| 1. |
- Edelbroek, Bart
(författare)
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Function and Evolution of Small Regulatory RNAs and their Associated Proteins : A Journey from Genome to Proteome
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Organisms throughout the tree of life have evolved distinct ways to regulate gene expression. Some of these processes involve non-coding RNAs (ncRNAs), which are not translated but functional nonetheless. These ncRNAs are of utmost importance, with dysregulation of some causing severe developmental effects or even being lethal.In order to get a better fundamental understanding of gene regulation, and the ncRNAs that evolved to regulate gene expression, we study this in Amoebozoa. Members of this taxon vary greatly in lifestyle and organismal complexity. Some are strictly unicellular, free-living, whereas others, such as the social amoeba Dictyostelium discoideum can transition between unicellular and multicellular lifestyles. D. discoideum features a variety of small ncRNAs. Among these are the microRNAs. microRNAs have mostly been studied in plants and animals, where they are believed to have evolved convergently, and hypothesized to have played a role when these taxa evolved multicellular lifestyles. At what point the D. discoideum microRNAs evolved, how they function, and if they are involved in its multicellular lifestyle are fundamental questions addressed in this thesis. Here, we studied the evolution and function of microRNAs in a broad set of species belonging to Amoebozoa. We could identify microRNAs in all studied amoebae, and concluded that they are probably not involved in the evolution of multicellularity. To in detail investigate the evolution of microRNAs, we performed comparative genomics using D. discoideum and the close relative Dictyostelium firmibasis. For this, we sequenced, assembled and annotated the genome of the latter. At this point, our findings suggest that the microRNAs evolved several times in Amoebozoa, although we cannot rule out if they have a deep evolutionary history.The Class I RNAs are another type of ncRNAs. These, on the other hand, are only present in the social amoebae. They are hypothesized to regulate the transition from unicellular to multicellular in these species, potentially in a post-transcriptional manner. In order to investigate this, it is essential to understand to what extent the proteome and transcriptome correlate. Hence, we performed paired transcriptomics and proteomics in a time-series during multicellular development. By including a strain in which a specific Class I RNA is knocked out, we have initiated studies of its role during the transition to multicellularity.In conclusion, we were able to answer broad evolutionary and functional questions about gene regulation and ncRNAs by studying Amoebozoa from genome to proteome.
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| 2. |
- Hong, Kuk-ki, 1976
(författare)
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Advancing Metabolic Engineering through Combination of Systems Biology and Adaptive Evolution
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Understanding evolutionary strategies of microorganisms may provide opportunities foradvanced strain development with the aim to produce valuable bio-products from renewablebiomass resources. Through evolutionary processes, microorganisms can attain new traitsassociated with genetic changes that may be useful for the construction of improved strains.Therefore, the characterization of evolutionary strategies may result in identification of themolecular and genetic changes underlying newly obtained traits, and can hereby become anessential step in strain development. However, so far the depth of analysis has limited the rangeof comprehension. This thesis applied genome-wide analyses such as transcriptome, metabolomeand whole-genome sequencing to investigate the evolutionary strategies of the yeastSaccharomyces cerevisiae. Three evolved mutants were independently generated by adaptiveevolution on galactose minimal media to obtain the trait of improved galactose utilization byyeast. Those strains expressed higher galactose utilization rates than a reference strain in terms ofboth maximum specific growth rate and specific galactose uptake rate. Application of thegenome-scale comparative analyses employing engineered strains as controls elucidated uniquechanges obtained by adaptive evolution. Molecular bases referred from the changes oftranscriptome and metabolome were located around galactose metabolism, while genetic basesfrom whole-genome sequencing showed no mutations in those changes. Common mutationsamong the evolved mutants were identified in the Ras/PKA signaling pathway. Those mutationswere placed on the reference strain background and their effects were evaluated by comparisonwith the evolved mutants. One of the site-directed mutants showed even higher specific galactoseuptake rate than the evolved mutants, and just few number of genetic and molecular changes wereenough to recover complete the adaptive phenotype. These results indicate that identification ofkey mutations provide new strategies for further metabolic engineering of strains. In addition, thepleiotropy of obtained phenotype that is improved galactose availability was tested. When thegalactose-evolved mutants were cultured on glucose that is the most favorite carbon source ofyeast, those mutants showed reduction of glucose utilization. Genome-wide analyses and sitedirectedmutagenesis were applied again to understand underlying molecular and genetic bases ofthis trade-off in carbon utilization. The results indicated that loosening of tight glucose regulationwas likely the reason of increased galactose availability. The implications of evolutionarystrategies and the impact of genome-scale analyses on characterization of evolved mutants arediscussed.
