| 1. |
- Görnerup, Olof, 1977
(author)
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Inference of Hierarchical Structure in Complex Systems
- 2008
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Doctoral thesis (other academic/artistic)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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| 2. |
- Wieloch, Thomas, 1979-
(author)
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Intramolecular isotope analysis reveals plant ecophysiological signals covering multiple timescales
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Our societies' wellbeing relies on stable and healthy environments. However, our current lifestyles, growth-oriented economic policies and the population explosion are leading to potentially catastrophic degradation of ecosystems and progressive disruption of food chains. Hopefully, more clarity about what the future holds in store will trigger stronger efforts to find, and adopt, problem-focused coping strategies and encourage environmentally friendly lifestyles.Forecasting environmental change/destruction is complicated (inter alia) by lack of complete understanding of plant-environment interactions, particularly those involved in slow processes such as plant acclimatisation and adaptation. This stems from deficiencies in tools to analyse such slow processes. The present work aims at developing tools that can provide retrospective ecophysiological information covering timescales from days to millennia.Natural archives, such as tree-rings, preserve plant metabolites over long timescales. Analyses of intramolecular isotope abundances in plant metabolites have the potential to provide retrospective information about metabolic processes and underlying environmental controls. Thus, my colleagues and I (hereafter we) analysed intramolecular isotope patterns in tree rings to develop analytical tools that can convey information about clearly-defined plant metabolic processes over multiple timescales. Such tools might help (inter alia) to constrain plants' capacities to sequester excess amounts of anthropogenic CO2; the so-called CO2 fertilisation effect. This, in turn, might shed light on plants' sink strength for the greenhouse gas CO2, and future plant performance and growth under climate change.In the first of three studies, reported in appended papers, we analysed intramolecular 13C/12C ratios in tree-ring glucose. In six angiosperm and six gymnosperm species we found pronounced intramolecular 13C/12C differences, exceeding 10‰. These differences are transmitted into major global C pools, such as soil organic matter. Taking intramolecular 13C/12C differences into account might improve isotopic characterisation of soil metabolic processes and soil CO2 effluxes. In addition, we analysed intramolecular 13C/12C ratios in a Pinus nigra tree-ring archive spanning the period 1961 to 1995. These data revealed new ecophysiological 13C/12C signals, which can facilitate climate reconstructions and assessments of plant-environment interactions at higher resolution; thus providing higher quality information. We proposed that 13C/12C signals at glucose C-1 to C-2 derive from carbon injection into the Calvin-Benson cycle via the oxidative pentose phosphate pathway. We concluded that intramolecular 13C/12C measurements provide valuable new information about long-term metabolic dynamics for application in biogeochemistry, plant physiology, plant breeding, and paleoclimatology.In the second study, we developed a comprehensive theory on the metabolic and ecophysiological origins of 13C/12C signals at tree-ring glucose C-5 and C-6. According to this theory and theoretical implications of the first study on signals at C-1 to C-3, analysis of such intramolecular signals can provide information about several metabolic processes. At C-3, a well-known signal reflecting CO2 uptake is preserved. The glucose-6-phosphate shunt around the Calvin-Benson cycle affects 13C/12C compositions at C-1 and C-2, while the 13C/12C signals at C-5 and C-6 reflect carbon fluxes into downstream metabolism. This theoretical framework enables further experimental studies to be conducted in a hypothesis-driven manner. In conclusion, the intramolecular approach provides information about carbon allocation in plant leaves. Thus, it gives access to long-term information on key ecophysiological processes, which could not be acquired by previous approaches.The abundance of the hydrogen isotope deuterium, δD, is important for linking the water cycle with plant ecophysiology. The main factors affecting δD in plant organic matter are commonly assumed to be the δD in source water and leaf-level evaporative enrichment. Current δD models incorporate biochemical D fractionations as constants. In the third study we showed that biochemical D fractionations respond strongly to low ambient CO2 levels and low light intensity. Thus, models of δD values in plant organic matter should incorporate biochemical fractionations as variables. In addition, we found pronounced leaf-level δD differences between α-cellulose and wax n-alkanes. We explained this by metabolite-specific contributions of distinct hydrogen sources during biosynthesis.Overall, this work advances our understanding of isotope distributions and isotope fractionations in plants. It reveals the immense potential of intramolecular isotope analyses for retrospective assessment of plant metabolism and associated environmental controls.
