| 1. |
- Aguirre Rivera, Javier, 1989-
(författare)
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Tracking single molecules in uncharted territory : A single-molecule method to study kinetics in live bacteria
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The synthesis of proteins, also known as translation, is a fundamental process in every living organism. The steps in the translation of genetic information to functional proteins have been meticulously studied, mostly using in vitro techniques, yielding a detailed model of their mechanisms. However, the use of minimal cell-free systems allows for the possibility to miss interactions from absent components or that reactions are affected by the buffer composition. The work presented in this thesis opens a way to study the kinetics of complex molecular processes, like protein synthesis, directly inside live bacterial cells in real time. We developed and optimized a method to deliver dye-labeled macromolecules inside live cells and generate a kinetic model of the particle’s interactions based on its diffusion inside the cell.This method facilitated the study of translation elongation and initiation directly in live cells. Our measurements of reaction times of tRNA in the ribosome, agree with previous reports from in vitro techniques. We further applied the method to examine the effects of three aminoglycoside antibiotics and erythromycin directly in live cells. The aminoglycoside antibiotics slowed-down protein synthesis 2- to 4-fold, while the number of elongation cycles per initiation event decreased significantly. In the case of erythromycin, cells showed a 4-fold slower protein synthesis. Additionally, we measured the kinetics of sequence-specific effects of erythromycin: translational arrest, and peptidyl-tRNA drop-off; these in vivo measurements revealed a complex mechanism of action of the drug, in agreement with models suggested by previous experiments. Additionally, we applied the method to measure the effects, on the kinetics of protein synthesis, caused by modifications in the C-terminal tail of the S13 ribosomal protein. Our measurements showed that specific mutations led to different changes in the occupancy and dwell-time of labeled-tRNA in the ribosome.To summarize, the present work will guide the reader through the development of a method to study the kinetics of protein synthesis directly in live bacterial cells, as well as its application to characterize the effects of different antibiotics within the complex environment of a living organism.
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| 2. |
- Alghamdi, Fayiq, 1985-
(författare)
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Dimensions of Professionalism : A Study of Computer Science Teaching in Saudi Arabia
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In Saudi Arabia, new computing education programs have been introduced in alignment with the Saudi Vision 2030, which is a plan launched in 2017 to reduce Saudi Arabia's reliance on oil, diversify its economy, and develop its health, education, recreation, infrastructure and tourism. Computer science is a rapidly changing area, which places high demands on teachers in the subject to develop both their subject and pedagogical competence. This thesis explores computer science teachers’ perspectives on professional development from three viewpoints—the Saudi Teaching Competencies Standard, engagement in teachers’ awards and self-directed learning. The thesis examines the efforts of computer science teachers as they develop new pedagogies during their teaching careers as a result of the new regulations. The main question is ‘How do Saudi Arabian computer science teachers develop their teaching professionalism?’ Conclusions draw on the outcomes of four sub-studies. A mixed-methods approach consisting of interviews and questionnaires was used to collect data. The participants comprised 389 computer science teachers from different Saudi Arabian cities with different demographics and different teaching experience. The analysis drew on a theoretical framework that integrates elements of the Theory of Reasoned Action, the Theory of Planned Behaviour and the Adult Learning Theory. A model for pedagogical change was developed and used to understand how and why computer science teachers change their educational pedagogy. The model explains the teachers’ shift in pedagogy and answers the question of how and why computer science teachers adopt a new pedagogical strategy. The studies show that both internal and external factors motivate the study participants to engage in competency development. In the Saudi model, the Saudi Teaching Competencies Standard and awards are external factors as they include a preparatory period of intensive skills development. Teachers' experience from this informs the picture of Saudi teachers' training that is presented in the dissertation. Indeed, the trial participants stated that they mainly used self-directed learning for their competence development, drawing on internal motivation. One reason for this was that they felt that many of the skills development programs offered lacked timeliness and relevance. The studies on which the dissertation is based have been conducted in Saudi Arabia, but the results also provide insights into general challenges associated with regulating teachers' competence and the design of in-service training for teachers. The results clearly point out the importance of teachers' participation in the development of the profession in order for changes to be accepted and incorporated into their profession. Behavior change theories can be used to understand and predict how new regulations and pedagogical strategies will be received, and if they are likely to be accepted or rejected by teachers. These theories, therefore, constitute a useful tool in regulating teaching and the teaching profession.
