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The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S. pastorianus remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple non-targeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same set-up yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid S. pastorianus genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement., BT/Industrial Microbiology, BT/Biotechnology

The first production of alcoholic beverages by fermentation of plant-derived materials occurred many thousands of years ago. In the 15th century, production of lager beer with pale barley malt, soft water and Saaz noble hops, first resulted in pilsner, a clear golden, crispy tasting beer. Today, this type of lager beer is the most produced alcoholic beverage in the world. The yeast responsible for conversion of wort sugars into ethanol, CO2 and aromatic compounds is S. pastorianus, a hybrid of S. cerevisiae and the recently discovered cryotolerant species S. eubayanus. Properties from both parents make S. pastorianus a superior fermenter of brewer’s wort at temperatures below 15 °C. Since S. cerevisiae is not a cryotolerant yeast, the low-temperature performance of S. pastorianus hybrids has been attributed to the S. eubayanus sub-genome. However, the geographical origin of the S. eubayanus sub-genome of S. pastorianus hybrids, as well as its contribution to metabolism of key oligosaccharides in brewer’s wort, is not fully understood. The research described in this thesis therefore focused on the genetics of the metabolism of maltose and maltotriose, two key sugars in brewer’s wort, in S. eubayanus., BT/Industrial Microbiology

Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer's wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357T, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study., BT/Industrial Microbiology, BT/Cell Systems Engineering

Trees are dying in response to gradual changes in climate and extreme climatic events

Background: The lager brewing yeast, S. pastorianus, is a hybrid between S. cerevisiae and S. eubayanus with extensive chromosome aneuploidy. S. pastorianus is subdivided into Group 1 and Group 2 strains, where Group 2 strains have higher copy number and a larger degree of heterozygosity for S. cerevisiae chromosomes. As a result, Group 2 strains were hypothesized to have emerged from a hybridization event distinct from Group 1 strains. Current genome assemblies of S. pastorianus strains are incomplete and highly fragmented, limiting our ability to investigate their evolutionary history. Results: To fill this gap, we generated a chromosome-level genome assembly of the S. pastorianus strain CBS 1483 from Oxford Nanopore MinION DNA sequencing data and analysed the newly assembled subtelomeric regions and chromosome heterozygosity. To analyse the evolutionary history of S. pastorianus strains, we developed Alpaca: a method to compute sequence similarity between genomes without assuming linear evolution. Alpaca revealed high similarities between the S. cerevisiae subgenomes of Group 1 and 2 strains, and marked differences from sequenced S. cerevisiae strains. Conclusions: Our findings suggest that Group 1 and Group 2 strains originated from a single hybridization involving a heterozygous S. cerevisiae strain, followed by different evolutionary trajectories. The clear differences between both groups may originate from a severe population bottleneck caused by the isolation of the first pure cultures. Alpaca provides a computationally inexpensive method to analyse evolutionary relationships while considering non-linear evolution such as horizontal gene transfer and sexual reproduction, providing a complementary viewpoint beyond traditional phylogenetic approaches., Pattern Recognition and Bioinformatics, BT/Industrial Microbiology, BT/Biotechnology

Saccharomyces pastorianus lager-brewing yeasts are domesticated hybrids of S. cerevisiae x S. eubayanus that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid of S. cerevisiae and S. eubayanus was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploid S. pastorianus brewing strains. In contrast to the extensive aneuploidy of domesticated S. pastorianus strains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilization and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticated S. pastorianus strains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation of S. cerevisiae × S. eubayanus hybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids., BT/Industrial Microbiology, Pattern Recognition and Bioinformatics

