Perception of competition : A measurement of competition from the perspective of the firm

In this report, we study competition from a cognitive psychology, marketing and strategic management perspective and hope to contribute to the notion of competition and competitive processes. In addition, we propose a new method to measure competition that is based on these more psychological insights.��

Ultrafiltration of protein solutions; the role of protein association in rejection and osmotic pressure

The monomer-dimer equilibrium of the protein β-lactoglobulin under neutral conditions appears to influence the rejection and the osmotic pressure build-up, both phenomena closely related to ultrafiltration. Rejection measurements indicate different rejections for the β-lactoglobulin monomers and dimers: the membrane rejects the dimer almost completely and the monomer only partially. The osmotic pressure turns out to be highly dependent on the protein concentration. A good agreement, up to high concentrations, is found between experimental data and theoretical osmotic pressures, calculated by taking into account the state of association, the excluded volume and the Donnan effects. The effect of changes in pH on the osmotic pressure has been measured: a minimum was found around pH = 4.5, where according to the literature, maximum protein-protein interaction occurs

Exploring the potential of metabolic models in the study of microbial ecosystems

Teusink, B. [Promotor]Roling, W.F.M. [Copromotor

Abundance of conserved CRISPR-Cas9 target sites within the highly polymorphic genomes of Anopheles and Aedes mosquitoes.

A number of recent papers report that standing genetic variation in natural populations includes ubiquitous polymorphisms within target sites for Cas9-based gene drive (CGD) and that these "drive resistant alleles" (DRA) preclude the successful application of CGD for managing these populations. Here we report the results of a survey of 1280 genomes of the mosquitoes Anopheles gambiae, An. coluzzii, and Aedes aegypti in which we determine that ~90% of all protein-encoding CGD target genes in natural populations include at least one target site with no DRAs at a frequency of ≥1.0%. We conclude that the abundance of conserved target sites in mosquito genomes and the inherent flexibility in CGD design obviates the concern that DRAs present in the standing genetic variation of mosquito populations will be detrimental to the deployment of this technology for population modification strategies

Structural studies on dihydrolipoyl transacetylase : the core component of the pyruvate dehydrogenase complex of Azotobacter vinelandii

The studies described in this thesis deal with the structure of the Azotobactervinelandii dihydrolipoyl transacetylase, the core component (E 2 ) of the pyruvate dehydrogenase complex. in all organisms the pyruvate dehydrogenase complex is closely related to the 2-oxoglutarate dehydrogenase complex and, if present, the branched-chain 2-oxoacid dehydrogenase complex. These enzyme complexes are large multimeric structures. The smallest known is the pyruvate dehydrogenase complex from A.vinelandii , Upon resolution of the other components, the tetrameric core component of this complex aggregates to a welldefined multimeric structure, resembling the structure from the large complexes from other organisms.. Therefore, it seems likely that the A.vinelandii complex could represent the model for the building unit of the large complexes from other organisms. Since the core component (E 2 ) carries all the information concerning the quaternary structure of the complex, we focussed our attention on this intriguing enzyme.The domain structure of E 2 has been examined by limited proteolysis of E 2 , as described in chapter 2. After limited proteolysis with trypsin two stable domains were obtained. The lipoyl domain carries the lipoyl groups which are concerned with the transport of the substrates between the active sites of the different components. The catalytic domain possesses the transacetylase active site and the E 2 -intersubunit binding sites, responsible for the quaternary structure of E 2 . The binding sites for the E 1 and E 3 components are lost during proteolysis.The cloning and sequencing of the gene encoding dihydrolipoyl transacetylase have been described in chapter 3. The gene, located downstream of the gene encoding the PDC E 1 component, does not possess an own promoter, but is probably regulated by the E 1 -promoter. The gene possesses a strong terminating sequence. Downstream the gene encoding E 2 no open reading frame, that codes for the E 3 component, has been identified, as has been found in E.coli . The primary structure of E 2 , derived from the DNA sequence, is homologous to that of E 2 from E.coli . The lipoyl domain, located at the N-terminus, is built from three repeating sequences, separated by regions which are very rich in alanine and proline residues. The catalytic domain, located at the C-terminus, comprises the transacetylase active site and the E 2 intersubunit binding sites. The region, located between the lipoyl and the catalytic domain contains many charged amino acid residues and is thought to possess the E 1 and E 3 binding sites. The expression of the gene encoding E 2 , located on plasmid pRA282 and cloned in E.coli , has been described in chapter 4. A high production of E 2 was obtained. The production raised dramatically when the cells were in the stationery phase of the growth-cycle. The percentage active E 2 varied strongly per culture. The inactivation was found to be caused by formation of intramolecular or intermolecular S-S-bridges, resulting in incorrect folding of the catalytic domain. An activation and an isolation procedure have been described.Mobility of the repeating units within the lipoyl domain has been studied using time-resolved fluorescence, as described in chapter 5. It has been shown that the repeats show no independent rotational mobility, but rotate as one unit, serving the active sites of the different components.Internal mobility within the lipoyl domain has been observed by 1H-NMR experiments, as described in chapter 6. Probably this internal mobility, that is ascribed to the alanine-proline rich region, does not result into an independent mobility of the three repeats. The catalytic domain, despite its compact structure, still possesses a certain amount of internal mobility. This can partly be ascribed to alanine and proline residues, probably the N-terminal region of the domain, which is rich in these residues. In the spectrum of E 2 sharp resonances have been observed that can be ascribed to mobility of the E 1 and E 3 binding domain. Such mobility has not been found after binding of E 1 and E 3 components, in the whole complex.The molecular mass of the native catalytic domain and of the single polypeptide chain have been determined, and from this and light-scattering and crosslinking experiments it has been concluded that the large multimeric structure of the isolated catalytic domain (and of E 2 ) is built from 24 subunits in contrast to a 32-meric structure as proposed previously. A model has been presented for the quaternary structure of E 2 , in which it is assumed that the multimeric E 2 -core is built from six tetrameric morphological subunits, forming the lateral faces of the cubic 24-mer.These tetrameric subunits represent the E2-core of the intact complex. Compared to other 2-oxoacid dehydrogenase complexes, the A.vinelandii PDC contains one additional binding site for E 1 per E 2 tetramer. It is assumed that this extra binding site becomes available during dissociation, resulting in the unique small PDC of A.vinelandii .</TT