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| 3. |
- Ali, Hasan, 1985-
(författare)
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Towards atomically resolved magnetic measurements in the transmission electron microscope : A study of structure and magnetic moments in thin films
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The magnetic properties of thin metallic films are significantly different from the bulk properties due to the presence of interfaces. The properties shown by such thin films are influenced by the atomic level structure of the films and the interfaces. Transmission electron microscope (TEM) has the potential to analyse the structure and the magnetic properties of such systems with atomic resolution. In this work, the TEM is employed to characterize the structure of the Fe/V and Fe/Ni multilayers and the technique of electron magnetic circular dichroism (EMCD) is developed to obtain the quantitative magnetic measurements with high spatial resolution.From TEM analysis of short period Fe/V multilayers, a coherent superlattice structure is found. In short period Fe/Ni multilayer samples with different repeat frequency, only the TEM technique could verify the existence of the multilayer structure in the thinnest layers. The methods of scanning TEM imaging and electron energy loss spectroscopy (EELS) results were used and refined to determine interdiffusion at the interfaces. The confirmation of the multilayer structure helped to explain the saturation magnetization of these samples.Electron magnetic circular dichroism (EMCD) has the potential to quantitatively measure the magnetic moments of the materials with atomic resolution, but the technique presents several challenges. First, the EMCD measurements need to acquire two EELS spectra at two different scattering angles. These spectra are mostly acquired one after the other which makes it difficult to guaranty the identical experimental conditions and the spatial registration between the two acquisitions. We have developed a technique to simultaneously acquire the two angle-resolved EELS spectra in a single acquisition. This not only ensures the accuracy of the measurements but also improves the signal to noise ratio as compared to the previously used methods. The second important question is the effect of crystal orientations on the measured EMCD signals, considering the fact that the crystal orientation of a real crystal does not remain the same in the measured area. We developed the methodology to simultaneously acquire the EMCD signals and the local crystal orientations with high precision and experimentally showed that the crystal tilt significantly changes the magnetic signal. The third challenge is to obtain EMCD measurements with atomic resolution which is hampered by the need of high beam convergence angles. We further developed the simultaneous acquisition technique to obtain the quantitative EMCD measurements with beam convergence angles corresponding to atomic size electron probes.
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| 4. |
- Ali, Muhammad, 1990-
(författare)
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Identification of SLiMs: Mapping and characterizing motif-based protein interactions
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During the last twenty years it has become evident that about 35-40% of amino acids in the proteome are in regions that have evolved to remain unstructured. These intrinsically disordered regions contain short linear motifs (SLiMs), which serve as docking sites for protein-protein interactions. SLiMs often mediate low-to-medium affinity interactions that are transient in their nature. The characteristics of SLiM-based interactions make them difficult to be captured using conventional approaches like affinity-purification coupled to mass spectrometry or yeast-two-hybrid. We therefore used and developed a dedicated method for large-scale screening of SLiM-based interactions termed proteomic peptide phage display (ProP-PD).Using ProP-PD, We identified large sets of ligands, for the binding pocket of shank1 PDZ domain, containing C-terminal or internal binding motifs and established the consensus motifs to be xTxL/F-COOH and xTxFx respectively. We further validated interactions using biophysical affinity determinations and pulldown experiments. Using X-ray crystallization, we uncovered that shank1 PDZ binds to internal xTxFx motifs using a binding mode similar to that for C-terminal peptides.Adding a level of complexity, we explored interactions of the multiple binding pocket containing FERM domains from four closely related proteins: ezrin, radixin, moesin and merlin. We found hundreds of FERM ligands, which contained binding motifs of at least four different classes. By combining docking simulations with experiments, we established ligands binding to different pockets, and uncovered a complex interplay between distinct pockets.We further developed an optimized version of a phage library that displays intrinsically disordered regions of the human proteome. We benchmarked the library using a set of protein domains and reported better recovery of known SLiM-based interactions. Furthermore, we highlighted the functional aspects of identified SLiMs, in the case of nuclear localization signals, found for binding to importin-subunit alpha-3. Finally, we validated predicted binding of SLiMs in the Sars-CoV-2 host receptor ACE2, which illustrates the importance of fundamental knowledge for SLiMs and their binding partners.This work, taken together, contributes with method development for expansion of motifs based interactomes and provide insights into the plastic yet selective nature of peptide binding proteins.