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| 3. |
- Ariöz, Candan, 1983-
(author)
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Exploring the Interplay of Lipids and Membrane Proteins
- 2014
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Doctoral thesis (other academic/artistic)abstract
- The interplay between lipids and membrane proteins is known to affect membrane protein topology and thus have significant effect (control) on their functions. In this PhD thesis, the influence of lipids on the membrane protein function was studied using three different membrane protein models.A monotopic membrane protein, monoglucosyldiacylglyecerol synthase (MGS) from Acholeplasma laidlawii is known to induce intracellular vesicles when expressed in Escherichia coli. The mechanism leading to this unusual phenomenon was investigated by various biochemical and biophysical techniques. The results indicated a doubling of lipid synthesis in the cell, which was triggered by the selective binding of MGS to anionic lipids. Multivariate data analysis revealed a good correlation with MGS production. Furthermore, preferential anionic lipid sequestering by MGS was shown to induce a different fatty acid modeling of E. coli membranes. The roles of specific lipid binding and the probable mechanism leading to intracellular vesicle formation were also investigated.As a second model, a MGS homolog from Synechocystis sp. PCC6803 was selected. MgdA is an integral membrane protein with multiple transmembrane helices and a unique membrane topology. The influence of different type of lipids on MgdA activity was tested with different membrane fractions of Synechocystis. Results indicated a very distinct profile compared to Acholeplasma laidlawii MGS. SQDG, an anionic lipid was found to be the species of the membrane that increased the MgdA activity 7-fold whereas two other lipids (PG and PE) had only minor effects on MgdA. Additionally, a working model of MgdA for the biosynthesis and flow of sugar lipids between Synechocystis membranes was proposed.The last model system was another integral membrane protein with a distinct structure but also a different function. The envelope stress sensor, CpxA and its interaction with E. coli membranes were studied. CpxA autophosphorylation activity was found to be positively regulated by phosphatidylethanolamine and negatively by anionic lipids. In contrast, phosphorylation of CpxR by CpxA revealed to be increased with PG but inhibited by CL. Non-bilayer lipids had a negative impact on CpxA phosphotransfer activity.Taken together, these studies provide a better understanding of the significance of the interplay of lipids and model membrane proteins discussed here.
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| 4. |
- Andersson, John, 1993
(author)
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Functional polymer brush coatings for nanoscale devices
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Nanobiotechnology is an interdisciplinary field that has garnered considerable attention for offering exciting new opportunities of studying and manipulating biomolecules at the nanoscale. This prospect bears large potential benefits in the field of medicine and the whole life science sector in general. Fabrication of different nanostructure devices that can handle liquids at the scale of biomolecules, such as nanochannels or nanopores, provide a good basis within nanobiotechnology. However, the materials of nanostructures tend to not interact with complex biomolecules in ways that are sufficiently specific or controlled. This issue can be avoided by functionalising the surface of nanostructures with different organic coatings, and polymer brushes have shown a diverse range of functionality in this regard. This thesis summarises efforts towards designing functional polymer brush coatings for nanoscale devices. Surface sensitive techniques are used to characterise the grafting of dense poly(ethylene glycol) brushes to various noble metals and silicon dioxide. The new functionalisation protocol for polymer brushes on silicon dioxide provides excellent biofunctionality and is demonstrated to be compatible with two different nanostructures. The specific hydrogen-bond mediated interaction between a poly(ethylene glycol) brush and poly(methacrylic acid) in solution at low pH is shown to make the polymer brush reversibly stimuli-responsive. Preliminary results further demonstrate how this interaction can be controlled electrochemically and indicates its suitability as a macromolecular gating mechanism for nanosized openings. Finally, characterisation and fabrication of plasmonic nanopore arrays with separately functionalisable compartments using electron beam lithography techniques is presented.