Kansen en bedreigingen voor de grote steden in een aantal bedrijfstakken

Economic

Generation of bacteriophage-insensitive mutants of Streptococcus thermophilus using an antisense RNA CRISPR-Cas silencing approach

Predation of starter lactic acid bacteria such as Streptococcus thermophilus by bacteriophages is a persistent and costly problem in the dairy industry. CRISPR-mediated Bacteriophage Insensitive Mutants (BIMs), while straightforward to generate and verify, can quickly be overcome by mutant phages. The aim of this study was to develop a tool allowing the generation of derivatives of commercial S. thermophilus strains which are resistant to phage attack through a non-CRISPR-mediated mechanism, with the objective of generating BIMs exhibiting stable resistance against a range of isolated lytic S. thermophilus phages. To achieve this, standard BIM generation was complemented by the use of the wild-type (WT) strain which had been transformed with an antisense mRNA-generating plasmid (targeting a crucial CRISPR-associated [cas] gene], in order to facilitate the generation of non-CRISPR-mediated BIMs. Phage sensitivity assays suggest that non-CRISPR-mediated BIMs exhibit some advantages compared to CRISPR-mediated BIMs derived from the same strain.Importance: The outlined approach reveals the presence of a powerful host-imposed barrier for phage infection in S. thermophilus. Considering the detrimental economic consequences of phage infection in the dairy processing environment, the developed methodology has widespread applications, particularly where other methods may not be practical or effective in obtaining robust, phage-tolerant S. thermophilus starter strains

Mobile sequences in the pyruvate dehydrogenase complex, the E2 component, the catalytic domain and the 2-oxoglutarate dehydrogenase complex of Azotobacter vinelandii, as detected by 600 MHz 1H-NMR spectroscopy

Abstract600 MHz 1H-NMR spectroscopy demonstrates that the pyruvate dehydrogenase complex of Azotobacter vinelandii contains regions of the polypeptide chain with intramolecular mobility. This mobility is located in the E2 component and can probably be ascribed to alanine-proline-rich regions that link the lipoyl subdomains to each other as well as to the E1 and E3 binding domain. In the catalytic domain of E2, which is thought to form a compact, rigid core, also conformational flexibility is observed. It is conceivable that the N-terminal region of the catalytic domain, which contains many alanine residues, is responsible for the observed mobility. In the low-field region of the 1H-NMR spectrum of E2 specific resonances are found, which can be ascribed to mobile phenylalanine, histidine and/or tyrosine residues which are located in the E1 and E3 binding domain that links the lipoyl domain to the catalytic domain. In the 1H-NMR spectrum of the intact complex, these resonances cannot be observed, indicating a decreased mobility of the E1 and E3 binding domain