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| 5. |
- Alipour, Mehdi
(författare)
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Rethinking Dynamic Instruction Scheduling and Retirement for Efficient Microarchitectures
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Out-of-order execution is one of the main micro-architectural techniques used to improve the performance of both single- and multi-threaded processors. The application of such a processor varies from mobile devices to server computers. This technique achieves higher performance by finding independent instructions and hiding execution latency and uses the cycles which otherwise would be wasted or caused a CPU stall. To accomplish this, it uses scheduling resources including the ROB, IQ, LSQ and physical registers, to store and prioritize instructions.The pipeline of an out-of-order processor has three macro-stages: the front-end, the scheduler, and the back-end. The front-end fetches instructions, places them in the out-of-order resources, and analyzes them to prepare for their execution. The scheduler identifies which instructions are ready for execution and prioritizes them for scheduling. The back-end updates the processor state with the results of the oldest completed instructions, deallocates the resources and commits the instructions in the program order to maintain correct execution.Since out-of-order execution needs to be able to choose any available instructions for execution, its scheduling resources must have complex circuits for identifying and prioritizing instructions, which makes them very expansive, therefore, limited. Due to their cost, the scheduling resources are constrained in size. This limited size leads to two stall points respectively at the front-end and the back-end of the pipeline. The front-end can stall due to fully allocated resources and therefore no more new instructions can be placed in the scheduler. The back-end can stall due to the unfinished execution of an instruction at the head of the ROB which prevents other resources from being deallocated, preventing new instructions from being inserted into the pipeline.To address these two stalls, this thesis focuses on reducing the time instructions occupy the scheduling resources. Our front-end technique tackles IQ pressure while our back-end approach considers the rest of the resources. To reduce front-end stalls we reduce the pressure on the IQ for both storing (depth) and issuing (width) instructions by bypassing them to cheaper storage structures. To reduce back-end stalls, we explore how we can retire instructions earlier, and out-of-order, to reduce the pressure on the out-of-order resource.
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| 6. |
- Ament-Velásquez, Sandra Lorena, M.Sc. 1988-
(författare)
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Drivers of evolutionary change in Podospora anserina
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Genomic diversity is shaped by a myriad of forces acting in different directions. Some genes work in concert with the interests of the organism, often shaped by natural selection, while others follow their own interests. The latter genes are considered “selfish”, behaving either neutrally to the host, or causing it harm. In this thesis, I focused on genes that have substantial fitness effects on the fungus Podospora anserina and relatives, but whose effects are very contrasting. In Papers I and II, I explored the evolution of a particular type of selfish genetic elements that cause meiotic drive. Meiotic drivers manipulate the outcome of meiosis to achieve overrepresentation in the progeny, thus increasing their likelihood of invading and propagating in a population. In P. anserina there are multiple meiotic drivers but their genetic basis was previously unknown. In Paper I, we demonstrated that drive is caused by members of the Spok gene family. We discovered two new Spok genes, Spok3 and Spok4, which locate in different chromosomes in different strains. In Paper II, we showed that Spok3 and Spok4 are found on a gigantic (up to 247 Kb long) variant of Enterprise, a Crypton-like transposable element. Enterprise likely mobilize through the action of a putative tyrosine-recombinase that we call Kirc. When carrying the Spoks, this element has double selfish properties: transposition and meiotic drive. In addition, we found that homologs of the Spoks (Paper I) and of Kirc (Paper II) are widespread in fungi but their phylogenies are discordant with that of the species, suggesting that they have undergone horizontal gene transfer. In Papers III and IV, I turned the focus into genes that have an adaptive function. In fungi, the het genes control conspecific self/non-self recognition. Such genes are expected to evolve under frequency-dependent balancing selection. In Paper III, we found evidence of balancing selection acting on some het genes across the P. anserina species complex. Unexpectedly, we also discovered that het genes of the HNWD gene family are duplicated in a transposon-like manner, broadening our understanding of their potential fitness effects. Finally, in Paper IV we show how het genes with pleiotropic effects on sexual recognition lead to the evolution of strong reproductive isolation, and hence speciation. Overall, the results of my thesis highlight the functional intersection between mobile selfish genetic elements and other genes, either selfish or adaptive, and their effects on genome architecture and population structure.
