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Coming dissertations at Uppsala university

  • Evolution of streamlined genomes in ultra-small aquatic bacteria Author: Weizhou Zhao Link: Publication date: 2019-10-15 11:51

    This thesis investigates the evolutionary processes of streamlined genomes from aquatic bacteria adapting to different salinities, using two groups of ultra-small aquatic bacteria (LD12 Alphaproteobacteria and acI Actinobacteria). Due to difficulties in obtaining pure cultures of these bacteria, culture-free approaches (single-cell genomics and metagenomics) were used to construct and compare genomes, and to study the mechanisms and selective forces of adaptation to freshwater, brackish, and marine ecosystems.

    A study of single-cell amplified genomes (SAGs) from freshwater LD12 Alphaproteobacteria revealed that LD12 forms a clade embedded within the globally dominant marine Alphaproteobacteria SAR11, and subclades were organized into distinct microclusters. LD12 genomes had a very low ratio of recombination to point mutations, in contrast to their marine relatives which had a very high ratio of recombination to mutation. We suggested that the transition from marine to freshwater was a bottleneck event, resulting in reduced opportunities for recombination.

    In a separate study, we analyzed complete genomes and SAGs from acI Actinobacteria abundant in freshwater ecosystems, and found overall low rates of sequence divergence with however a dramatic acceleration near genomic island 1 (GI-1). We also identified a type IV topoisomerase, the delta subunit of DNA polymerase, and an RNA polymerase sigma factor near GI-1. Based on these results, we proposed a model for the evolution and expression of novel genes in these genomes.

    We also isolated and analyzed the genomes of single cells from a marine Actinobacteria (subclass Candidatus Actinomarinidae). These were not related to acI, but to Acidimicrobiia, which suggested salinity barriers have been crossed several times by Actinobacteria.

    To further understand the transition to different salinities, we obtained acI SAGs from three different intermediate-salinity Baltic Sea locations. We took sequence reads from 21 metagenomes taken along the salinity gradient, and recruited these fragments to both the freshwater and brackish acI reference genomes. These results indicated that transitions between fresh and brackish waters have occurred multiple times in acI Actinobacteria and some of these strains are globally present in coastal waters.

  • Prospective Applications of Microwaves in Medicine : Microwave Sensors for Orthopedic Monitoring and Burn Depth Assessment Author: Syaiful Redzwan Mohd Shah Link: Publication date: 2019-10-15 10:13

    In recent years, the use of microwave techniques for medical diagnostics has experienced impressive developments. It has demonstrated excellent competencies in various modalities such as using non-ionizing electromagnetic waves, providing non-invasive diagnoses, and having the ability to penetrate human tissues within the GHz range. However, due to anatomical, physiological, and biological variations in the human body, certain obstacles are present. Moreover, there are accuracy problems such as the absence of numerical models and experimental data, difficulty in conducting tests due to safety issues with human subjects, and also practical restrictions in clinical implementation. With the presence of these issues, a better understanding of the microwave technique is essential to further improve its medical application and to introduce alternative diagnostic methods that can detect and monitor various medical conditions in real time.

    The first part of this thesis focuses on measurement systems for the microwave technique in terms of sensor design and development, numerical analysis, permittivity measurement, and phantom fabrication. The aim is to investigate the feasibility of flexible systems with different fields of application including a microwave sensor system for measuring the healing progression of bone defects present in lower extremity trauma, bone regeneration in craniotomy for craniosynostosis treatments, and dielectric variation for burn injuries. The microwave sensor which utilizes the contrast in dielectric constant between various tissues was used as the primary sensor for the proposed application. This involved detailed optimization of the sensor for greater sensitivity. The experimental work carried out in the lab environment showed that the microwave sensor was able to detect the contrast in dielectric properties so that it can give an indication of the healing status for actual clinical scenarios.

    The second part of the thesis is making a significant step towards its practical implementation by establishing a system that can detect and monitor the rate of healing progression with fast data acquisition speed of microseconds, and developing an efficient user interface to convert raw microwave data into legible clinical information in terms of bone healing and burn injuries. As an extension to this thesis, clinical studies were conducted and ethical approval for conducting tests on human subjects was obtained for the development of a microwave medical system. The results showed a clear difference in healing progressions due to high detection capability in terms of dielectric properties of different human tissues. All of these contributions enable a portable system to complement existing medical applications with the aim of providing more advanced healthcare systems.

  • The birth of the hydrogenase : Studying the mechanism of [FeFe] hydrogenase maturation Author: Brigitta Németh Link: Publication date: 2019-10-11 14:07

    The [FeFe] hydrogenases are ancient metalloenzymes that catalyse the reversible interconversion between protons, electrons and molecular hydrogen. Despite the large structural variability within the [FeFe] hydrogenase family, the active site, the so called “H-cluster” is present in every representative. The H-cluster is composed by a four cysteine coordinated [4Fe4S] cluster, ligated via a shared cysteine to a biologically unique [2Fe] subsite decorated with CO and CN ligands and an azadithiolate bridging ligand. The biosynthesis of the [2Fe] subsite requires a maturation machinery, composed of at least three maturase enzymes, denoted HydG, HydE, and HydF. HydE and HydG are members of the radical SAM enzyme family, and are responsible for the construction of a pre-catalyst on HydF. This pre-catalyst is finally transferred from HydF to HydA, where it becomes part of the H-cluster.

    Recently, a pioneer study combined synthetic chemistry and biochemistry in order to create semi-synthetic HydF proteins. Synthetic mimics of the [2Fe] subsite were introduced to HydF, and this resulting semi-synthetic HydF was used to activate the unmatured hydrogenase (apo-HydA). This technique ushered in a new era in [FeFe] hydrogenase research.

    This thesis work is devoted to a deeper understanding of H-cluster formation and [FeFe] hydrogenase maturation, and this process is studied using standard molecular biological and biochemical techniques, and EPR, FTIR, XAS and GEMMA spectroscopic techniques combined with this new type of chemistry mentioned above. EPR spectroscopy was employed to verify the construction of a semi-synthetic [FeFe] hydrogenase inside living cells. The addition of a synthetic complex to cell cultures expressing apo-HydA resulted in a rhombic EPR signal, attributable to an Hox-like species. Moreover, the assembly mechanism of the H-cluster was probed in vitro using XAS, EPR, and FTIR spectroscopy. We verified with all three techniques that the Hox-CO state is formed on a time-scale of seconds, and this state slowly turns into the catalytically active Hox via release of a CO ligand. Furthermore, a semi-synthetic form of the HydF protein from Clostridium acetobutylicum was prepared and characterized in order to prove that such semi-synthetic forms of HydF are biologically relevant. Finally,GEMMA measurements were performed to elucidate the quaternary structure of the HydF-HydA interaction, revealing that dimeric HydF is interacting with a monomeric HydA. However, mutant HydF proteins were prepared, lacking the dimerization (as well as its GTPase) domain, and these severely truncated forms of HydF was found to still retain the capacity to both harbor the pre-catalyst as well as transferring it to apo-HydA. These observations highlight the multi-functionality of HydF, where different domains are critical in different steps of the maturation, that is the dimerization and GTPase domain are rather involved in pre-catalyst assembly rather than its transfer to apo-HydA.