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| 5. |
- Ekelund Ugge, Gustaf Magnus Oskar, 1990-
(author)
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Transcriptional biomarkers of toxicity – powerful tools or random noise? : An applied perspective from studies on bivalves
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Aquatic organisms are constantly at risk of being exposed to potentially harmful chemical compounds of natural or anthropogenic origin. Biological life can for instance respond to chemical stressors by changes in gene expression, and thus, certain gene transcripts can potentially function as biomarkers, i.e. early warnings, of toxicity and chemical stress. A major challenge for biomarker application is the extrapolation of transcriptional data to potential effects at the organism level or above. Importantly, successful biomarker use also requires basal understanding of how to distinguish actual responses from background noise. The aim of this thesis is, based on response magnitude and variation, to evaluate the biomarker potential in a set of putative transcriptional biomarkers of general toxicity and chemical stress.Specifically, I addressed a selection of six transcripts involved in cytoprotection and oxidative stress: catalase (cat), glutathione-S-transferase (gst), heat shock proteins 70 and 90 (hsp70, hsp90), metallothionein (mt) and superoxide dismutase (sod). Moreover, I used metal exposures to serve as a proxy for general chemical stress, and due to their ecological relevance and nature as sedentary filter-feeders, I used bivalves as study organisms.In a series of experiments, I tested transcriptional responses in the freshwater duck mussel, Anodonta anatina, exposed to copper or an industrial wastewater effluent, to address response robustness and sensitivity, and potential controlled (e.g. exposure concentration) and random (e.g. gravidness) sources of variation. In addition, I performed a systematic review and meta-analysis on transcriptional responses in metal exposed bivalves to (1) evaluate what responses to expect from arbitrary metal exposures, (2) assess the influence from metal concentration (expressed as toxic unit), exposure time and analyzed tissue, and (3) address potential impacts from publication bias in the scientific literature.Response magnitudes were generally small in relationship to the observed variation, both for A. anatina and bivalves in general. The expected response to an arbitrary metal exposure would generally be close to zero, based on both experimental observations and on the estimated impact from publication bias. Although many of the transcripts demonstrated concentration-response relationships, large background noise might in practice obscure the small responses even at relatively high exposures. As demonstrated in A. anatina under copper exposure, this can be the case already for single species under high resolution exposures to single pollutants. As demonstrated by the meta-regression, this problem can only be expected to increase further upon extrapolation between different species and exposure scenarios, due to increasing heterogeneity and random variation. Similar patterns can also be expected for time-dependent response variation, although the meta-regression revealed a general trend of slightly increasing response magnitude with increasing exposure times.In A. anatina, gravidness was identified as a source of random variability that can potentially affect the baseline of most assessed biomarkers, particularly when quantified in gills. Response magnitudes and variability in this species were generally similar for selected transcripts as for two biochemical biomarkers included for comparison (AChE, GST), suggesting that the transcripts might not capture early warnings more efficiently than other molecular endpoints that are more toxicologically relevant. Overall, high concentrations and long exposure durations presumably increase the likelihood of a detectable transcriptional response, but not to an extent that justifies universal application as biomarkers of general toxicity and chemical stress. Consequently, without a strictly defined and validated application, this approach on its own appears unlikely to be successful for future environmental risk assessment and monitoring. Ultimately, efficient use of transcriptional biomarkers might require additional implementation of complementary approaches offered by current molecular techniques.