Saccharomyces pastorianus lager brewing yeasts are domesticated hybrids of Saccharomyces cerevisiae and cold-tolerant Saccharomyces eubayanus. To understand the contribution of both parental genomes to maltose metabolism in brewing wort, this study focuses on maltose transport in the S. eubayanus type strain CBS 12357T/FM1318. To obtain complete sequences of the MAL loci of this strain, a near-complete genome assembly was generated using the Oxford Nanopore Technology MinION sequencing platform. Except for CHRXII, all sixteen chromosomes were assembled as single contigs. Four loci harboring putative maltose transporter genes (SeMALT1-4), located in subtelomeric regions of CHRII, CHRV, CHRXIII, and CHRXVI, were completely resolved. The near-identical loci on CHRV and CHRXVI strongly resembled canonical S. cerevisiae MAL loci, while those on CHRII and CHRXIII showed different structures suggestive of gene loss. Overexpression of SeMALT1-4 in a maltose-transport-deficient S. cerevisiae strain restored growth on maltose, but not on maltotriose, indicating maltose-specific transport functionality of all four transporters. Simultaneous CRISPR-Cas9-assisted deletion of only SeMALT2 and SeMALT4, which shared 99.7% sequence identity, eliminated growth of S. eubayanus CBS 12357T on maltose. Transcriptome analysis of S. eubayanus CBS 12357T established that SeMALT1 and SeMALT3, are poorly expressed in maltose-grown cultures, while SeMALT2 and SeMALT4 were expressed at much higher levels than SeMALT1 and SeMALT3, indicating that only SeMALT2/4 are responsible for maltose consumption in CBS 12357T. These results represent a first genomic and physiological characterization of maltose transport in S. eubayanus CBS 12357T and provides a valuable resource for further industrial exploitation of this yeast., BT/Industrial Microbiology, Applied Sciences, Medezeggenschapsondersteuning, BT/Biotechnology

The haploid Saccharomyces cerevisiae strain CEN.PK113–7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C. However, these assemblies contain large sequence gaps, particularly in subtelomeric regions, and the assumption of perfect homology to S288C for scaffolding introduces bias. In this study, we obtained a near-complete genome assembly of CEN.PK113–7D using only Oxford Nanopore Technology's MinION sequencing platform. Fifteen of the 16 chromosomes, the mitochondrial genome and the 2-μm plasmid are assembled in single contigs and all but one chromosome starts or ends in a telomere repeat. This improved genome assembly contains 770 Kbp of added sequence containing 248 gene annotations in comparison to the previous assembly of CEN.PK113–7D. Many of these genes encode functions determining fitness in specific growth conditions and are therefore highly relevant for various industrial applications. Furthermore, we discovered a translocation between chromosomes III and VIII that caused misidentification of a MAL locus in the previous CEN.PK113–7D assembly. This study demonstrates the power of long-read sequencing by providing a high-quality reference assembly and annotation of CEN.PK113–7D and places a caveat on assumed genome stability of microorganisms., Pattern Recognition and Bioinformatics, BT/Industrial Microbiology

Summary Anthropogenic global change compromises forest resilience, with profound impacts to ecosystem functions and services. This synthesis paper reflects on the current understanding of forest resilience and potential tipping points under environmental change and explores challenges to assessing responses using experiments, observations and models. Forests are changing over a wide range of spatio-temporal scales, but it is often unclear whether these changes reduce resilience or represent a tipping point. Tipping points may arise from interactions across scales, as processes such as climate change, land-use change, invasive species or deforestation gradually erode resilience and increase vulnerability to extreme events. Studies covering interactions across different spatio-temporal scales are needed to further our understanding. Combinations of experiments, observations and process-based models could improve our ability to project forest resilience and tipping points under global change. We discuss uncertainties in changing CO2 concentration and quantifying tree mortality as examples. Synthesis. As forests change at various scales, it is increasingly important to understand whether and how such changes lead to reduced resilience and potential tipping points. Understanding the mechanisms underlying forest resilience and tipping points would help in assessing risks to ecosystems and presents opportunities for ecosystem restoration and sustainable forest management.

We used a spatially explicit model of forest dynamics, supported by empirical field data and socioeconomic data, to examine the impacts of human disturbances on a protected forest landscape in Kyrgyzstan. Local use of 27 fruit and nut species was recorded and modeled. Results indicated that in the presence of fuelwood cutting with or without grazing, species of high socioeconomic importance such as Juglans regia, Malus spp., and Armeniaca vulgaris were largely eliminated from the landscape after 50-150 yr. In the absence of disturbance or in the presence of grazing only, decline of these species occurred at a much lower rate, owing to competitive interactions between tree species. This suggests that the current intensity of fuelwood harvesting is not sustainable. Conversely, current grazing intensities were found to have relatively little impact on forest structure and composition, and could potentially play a positive role in supporting regeneration of tree species. These results indicate that both positive and negative impacts on biodiversity can arise from human populations living within a protected area. Potentially, these could be reconciled through the development of participatory approaches to conservation management within this reserve, to ensure the maintenance of its high conservation value while meeting human needs.

