biscuits, fruit juice, and yellow fats) during 52 weeks were analyzed. All the brands purchased were classified according to the level of informational and utilitarian reinforcement they were programmed to offer. An adaptation of the generalized matching law was adopted, in which the amount of money spent was a power function of the quantity bought, informational level of the brand bought, utilitarian level of the brand bought, and a measure of price promotion

Determination of Odorants in Varietal Wines from International Grape Cultivars (Vitis vinífera) Grown in NW Spain

This work was carried out to investigate the odorants found in ten varietal wines from different internationalgrape cultivars (Merlot, Cabernet Sauvignon, Pinot noir, Tempranillo, Sauvignon blanc, Riesling,Chardonnay, Pinot gris, Pinot blanc and Gewürztraminer) grown in northwest Spain. Monoterpenes,alcohols, fatty acids, ethyl esters, acetates and volatile phenols were determined by gas chromatographymassspectrometry (GC-MS). The results showed that Gewürztraminer white wines had the highestconcentration of volatile compounds (35.7 mg/L). Monoterpenes, linalool, terpineol, citronellol and nerolwere detected only in Riesling and Gewürztraminer white wines. In the red wines, Cabernet Sauvignonfollowed by Merlot wines showed the highest concentration of total volatile composition (55.60 mg/L and50.90 mg/L respectively), characterised by a higher concentration of alcohols. Based on the individual odourthreshold, white Gewürztraminer and red Pinot noir wines showed the highest total OAV value. ANOVAhas shown significant differences among wines. Principal component analysis performed a grouping ofthe monovarietal wines – Sauvignon blanc-Pinot blanc-Riesling and Pinot gris gris-Gewürztraminer in thewhite wines, and Cabernet Sauvignon-Tempranillo in the red wines

Roughness of Sandpile Surfaces

We study the surface roughness of prototype models displaying self-organized criticality (SOC) and their noncritical variants in one dimension. For SOC systems, we find that two seemingly equivalent definitions of surface roughness yields different asymptotic scaling exponents. Using approximate analytical arguments and extensive numerical studies we conclude that this ambiguity is due to the special scaling properties of the nonlinear steady state surface. We also find that there is no such ambiguity for non-SOC models, although there may be intermediate crossovers to different roughness values. Such crossovers need to be distinguished from the true asymptotic behaviour, as in the case of a noncritical disordered sandpile model studied in [10].Comment: 5 pages, 4 figures. Accepted for publication in Phys. Rev.

Consumer Behaviour Analysis and Consumer Brand Choice

This is the SECOND of TWO linked articles on consumer behavioural analysis dealing with the Behavioural Perspective Model, which locates consumer behaviour at the intersection of the consumer’s learning history and the consumer situation. As an example of research inspired by the Model, this article presents investigations into consumers’ patterns of brand choice, showing how brand repertoires are formed and how brands are selected within those repertoires