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| 7. |
- Amini, Samar
(författare)
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Source analysis of multiplet earthquakes (two case studies in Iran)
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Multiplet earthquakes are large earthquakes of similar magnitude which occur close in time in the same limited geographical area. They are not common but they considerably increase the potential hazard in the area in which they occur. This thesis studies source properties and triggering mechanisms of two sets of multiplet events in Iran, which both occurred in unexpected areas, but close to some major active fault systems. The first multiplet is an earthquake doublet (Mw 6.5 and Mw 6.4) which occurred in northwestern Iran and caused more than 300 fatalities and significant injuries. In paper I, a teleseismic body-waveform inversion was used to deduce the slip distribution pattern on the fault plane of the first mainshock. The estimated slip pattern was utilized to calculate the Coulomb stress changes on the second fault plane and on the following aftershocks. Based on this analysis, the ambiguity between the primary and auxiliary fault plane of the second mainshock could be resolved. The second set of events is a triplet (Mw 6.1 - 6.0) that occurred in eastern Iran, close to the Kerman province. In paper II, the rupture propagation patterns of the three mainshocks were analyzed using Empirical Green’s Function (EGF) deconvolution. Two different approaches were used: One, the analysis of the azimuthal variation of the apparent rupture duration based on the width of the observed relative source time functions, and the second, the analysis of along-strike rupture directivity by assessing azimuthal variations of the relative amplitude spectra. The second approach was also used to investigate the rupture directivity of the largest aftershocks in the sequence (Mw 5 - 5.5). A clear tendency for rupture propagation towards the northwest was observed for the sequence, which suggests that the regional stress field has a central role in controlling the rupture propagation direction. In paper III, the slip distribution patterns of the triplet earthquakes were analyzed using teleseismic body-waveform inversion, and the similarities and differences in the rupture processes of the three mainshocks were investigated. Using the Coulomb stress analyses, the stress interactions between the mainshocks were examined, leading to identification of the primary and auxiliary planes. Finally, we suggest that the hazard estimates in complex continental regions such as Iran need to consider the probability of multiplets, which might allow a reduction of the seismic risk associated to the occurrence of further large earthquakes subsequent to a devastating earthquake.
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| 8. |
- Baião, Guilherme Costa, 1984-
(författare)
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Genomic and transcriptomic investigation of reproductive incompatibility in Drosophila
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Both nuclear and cytoplasmic elements can contribute to the emergence of reproductive incompatibilities that influence evolution and speciation. In the projects that compose this thesis, we use genomics and transcriptomics to study some of those elements in Drosophila.In the first study, we show that Wolbachia, an endosymbiotic bacterium known to cause reproductive alterations in its hosts, influences gene expression in D. paulistorum. Affected genes were associated with biological functions such as metabolism, immunity, reproduction, and chemical communication. Our results indicate that Wolbachia accentuates the differences in expression profiles between semispecies and suggest that the symbiont influences host pre-and postmating isolation. In the second paper, we uncover widespread persistent heteroplasmy in D. paulistorum. We reveal that D. paulistorum mitochondria are polyphyletic, with two divergent mitotypes, and that the heteroplasmy likely originated through introgression. One of the mitotypes shows biparental inheritance, non-responsiveness to host energy demands and rapid titer increase in the early embryo. We hypothesize that such selfish traits evolved in response to competition between mitotypes.In the third project, we show that differentially expressed genes between D. paulistorum semispecies are associated with a variety of biological processes, especially broad regulatory functions that occur via variability in transcription, translation and ubiquitination of post-translational modification. We reveal that the expression profile of F1 inter-semispecies hybrids is markedly similar to that of the maternal line, and that Wolbachia has a small but potentially significant interaction with genes that are differentially expressed in semispecies and F1 hybrids.Finally, we use comparative genomics to study the evolution of closely related Wolbachia strains with known reproductive phenotypes. We confirm previous observations that Wolbachia genomes are very dynamic and that phage-associated regions are particularly variable and likely involved in horizontal transfer of genes linked to reproductive phenotypes. An in-depth screen for genetic elements potentially involved in Wolbachia-induced cytoplasmic incompatibility recovers genes previously known to be involved in the phenotype and novel candidates.In conclusion, this thesis contributes to our understanding of genetic factors that affect Drosophila evolution, particularly those leading to reproductive incompatibility in D. paulistorum and associated with Wolbachia.