The increasing ubiquity of the modern smartphone, coupled with its technical capabilities in terms of processing power, internet connectivity, and sensor richness, make it an ideal platformfor sensing all kinds of information about ourselves and the world around us. The emerging field of Mobile Phone Sensing (MPS) is taking advantage of this new technology, with applications in health care, traffic planning, social science, and more. However, with these new capabilities come new challenges as well, several of which we address in this dissertation. First, accurate, continuous, and instantaneous localization is needed both as a primary sensor of human mobility, and to place other sensor source into a spatial context. Second, scaling up experiments to potentially thousands of devices poses unique organizational and technological challenges. Third, energy consumption is of paramount importance in mobile devices where battery life time is a major constraint on what can be achieved, yet accurate, fine-grained energy analysis on mobile devices is still very difficult. We studied two aspects of smartphone localization. First, we explored howwell three different sensor modalities (GPS,Wi-Fi, and Geolocation) performon the task of finding when and where people dwell. We found that each of these offer roughly equal performance, but only when the signal is of sufficient quality. Therefore, coverage is an important issue. Another consequence of this study is that where available,Wi-Fi is a good substitute for the more energy-hungry GPS receiver on these tasks. Second, encouraged by this last result, we designed a novelWi-Fi scanning algorithm that reduces the energy consumption of this task by roughly half on modern smartphones. It achieves this by scanning as few channels as possible, thereby reducing the time taken by a scan, and thus the energy consumed. The algorithm visits the available channels in sequence of popularity, and terminates early when enough information has bee, Software Computer Technology, Electrical Engineering, Mathematics and Computer Science

The complex problems associated with global change processes calls for close collaboration between science disciplines to create new, integrated knowledge. In the wake of global change processes, forests and other natural environments have been rapidly changing, highlighting the need for collaboration and integrative research development. Few tools are available to explore the potential for collaborations in research ventures that are just starting up. This study presents a useful approach for exploring and fostering collaborations between academics working in research teams and organizations comprising multiple science disciplines (i.e., multi-disciplinary). The research aim was to reveal potential barriers, common ground, and research strengths between academics working in a new centre focused on forest and climate change research. This aim was based on the premise that raising awareness and working with this acquired knowledge fosters collaborations and integrative research development. An email survey was deployed amongst the academics to obtain: (i) their understanding of common themes (e.g., climate change, scale of investigation, woodland/forest health/decline); (ii) descriptions of the spatial and temporal scales of their research; and (iii) their approach and perceived contributions to climate change research. These data were analysed using a semi-quantitative content analysis approach. We found that the main potential barriers were likely to be related to differences in understanding of the common research themes, whilst similarities and disciplinary strengths provided critical elements to foster collaborations. These findings were presented and discussed amongst the centre academics to raise awareness and create a dialogue around these issues. This process resulted in the development of four additional research projects involving multiple disciplines. The approach used in this study provides a useful methodology of broader benefit to similar multi-discipl

Mediterranean regions are under increasing pressure from global climate changes. Many have experienced more frequent extreme weather events such as droughts and heatwaves, which have severe implications for the persistence of forest ecosystems. This study reports on a landscape-scale assessment investigating potential associated factors of crown dieback in dominant tree species following an extreme dry and hot year/summer of 2010/11 in the Northern Jarrah Forest of Western Australia. Analyses focussed on the influence of (i) geology, (ii) topography, (iii) climate, and (iv) fire history. The results showed that trees on specific soils were more likely to show canopy dieback. Generally, trees on rocky soils with low water holding capacity were found to be affected more frequently. Other explanatory factors identified that dieback occurred (i) on sites that were close to rock outcrops, (ii) in areas that received a slightly higher amount of annual rainfall compared to the surrounding landscape, (iii) on sites at high elevations and (vi) on steep slopes, and (v) in areas that were generally slightly warmer than their surroundings. These results expand our understanding of how landscape-scale factors contribute to the effects of an extreme drought and heating event in Mediterranean forest ecosystems, and give indications of where changes are likely to occur within the landscape in the future. The analogues with other Mediterranean climate regions make the results of this study transferable and a starting point for further investigations.