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| 3. |
- Nandakumar, Mridula
(författare)
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Pathogen-mediated selection in the immune system of rodents : Exploring selection targets, functional effects and trade-offs
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pathogens cause disease and play an important role in shaping evolution of the host immune system. They create pressure on host immunity to evolve in numerous ways, most commonly by increasing divergence between species (positive selection) or increasing polymorphisms within a population (balancing selection). Especially with balancing selection, trade-offs between different traits, for example responses to different pathogens, are essential. Across five papers, questions related to what immune genes are under selection, how this translates to an effect on the immune response and what trade-offs occur, are addressed using rodents as study system. Paper I utilised genomes from 30 rodent species to identify signatures of positive selection in immune genes. In general, function of immune genes was a significant determinant for signs of positive selection. This effect was significant even after accounting for potential confounding factors like gene expression and protein-protein interactions. In Paper II, the focus is on a local population of bank voles in Sweden, to look for signatures of balancing selection in the complement system – a branch of innate immunity. One complement gene, FCNA, was found to be under strong balancing selection. In Paper III, FCNA polymorphism was linked to associations with natural infections of Borrelia afzelii, a common pathogen for bank voles. Papers IV and V look at how the immune response of bank voles of various genotypes differ on stimulation with B. afzelii and the human pathogen Streptococcus pyogenes, captured with transcriptome sequencing of spleen cells. In Paper IV, the analysis is focused on various genotypes of TLR2, an immune gene under balancing selection in bank voles and associated with infection prevalence of B. afzelii in the wild. A stimulation-specific effect of TLR2 on immune response was found, where the magnitude of immune response to B. afzelii, but not S. pyogenes, depends on TLR2 expression level in a TLR2 genotype-specific way. In Paper V, tradeoffs at the cis-regulatory level between the response to B. afzelii and S. pyogenes was tested by searching for polymorphisms where the alleles are expressed differently to these two stimulations. Abundant cis-regulatory variation for responses to the two bacteria was found, but there was no evidence for trade-offs. In summary, this work pushes our knowledge of what immune genes can be expected to be under pathogen-mediated selection, as heretofore understudied categories of immune function showed signs of selection. A novel basis – the combination of genotype and expression – was uncovered for functional effects of polymorphic genes. Finally, there was no evidence for trade-offs between responses to different pathogens. Investigating the nature of trade-offs in the immune system further would be necessary towards understanding the causes and consequences of pathogen-mediated selection.
