Coming dissertations at Uppsala university
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Advanced molecular tools for diagnostic analyses of RNA and antibodies in situ and in solution
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-522118
Advanced molecular diagnostics uses in vitro biological assays to detect nucleic acids or proteins even in low concentrations across samples, allowing for the identification of biomarkers, monitoring the course of the disease over time, and selection of appropriate therapy. In this thesis, I focus on development and early applications of several molecular tools of expected value in research, and eventually also clinically.
In papers I and II, proximity extension assay (PEA) was for the first time modified to measure specific antibody responses, rather than protein levels as in the standard PEA. We call the method AbPEA and the technique was used to sensitively measure antibody responses to the spike protein or the nucleocapsid of SARS-CoV-2. We demonstrated that AbPEA has high specificity, sensitivity, and broad dynamic range, along with multiplexing potential, offering performance similar to that of other methods for antibody measurements. We demonstrated utilization of blood and saliva samples in paper I and paper II, respectively, which further establish that our approach has great potential for large-scale screening and biobanking.
In paper III, we aimed to investigate how the protein composition of extracellular vesicles (EVs) differed among blood samples collected from healthy individual or ones with either mild or severe COVID-19. Proximity barcoding assay was applied to obtain a comprehensive overview of the protein composition of large numbers of individual EVs, demonstrating interesting differences.
In paper IV, we enhanced padlock-RCA-based RNA genotyping in situ by using another newly developed technology for highly selective detection of DNA or RNA sequence variants, referred to as super RCA (sRCA). Our analysis showed that this approach can improve the selectivity for sequence variants during in situ detection of mutant or wild-type transcripts, and the signals representing superRCA reaction products are prominent and easily distinguished from any background.
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Exploring Reaction Pathways in Li-ion Batteries with Operando Gas Analysis
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-522294
The reliance on Li-ion batteries is increasing as we transition from fossil fuels to renewable energy sources. Despite their widespread use, a gap remains in understanding certain processes within these batteries, especially regarding the solid electrolyte interphase (SEI) and the impact of side reactions on Li-ion batteries. A custom-made Online Electrochemical Mass Spectrometry (OEMS) instrument was designed to explore these aspects. The OEMS instrument was validated through the study of gas-evolving reactions in the classic LiCoO2 | Graphite system. In-depth studies focusing on the reaction pathways of ethylene carbonate, the archetype Li-ion battery electrolyte solvent, identified the specific reaction pathways contributing to SEI formation. Moreover, ethylene carbonate’s interaction with residual contaminants like OH– from H2O reduction was explored. It was revealed that the integrity of the SEI can be compromised by minor amounts of contaminants, establishing a competitive dynamic at the negative electrode surface between ethylene carbonate and residual contaminants such as H2O and HF. Additionally, the roles of additives like vinylene carbonate and lithium bis(oxolato) borate in SEI formation were explored. Vinylene carbonate was shown to form a layer on the negative electrode, but also scavenge protons and H2O, revealing that it is a multi-functional additive. Lithium bis(oxolato) borate on the other hand formed an SEI layer before H2O reduction, blocking the residual contaminant and ethylene carbonate from reaching the electrode surface. By providing insights into the negative electrode’s interphase and SEI formation through a custom-made OEMS instrument, this research underscores the complexity of reaction pathways and the necessity of considering both major and minor, as well as, primary and secondary reactions for a holistic understanding of Li-ion batteries.
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Function and Evolution of Small Regulatory RNAs and their Associated Proteins : A Journey from Genome to Proteome
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-522164
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.