The State Centre of Excellence for Climate Change, Woodland and Forest Health is dedicated to provide up-to-date and scientifically sound science and information to underpin the protection, health and sustainable management of Western Australia’s unique woodland and forest systems in a rapidly changing climate. This Centre is build around four distinct research programs including a range of natural and social sciences. One of the key goals of the Centre is to develop common research questions within an interdisciplinary framework. This is a necessary step to effectively work together and combine research results for further research development. However, differences in research traditions, not just between the social and the natural sciences but among the various sub-disciplines as well, make this a challenging task. The aim of this research is to provide guidelines and a framework that will help facilitate the interdisciplinary research approach, enhancing the quality of output and implementation of the results into future policy and conservation management practices. Broadening interdisciplinary teamwork to a transdisciplinary effort that includes researchers and decision makers at different levels will be a further step to take. A preliminary framework will be presented and future steps and challenges will be discussed.

Writing software for Wireless Sensor Networks (WSN) is hard, as programmers have to write robust, distributed, highly concurrent applications on extremely resource limited devices. Virtual machines offer among other things support for high-level object-oriented languages, dynamic memory management and protection, hardware abstraction, and efficient code distribution. The main challenge is to ensure good programming tools and a minimal footprint for the virtual machine to match the limited amounts of memory available on typical WSN platforms. This thesis describes the design and implementation of Darjeeling, a virtual machine modelled after the Java VM and capable of executing a substantial subset of the Java language, but designed specifically to run on 8- and 16-bit microcontrollers with 2-10kB of RAM. The Darjeeling VM uses a 16- rather than a 32-bit architecture, which is more efficient on the targeted platforms. Darjeeling features a novel memory organisation with strict separation of reference from non-reference types that eliminates the need for run-time type analysis in the underlying precise garbage collector. Darjeeling also includes a linked stack model that provides light-weight threads, compacting garbage collection, and synchronization. The VM has been implemented on three different platforms, and was evaluated with micro benchmarks as well as a real-world monitoring application. The latter includes a pure Java implementation of the Collection Tree Protocol (CTP) conveniently programmed as a set of cooperating threads, and a reimplementation of an existing environmental monitoring application. The results show that Darjeeling is a viable solution for deploying large-scale, heterogeneous sensor networks., Embedded Software, Electrical Engineering, Mathematics and Computer Science

The acronym DIPLOID stands for DIstributed P2P LOcation-based Information Database. The development of DIPLOID is the main assignment of this BSc project. The purpose of this information storage system is to store location-based GMapWiki-information in a flexible extendible way using PIAX, a distributed storage system developed at Osaka University. To fulfill these goals, information is stored as a peer-to-peer system and the information is distributed over the peers in the system. In short, DIPLOID is a two way interface between a front-end application and a storage layer. Using DIPLOID, users should be able to create their own custom sensors, to add information to the location-based-wiki. The information added is stored on the sensor itself but it is indexed by the peer-to-peer system, to make the data accessible by all users in the system., Software Technology, Electrical Engineering, Mathematics and Computer Science

Alzheimer's disease (AD) is a genetically complex disorder, and several genes related to cholesterol metabolism have been reported to contribute to AD risk. To identify further AD susceptibility genes, we have screened genes that map to chromosomal regions with high logarithm of the odds scores for AD in full genome scans and are related to cholesterol metabolism. In a European screening sample of 115 sporadic AD patients and 191 healthy control subjects, we analyzed single nucleotide polymorphisms in 28 cholesterol-related genes for association with AD. The genes HMGCS2, FDPS, RAFTLIN, ACAD8, NPC2, and ABCG1 were associated with AD at a significance level of P < or = 0.05 in this sample. Replication trials in five independent European samples detected associations of variants within HMGCS2, FDPS, NPC2, or ABCG1 with AD in some samples (P = 0.05 to P = 0.005). We did not identify a marker that was significantly associated with AD in the pooled sample (n = 2864). Stratification of this sample revealed an APOE-dependent association of HMGCS2 with AD (P = 0.004). We conclude that genetic variants investigated in this study may be associated with a moderate modification of the risk for AD in some samples.