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| 4. |
- Nilsson, R. Henrik, 1976
(författare)
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Fungal taxonomy and systematics in the digital era, with an outlook on the cantharelloid clade (Basidiomycota)
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fungi form a large and ubiquitous group of organisms where species identification and delimitation on morphological grounds often fall short. DNA sequences have proved an invaluable information source for these pursuits and are now routinely used in most mycological laboratories. Newl generated DNA sequences are typically compared with the entries of the large INSD sequence database for inference of taxonomic affiliations and other properties using the sequence similarity search tool BLAST. This thesis highlights some practical difficulties in using BLAST for these purposes it is for example very sensitive to the length of the sequences - and shows that improper use of BLAST appears to have had considerable repercussion on the general level of taxonomic reliability of the fungal sequences in INSD, more than 10% of which may be incorrectly identified to species level. An initiative to build a new DNA sequence database for taxonomically reliable DNA-based identification of mycorrhizal (plant-mutualistic) fungi is described. The database differs from similar initiatives in that its entries are determined to species level by pertinent experts; it allows for integrative sequence annotations, including photos and morphological descriptions; and it employs new, phylogeny-based tools for sequence identification to alleviate the concerns with simplistic, similarity-based tools as arbiters of taxonomic affiliation. That phylogenetic analyses can be beneficial also to classification and nomenclatural projects is shown in the mor enterprise, which is a weekly automaton of Agaricomycetes (mushroom-forming fungi) phylogeny. All fungal sequences from the nuclear ribosomal large subunit gene are assembled on a weekly basis; automated phylogenetic analyses are undertaken; and the resulting phylogenetic trees are displayed and analyzed for changes in clade topology and inclusiveness. The enigmatic cantharelloid clade of the Agaricomycetes is studied using a four-gene phylogenetic approach. While this heterogeneous assembly of mushroom-like, resupinate, clavarioid, and lichen-forming fungi defies any morphological attempt at indicating a close relatedness for its species, the results from the molecular analyses show that there is indeed strong evidence to support that these fungi form a monophyletic group; a restrictive circumscription of the clade to include the genera Botryobasidium, Sistotrema, Multiclavula, Membranomyces, Hydnum, Clavulina, Cantharellus, and Craterellus is advocated. Stichic basidia, and to a lesser extent parenthesome ultrastructure, are found to be characteristic of the clade, and the previously reported divergent rates of evolution for the genera Cantharellus and Craterellus are shown to be limited to the nuclear ribosomal genes. The largely resupinate, and purportedly wood-decaying, genus Sistotrema is demonstrated to hold mycorrhizal lineages, and the molecular evidence to consider the genus polyphyletic is found to be very convincing.
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| 5. |
- Bag, Pushan, 1993-
(författare)
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How could Christmas trees remain evergreen? : photosynthetic acclimation of Scots pine and Norway spruce needles during winter
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Plants and other green organisms harvest sunlight by green chlorophyll pigments and covertit to chemical energy (sugars) and oxygen in a process called photosynthesis providing the foundation for life on Earth. Although it is unanimously believed that oceanic phytoplanktons are the main contributors to the global photosynthesis, the contribution of coniferous boreal forests distributed across vast regions of the northern hemisphere cannot be undermined. Hence boreal forests account signifificantly for social, economical and environmental sustainability. Not only do conifers thrive in the tundra regions with extreme climate, but they also maintain their needles green over the boreal winter. A question remains; what makes them so resilient? In this respect, we aimed to understand the remarkable winter adaptation strategies in two dominant boreal coniferous species,i.e., Pinus sylvestris and Picea abies. First, we mapped the transcriptional landscape in Norway spruce (Picea abies) needles over the annual cycle. Transcriptional changes in the nascent needles reflflected a sequence of developmental processes and active vegetative growth during early summer and summer. Later after maturation, transcriptome reflflected activated defense against biotic factors and acclimationin response to abiotic environmental cues such as freezing temperatures during winter. Secondly, by monitoring the photosynthetic performance of Scot pine needles, we found that the trees face extreme stress during the early spring (Feb-Mar) when sub-zero temperatures are accompanied by high solar radiation. At this time, drastic changes occur in the thylakoid membranes of the chloroplast that allows the mixing of photosystem I and photosystem II that typically remain laterally segregated. This triggers direct energy transfer from PSII to PSI and thus protects PSII from damage. Furthermore, we found that this loss of lateral segregation may be a consequence of triple phosphorylationof Lhcb1 (Light harvesting complex1 of photosystem II). The structural changes in thylakoid membranes also lead to changes inthe thylakoid macro domain organisationand pigment protein composition. Furthermore, we discovered that while PSII is protected by direct energy transfer, the protection of PSI is provided through photoreduction of oxygen by flavodiiron proteins, which in turn allows P700 to stay in an oxidised state necessary for direct energy transfer. These coordinated cascades of changes concomitantly protect both PSI and PSII to maintain the needles green over the winter.
