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- Mukherjee, Vaskar, 1986, et al.
(författare)
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High throughput screening of yeast strains for desirable stress tolerant traits for bioethanol production
- 2013
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Ingår i: Yeast. - : Wiley. - 0749-503X.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Implementation of very high gravity (VHG) fermentation technology in second generation bioethanol production using raw lignocellulosic biomass is fundamental to establish a commercially viable plant. However, so far the application of this technology is greatly restricted by the unavailability of a fermentative microorganism, resistant enough to the wide variety of stressors commonly encountered in VHG fermentation. In addition, the appropriate tools and knowledge to select such multi-stress tolerant microorganisms and to make a scientifically proven choice of the appropriate candidate strains have been lacking until recently. In this study we screened a large yeast culture collection, consisting of about 700 Saccharomyces cerevisiae and non-Saccharomyces strains from diverse origins, for different desirable traits for bioethanol production. These included, for example, osmotolerance, halotolerance, ethanol tolerance, thermotolerance, and tolerance against fermentation inhibitors like furfural and hydroxymethyl furfural as well as some heavy metals. To this end, a high throughput semi-automated robot was used for spotting up to 96 strains per screening plate. After incubation, plates were scanned and growth was recorded and analyzed using dedicated software. Cluster analysis showed clear differences in tolerance among species and among strains of the same species. In addition, strains showing co-tolerance against different traits could be identified. As such, our study enabled to efficiently select top candidate strains having desirable traits for VHG bioethanol production.
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| 2. |
- Philip, Philge, 1983-
(författare)
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Mining DNA elements involved in targeting of chromatin modifiers
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: In all higher organisms, the nuclear DNA is condensed into nucleosomes that consist of DNA wrapped around a core of highly conserved histone proteins. DNA bound to histones and other structural proteins form the chromatin. Generally, only few regions of DNA are accessible and most of the time RNA polymerase and other DNA binding proteins have to overcome this compaction to initiate transcription. Several proteins are involved in making the chromatin more compact or open. Such chromatin-modifying proteins make distinct post-translational modifications of histones – especially in the histone tails – to alter their affinity to DNA. Aim: The main aim of my thesis work is to study the targeting of chromatin modifiers important for correct gene expression in Drosophila melanogaster (fruit flies). Primary DNA sequences, chromatin associated proteins, transcription, and non-coding RNAs are all likely to be involved in targeting mechanisms. This thesis work involves the development of new computational methods for identification of DNA motifs and protein factors involved in the targeting of chromatin modifiers. Targeting and functional analysis of two chromatin modifiers, namely male-specific lethal (MSL) complex and CREB-binding protein (CBP) are specifically studied. The MSL complex is a protein complex that mediates dosage compensation in flies. CBP protein is known as a transcriptional co-regulator in metazoans and it has histone acetyl transferase activity and CBP has been used to predict novel enhancers. Results: My studies of the binding sites of MSL complex shows that promoters and coding sequences of MSL-bound genes on the X-chromosome of Drosophila melanogaster can influence the spreading of the complex along the X-chromosome. Analysis of MSL binding sites when two non-coding roX RNAs are mutated shows that MSL-complex recruitment to high-affinity sites on the Xchromosome is independent of roX, and the role of roX RNAs is to prevent binding to repeats in autosomal sites. Functional analysis of MSL-bound genes using their dosage compensation status shows that the function of the MSL complex is to enhance the expression of short housekeeping genes, but MSL-independent mechanisms exist to achieve complete dosage compensation. Studies of the binding sites of the CBP protein show that, in early embryos, Dorsal in cooperation with GAGA factor (GAF) and factors like Medea and Dichaete target CBP to its binding sites. In the S2 cell line, GAF is identified as the targeting factor of CBP at promoters and enhancers, and GAF and CBP together are found to induce high levels of polymerase II pausing at promoters. In another study using integrated data analysis, CBP binding sites could be classified into polycomb protein binding sites, repressed enhancers, insulator protein-bound regions, active promoters, and active enhancers, and this suggested different potential roles for CBP. A new approach was also developed to eliminate technical bias in skewed experiments. Our study shows that in the case of skewed datasets it is always better to identify non-altered variables and to normalize the data using only such variables.
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