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| 6. |
- Bag, Pushan, 1993-
(author)
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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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Doctoral thesis (other academic/artistic)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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| 7. |
- Ghirmai, Semhar, 1993
(author)
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Prevention of hemolysis as a novel strategy to limit hemoglobin-mediated lipid oxidation in fish - towards a more sustainable use of fish raw materials
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Hemoglobin (Hb) has been recognized as a main pro-oxidant in fish, causing fast and intense lipid oxidation. This thesis explores the hypothesis that maintaining red blood cells (RBCs) intact for an extended time during fish processing could delay Hb-mediated lipid oxidation. The aims were to (i) unravel how selected endogenous and exogenous parameters affect fish RBC-stability, (ii) confirm the relation between hemolysis and lipid oxidation and (iii), evaluate antioxidative strategies to prevent oxidation caused by released Hb. In Study I, washed and resuspended trout RBCs (wr-RBC) and whole trout blood (WB) were used to study how temperature, salinity and mechanic pressure affect hemolysis. Cold temperatures, 0-6 °C, and physiological saline were advantageous for the RBC stability while hyper- (3% NaCl) or hypotonic (tap water) conditions as well as simulated mechanic pressure hemolysis. Study II revealed that typical post-mortem pH’s, 6.4 and 6.8, yielded a higher hemolysis rate of wr-RBCs compared to pH 7.2-8.0, and stimulated lipid oxidation of the RBC membrane. Similarly, in RBC-spiked washed cod mince (WCM), pH 6.4/6.8 stimulated both lipid oxidation and hemolysis compared to pH 7.2/7.6 (Study III). Hb-analyses of the soluble phase revealed that only low levels of released Hb (7.1 µM) were needed to initiate lipid oxidation at pH 6.4-6.8, which was not the case at high pH, even when >50% of the Hb was in the metHb form. In experiments with wr-RBCs, re-addition of blood plasma (12-75% v/w) largely increased the RBC stability (Study I-II). The stabilizing effect was partly ascribed to glucose, albumin, and ascorbic acid (AA). Adding blood plasma to the RBC-spiked WCM system 3% (v/w), delayed the onset of lipid oxidation at pH 6.8 with 3-4 days without delaying hemolysis; the latter most likely due to the high plasma dilution. To clarify the effect of hemolysis on WCM lipid oxidation, different ratios of intact and lysed RBCs were added to a WCM system at pH 6.8 (Study III). Samples with 25-100% lysed RBCs oxidized rapidly within the first day of storage, whereas the sample with 100% intact RBCs provided a 1-day delay in the onset of lipid oxidation. Further, the sample with initially fully intact RBCs resulted in 59.5± 2.9% and 48.1± 2.9% reduced maximum peroxide value (PV) and thiobarbituric acid reactive substances (TBARS), respectively. Short pre-incubation of herring co-products in 0.9% NaCl removed 6.6-18.0% of their Hb and slightly reduced the intensity of TBARS development during ice storage of the minced product, compared to pre-incubation in tap water or no pre-incubation (Study IV). Refrigerated storage of herring co-products while submerged in tap water, 0.9% NaCl or phosphate buffered saline (PBS) (pH 6.5 or 7.5) also reduced the lipid oxidation intensity compared to air-storage; however, without any effect from pH or salinity. When adding the commercial antioxidant mixture Duralox-MANC to the pre-incubation or submerging solutions (2% vs 0.5%) the lipid oxidation lag phase was largely prolonged; e.g., from < 1 day in controls to >6 days. Direct fortification of minced herring co-products with 0.5 % Duralox-MANC confirmed its antioxidative efficiency as lipid stability was prolonged from <1 day to >8 days. Altogether, this thesis confirmed that delaying hemolysis can be a route to delay lipid oxidation. It also revealed that current conditions used in the early handling/processing of fish, such as subjection to crowding, pumping, refrigerated sea water (RSW)-storage or tap water rinsing do not favor RBC stability and call for adjustments to limit hemolysis; and thereby Hb-mediated lipid oxidation. Intact RBCs, presence of blood plasma and elevated pH’s delayed lipid oxidation in a WCM system, providing an important window of time which could allow for better utilization of blood-rich fish raw materials currently leaving the food chain. To further enhance the lipid stability of such materials, subjecting them to rosemary-derived antioxidants via pre-incubation, submerging or direct addition is recommended. Results presented can contribute to an increased robustness of our global food systems towards unprecedented challenges such as pandemics, war, and climate threats. They also contribute to the ongoing dietary protein shift in which aquatic foods as small pelagic fish and fish rest raw materials have an immense potential if correctly preserved by updated procedures.