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| 6. |
- Klevebring, Daniel, 1981-
(författare)
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On Transcriptome Sequencing
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci.
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| 7. |
- Froslev Nielsen, Jens Christian, 1987
(författare)
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Systems Biology of the Secondary Metabolism in Filamentous Fungi
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Filamentous fungi constitute a rich reservoir of pharmaceutically relevant bioactive small molecules. These compounds, commonly referred to as secondary metabolites, are widely used as antibiotics for the treatment of microbial infections, but also as other pharmaceuticals such as immunosuppressors, cholesterol lowering agents and anticancer drugs. Although fungal derived antibiotics have been known for almost a century, genome sequencing has revealed that the biosynthetic potential of fungi is not fully exhausted.The Penicillium genus consists of around 350 accepted species, and many of these are well-known producers of pharmaceuticals and industrially exploited for this. The genus as a whole, however, is grossly understudied at the genomic level. To assess the potential for secondary metabolite biosynthesis in the Penicillium genus, we sequenced the genomes of ten species that produce diverse arrays of secondary metabolites in culture. One of the sequenced isolates was described as a new species, and we mapped secondary metabolites detected in culture to the corresponding biosynthetic gene clusters. The ten sequenced genomes were analyzed together with published Penicillium genomes, altogether 24, and we developed a pipeline to group biosynthetic gene clusters and map them to known pathways. We found a large untapped potential for biosynthesis of secondary metabolites, encoded in the genomes of these species, that potentially could fill the drug discovery pipeline. Based on our predictions, we experimentally identified a novel compound from the antifungal class of antibiotics called yanuthones.Since heterologous expression of secondary metabolite pathways has proved troublesome, the ten genome-sequenced Penicillium species were evaluated as cell factories in controlled bioreactor fermentations. Compared to an industrially relevant strain, the ten Penicillium species showed growth characteristics that encourage further exploration of their industrial potential. Transcriptome analysis of six of the species enabled the identification of a metabolic network that is responsible for precursor formation of secondary metabolites. This network provides important insight into the further industrial development of Penicillium cell factories, and could be used in designing metabolic engineering strategies for optimization of secondary metabolite production.Altogether this thesis provides novel insights into genetic and metabolic aspects of fungal secondary metabolism. Our findings propose that industrial production of secondary metabolites can be effectively established on the basis of native producers. Penicillium species constitute a rich source of drug leads, and possess promising physiological characteristics to be established as industrial production platforms.
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| 8. |
- Görnerup, Olof, 1977
(författare)
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Inference of Hierarchical Structure in Complex Systems
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hierarchical organization is a central property of complex systems. It is even argued that a system is required to be hierarchical in order to evolve complexity within reasonable time. A hierarchy of a system is defined as the set of self-contained levels at which the system operates and can be described on. Given a dynamical system there are only specific levels that are valid. This thesis mainly concerns the definition and inference of such levels. Paper I describes an algorithm for finding hierarchical levels in stochastic processes. The method systematically explores the set of possible partitions of a process' state space and statistically determines which of the partitions that impose closed dynamics. It is applicable to moderately sized systems. In Paper II an alternative approach that applies to linear dynamical systems is presented. In this case the spectral properties of the matrix that defines a system's dynamics is utilized, which allows for analysis of large systems (with on the order of thousand states). The specification and analysis of an algorithm that is based on the results in Paper II is presented in Paper III. Paper IV applies the spectral method and a complementary agglomeration method to infer aggregated dynamics in a Markov model of codon substitutions in DNA. The standard genetic code is identified as a projection that gives the hierarchical level of amino acid substitutions. Further, higher order amino acid groups that are relatively conserved under substitutions are found to define other levels of dynamics. Paper V and VI relate hierarchical organization to primordial evolution in a conceptual model that is based on the RNA world hypothesis. A well-stirred system of processes that catalyze the production of other processes is shown to successively build higher levels of organization from simple and general-purpose components by autocatalysis.