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| 9. |
- Bedin, Michele, PhD, 1984-
(författare)
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Iron, Manganese and Iridium Complexes From Models of RNR and Catalase to Water Oxidation
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The focus of this thesis has been synthesise and study metal complexes that mimic the structure and function of the active site in two particular metalloenzymes, ribonucleotide reductase (RNR) and manganese catalase (MnCAT). These two metalloenzymes both have two transition metals ions in the cofactor: two manganese ions in MnCAT and either two iron, iron-manganese or two manganese ions in RNR.Three different ligands were synthetized to make model complexes for these cofactors. The first ligand, BPMP, can bind two metal ions and provides two symmetric pockets with two pyridine groups and one amine each, plus a phenolate group that can bridge the two metals. The second ligand DPCPMP had one carboxylate group instead of a pyridine group in one pocket, creating an asymmetric ligand, and the third ligand BPCPMP, had two carboxylate groups, one in each pocket. From the first and the second ligands it was possible to obtain six complexes: low-valent homometallic Mn/Mn and Fe/Fe complexes and a heterometallic complex for each ligand. For the third ligand, only the Fe/Fe complex was synthetized.All seven complexes were characterized by a number of spectroscopic methods. The presence of carboxylate groups in the ligand shifted the redox potential for the metal complexes towards more negative values, particularly in the case of the homometallic Fe/Fe complexes. Surprisingly, for the asymmetric ligand the placement of the metal ions in the two pockets was not dictated by the asymmetry. Additionally, the relative stability of the homometallic complexes versus the heterometallic complexes and the possibility to transform a homometallic complex into a heterometallic complex were investigated. By titrating one metal into a solution containing the other homometallic dimer it was possible to observe that Fe2+ added to a solution of a Mn/Mn complex led to the replacement of one Mn ion in the complex with a Fe ion.The manganese complex of DPCPMP was investigated as a functional model for MnCAT, catalysing the disproportionation of H2O2 to oxygen and water. In the presence of H2O2 this complex also forms a high-valent species with a di-µ-oxo bridge similar to the MnCAT and RNR.Finally, the methodology used for the study of these complexes was also applied to a set of Ir complexes that act as water oxidation catalysts, and we could show that the presence of a pendant group stabilizes the metal at higher oxidation states leading to higher activity for the catalyst.
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| 10. |
- Billinger, Erika, 1986-
(författare)
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Characterization of conjugated protease inhibitors
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The overall theme of this thesis is a step by step approach for the design and characterization of conjugated protease inhibitors. This involves both a new assay method for protease activity and protease inhibition (paper I), a study of the stoichiometry for protease inhibitor interaction (paper II), design of inhibitory peptides (paper IV) and the construction of inhibitor conjugates (paper III & IV).(I) A model based primarily on erosion in gelatin for protease activity and inhibition studies was designed. The model was also extended to a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and rate of erosion was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in the erosion rate. Kinetic analyses of a two-layer model with substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top “protective” layer.(II) The binding stoichiometry between pancreatic proteases and a serine protease inhibitor purified from potato tubers was determined by chromatography-coupled light scattering measurements. This revealed that the inhibitor was able to bind trypsin in a 2:1 complex, whereas the data for a-chymotrypsin clearly showed a limitation to 1:1 complex. The same experiment carried out with elastase and the potato inhibitor gave only weak indications of complex formation under the conditions used.(III) A serine protease inhibitor was extracted from potato tubers and conjugated to soluble, prefractionated dextran or inorganic particles. A certain degree of inhibitory activity was retained for both the dextran-conjugated and particle-conjugated inhibitor. The apparent Ki value of the dextran-conjugated inhibitor was found to be in the same range as that for free inhibitor. The dextran conjugate retained a higher activity than the free inhibitor after 1 month of storage at room temperature. Conjugation to oxide particles improved the heat stability of the inhibitor.(IV) New synthetic Leupeptin analogues, Ahx-Phe-Leu-Arg-COOH & Ahx-Leu-Leu-Arg-COOH, were synthesized with solid-phase peptide synthesis using Fmoc strategy. These tripeptide inhibitors were tight binding inhibitors to the target enzyme trypsin, similar to the natural occurring leupeptin. The phenylalanine containing synthetic analogue was conjugated to inorganic particles and agarose gel beads. In all cases, the inhibitory activity was well preserved.
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