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| 8. |
- Larsson, Daniel, 1981-
(author)
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Exploring the Molecular Dynamics of Proteins and Viruses
- 2012
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Doctoral thesis (other academic/artistic)abstract
- Knowledge about structure and dynamics of the important biological macromolecules — proteins, nucleic acids, lipids and sugars — helps to understand their function. Atomic-resolution structures of macromolecules are routinely captured with X-ray crystallography and other techniques. In this thesis, simulations are used to explore the dynamics of the molecules beyond the static structures.Viruses are machines constructed from macromolecules. Crystal structures of them reveal little to no information about their genomes. In simulations of empty capsids, we observed a correlation between the spatial distribution of chloride ions in the solution and the position of RNA in crystals of satellite tobacco necrosis virus (STNV) and satellite tobacco mosaic virus (STMV). In this manner, structural features of the non-symmetric RNA could also be inferred.The capsid of STNV binds calcium ions on the icosahedral symmetry axes. The release of these ions controls the activation of the virus particle upon infection. Our simulations reproduced the swelling of the capsid upon removal of the ions and we quantified the water permeability of the capsid. The structure and dynamics of the expanded capsid suggest that the disassembly is initiated at the 3-fold symmetry axis.Several experimental methods require biomolecular samples to be injected into vacuum, such as mass-spectrometry and diffractive imaging of single particles. It is therefore important to understand how proteins and molecule-complexes respond to being aerosolized. In simulations we mimicked the dehydration process upon going from solution into the gas phase. We find that two important factors for structural stability of proteins are the temperature and the level of residual hydration. The simulations support experimental claims that membrane proteins can be protected by a lipid micelle and that a non-membrane protein could be stabilized in a reverse micelle in the gas phase. A water-layer around virus particles would impede the signal in diffractive experiments, but our calculations estimate that it should be possible to determine the orientation of the particle in individual images, which is a prerequisite for three-dimensional reconstruction.
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| 9. |
- Hildebrandt, Franziska, 1994-
(author)
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Host-parasite interactions in space and time
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Unicellular parasites of the apicomplexan phylum have a considerable effect on global health and agriculture. Two prominent examples of this phylum include malaria causing parasites of the Plasmodium genus and the widely prevalent parasite Toxoplasma gondii. While sharing a common ancestor, these parasites occupy unique biological niches, follow distinct life cycles, and result in different courses and outcomes of disease. In response to the parasite, the mammalian host has developed efficient and effective defense strategies. However, both Plasmodium and Toxoplasma have evolved strategies to evade the host’s defense response. Plasmodium parasites infect distinct tissues and cell types whereas T. gondii parasites are highly promiscuous and infect all nucleated cells. The identification of key factors involved in the interaction between the host and parasite is crucial for disease intervention, prevention, and eventually eradication efforts.Next-generation sequencing technologies have proven effective tools to investigate the response in a tissue or cell population of an infected organism. Novel genomics methods such as single-cell RNA-seq and spatial transcriptomics have enabled the investigation of heterogeneous transcriptional responses of individual cells in a population as well as heterogeneous expression profiles at spatially distinct tissue positions across entire tissue sections. This thesis pioneers the exploration of these methods in discerning the enormous complexity underlying host-parasite interplay.In Paper I, we determine spatial components of naive mouse liver in its true tissue context. We define gene expression gradients of pericentral and periportal zones in the liver and predict vein types with ambiguous annotations, based on in situ transcriptional profiles. We further identify novel spatial structures with distinct transcriptional profiles, associated with tissue integrity and integrate cell type proportions across the tissue.In Paper II we investigate host-pathogen interactions in P. berghei infected liver sections with spatiotemporal resolution. We establish spatial gene expression gradients from infection sites exhibiting upregulation of lipid metabolism associated genes 38 hours post-infection, suggesting a potential role of these pathways in immune evasion. We further show that local and systemic inflammation are delayed but not ablated in salivary gland lysate challenged control livers and propose that local inflammatory hotspots may represent an important spatial component for parasite development in the liver.In Paper III we use dual scRNA-seq to investigate heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites. We show differential responses towards the two T. gondii genotypes in two distinct subpopulations of BMDCs over multiple time points post infection. Moreover, we generate co-expression networks that define host and parasite genes, which are likely involved in the modulation of host immunity.In summary, this thesis aims to characterize host-pathogen interactions of two major apicomplexan genera in two distinct cell niches of the murine host with spatiotemporal or single cell resolution. In detail, this encompasses the study of spatial structures of the host in the liver environment and the spatiotemporal consequences of an infection with P. berghei. Furthermore, the aims include deciphering heterogeneous interactions between two distinct T. gondii strains and infected BMDCs.