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| 9. |
- Chumnanpuen, Pramote, 1983
(författare)
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Systems Biology of Yeast Lipid Metabolism
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lipid metabolism plays an important role in the development of many different life-style related diseases, such as type 2 diabetes and atherosclerosis, and understanding the molecular mechanisms behind regulation of lipid biosynthesis and degradation may lead to development of new therapies. In this project we undertook a global study of lipid metabolism in the eukaryotic model organism Saccharomyces cerevisiae. The objective of this project is to quantify how the fluxes in lipid metabolism of eukaryotic cells are controlled by different component of the regulatory network. Using systems biology approaches there was established a global regulatory model for lipid metabolism, and it was quantified how the fluxes toward different lipid components are regulated. Using different mutants that carry deletion in genes encoding key transcriptional factors and protein kinases involved in lipid regulation, the fluxes towards the different lipid components was perturbed. The wild-type yeast strain CEN.PK113-7D and the yeast mutants opi1∆, snf1∆, tor1∆, ino2∆, ino4∆, and ino2∆ino4∆ were grown in chemostat cultures at carbon or nitrogen-limited conditions and also high or low inositol-choline (IC) condition at a dilution rate of 0.1 h-1. At steady state conditions samples were withdrawn for analysis of the transcriptome, the metabolome and the lipidome. There was also developed 3 high-throughput methods for lipid quantification, i) for storage lipid monitoring at single-cell level using CARS microscopy, ii) for lipid classes analysis based on microwave-assisted extraction, HPLC-CAD, and iii) for fatty acids species analysis based on microwave-assisted derivatization. Through combined measurements of the transcriptome, the metabolome, the lipidome and the fluxome it was possible to obtain a large dataset that could be used to identify correlations between the different components such as the co-influences of Snf1-IC effects, INO-level, and Snf1-TORC1 effects on yeast lipid metabolism.
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| 10. |
- Domenzain Del Castillo Cerecer, Iván, 1991
(författare)
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A systems biology understanding of protein constraints in the metabolism of budding yeasts
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fermentation technologies, such as bread making and production of alcoholic beverages, have been crucial for development of humanity throughout history. Saccharomyces cerevisiae provides a natural platform for this, due to its capability to transform sugars into ethanol. This, and other yeasts, are now used for production of pharmaceuticals, including insulin and artemisinic acid, flavors, fragrances, nutraceuticals, and fuel precursors. In this thesis, different systems biology methods were developed to study interactions between metabolism, enzymatic capabilities, and regulation of gene expression in budding yeasts. In paper I, a study of three different yeast species (S. cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus), exposed to multiple conditions, was carried out to understand their adaptation to environmental stress. Paper II revises the use of genome-scale metabolic models (GEMs) for the study and directed engineering of diverse yeast species. Additionally, 45 GEMs for different yeasts were collected, analyzed, and tested. In paper III, GECKO 2.0, a toolbox for integration of enzymatic constraints and proteomics data into GEMs, was developed and used for reconstruction of enzyme-constrained models (ecGEMs) for three yeast species and model organisms. Proteomics data and ecGEMs were used to further characterize the impact of environmental stress over metabolism of budding yeasts. On paper IV, gene engineering targets for increased accumulation of heme in S. cerevisiae cells were predicted with an ecGEM. Predictions were experimentally validated, yielding a 70-fold increase in intracellular heme. The prediction method was systematized and applied to the production of 102 chemicals in S. cerevisiae (Paper V). Results highlighted general principles for systems metabolic engineering and enabled understanding of the role of protein limitations in bio-based chemical production. Paper VI presents a hybrid model integrating an enzyme-constrained metabolic network, coupled to a gene regulatory model of nutrient-sensing mechanisms in S. cerevisiae. This model improves prediction of protein expression patterns while providing a rational connection between metabolism and the use of nutrients from the environment. This thesis demonstrates that integration of multiple systems biology approaches is valuable for understanding the connection of cell physiology at different levels, and provides tools for directed engineering of cells for the benefit of society.
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