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| 10. |
- Reymer, Anna, 1983
(author)
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Unveiling Mechanistic Details of Macromolecular Interactions: Structural Design and Molecular Modelling of DNA-Protein Systems in Their Active State
- 2012
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Doctoral thesis (other academic/artistic)abstract
- Molecular structure is fundamental for understanding mechanisms of molecular interactions. This applies not least to understanding biological function: every biological cell, whether bacterial or human, is an immensely complex system of thousands of molecules that exist in constant motion and interaction with each other. Structural properties of the two main classes of biological macromolecules, nucleic acids and proteins, have been studied in this Thesis focusing on functional interactions of both DNA and the DNA-binding enzyme human recombinase Rad51. DNA is a highly polymorphic molecule and its plasticity may be important for its function. Conformational mechanics of the DNA helix was addressed to understand interactions with dumbbell-shaped ruthenium(II) polypyridyl compounds, known for their remarkable ability to slowly thread one of their bulky centres through a tightly packed DNA stack, probably invoking large transient conformational rearrangements of the helix. Thread-intercalation rate is accelerated by several orders of magnitude if the DNA target sequence is a stretch of at least ten base pairs of AT, as well as by the hydrophobicity of the auxiliary “dumbbell” ligands: counterintuitively, a smaller and less hydrophobic compound takes longer times to thread. It is hypothesized that thread-intercalation might be facilitated by an A-like DNA conformation, induced by the outside binding of the Ru(II) compounds. An NMR study, aiming to solve a thread-intercalated structure of the binuclear Ru(II)-DNA complex, resulted in a groove-binding geometry, probably representing an initial binding mode preceding intercalation, a result emphasizing the elusiveness and immense complexity of the threading process. Turning back to simpler monomeric propeller-shaped Ru(II) compounds it was deduced that, despite acting as classic intercalators, they can “read out” the chirality of the DNA helix by enantiospecifically kinking it, in a fashion analogous to several families of operatory DNA binding proteins.Another operatory protein, human recombinase Rad51, that facilitates homologous recombination, the process of exchanging near identical-sequence DNA strands, is also mechanically acting on DNA, by stretching it. A 3-D high-resolution model structure of human Rad51 filament was solved by a combination of polarized-light spectroscopic data and molecular modelling. Highlighted by the model some interesting structural features could be addressed: strategic locations of two putative DNA binding loops inside the protein filament; as well as location of a putative ATP binding site at the interface between two protein subunits and in direct proximity to a supposed location of DNA – could hint about DNA docking mechanism and potential role of ATP in the protein function.The Rad51-DNA model has proven itself useful also in a follow-up study on the stimulatory role of Ca2+ in the strand exchange reaction by human Rad51. A mechanism is proposed involving a high affinity DNA binding state of the Rad51 filament induced by Ca2+, regulatorily crucial for the search of homology and subsequent DNA strands exchange.
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