Doctoral Theses / Doktorsavhandlingar Institutionen för kemi och molekylärbiologi

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A Holistic View on Aquaporins: Production, Structure, Function and Interactions
(2020-10-21) Schmitz, Florian
Aquaporins are specialised membrane proteins, which regulate the water homeostasis of cells. In eukaryotic organisms, this process is tightly regulated, and aberrations in aquaporin functionality lead to severe pathologies in humans. The aim of this thesis is to shed light on the aquaporin function and regulation, both as individual protein targets and in the cellular context, as well as exploring various applications for human aquaporin 4, specifically. A wide range of biochemical methods have been applied, ranging from the importance of robust protein production methods, for targets as well as for their complexes, to functional and structural characterization. For biochemical characterization and structural analysis, large amounts of pure, homogeneous and stable recombinant protein are needed. The methylotrophic yeast Pichia pastoris was utilized for the overproduction of the soluble protein Sirtuin2, an indirect up-regulator of Aquaporin4 in humans. The highest yet-reported yield of the protein (40 mg/l) was achieved, facilitating modulation trials of the potential drug target. The P. pastoris overproduction system was also employed for the expression of human AQP4, facilitating new research applications, such as improved Neuromyelitis Optica diagnosis, and a better understanding of the intermolecular binding between the monomeric subunits. In addition, the novel structural characteristics of AQP1 from the fish Anabas testudineus was studied in this thesis and key residues responsible for the molecular mechanisms for osmoregulation were identified by mutational analysis combined with functional studies. By combining stopped-flow assays and molecular dynamics simulations, a novel extracellular gating mechanism could be elucidated for this particular aquaporin isoform, being less efficient in water transport than AQP4 and phosphorylation of Tyrosine 107 leads to a closed conformation involving loop C. Functional studies were also performed for the development of a new method for testing the transport specificity of aquaporins regarding hydrogen peroxide. The transport rate can be standardized in relation to protein quantity, resulting in a more accurate determination of transport rates as compared to cell growth assays. Interactions between proteins are difficult to evaluate, but using bimolecular fluorescence complementation, membrane protein complexes could be quantified and screened in vivo in a high-throughput manner. During the course of this work, we standardized sample preparation and defined criteria which allow the discrimination between constructive and random interactions. Taken together, the results presented in this thesis lay the fundament for future screening for novel interaction partner using a cDNA library, a method that is not limited to aquaporins.
A Soot Transformation Study: Interactions Between Soot, Sulfuric Acid and Secondary Organic Aerosol (SOA)
(2018-01-19) Pei, Xiangyu
Atmospheric black carbon (BC), generally called soot, is the most important aerosol component that warms the Earth’s climate significantly, and reducing atmospheric soot level has been proposed as a strategy for near-term climate change mitigation. However, policy development is hampered by large uncertainties in models’ predictions regarding the global warming induced by BC. These uncertainties primarily result from a limited scientific understanding of the transformations soot undergoes upon interacting with other aerosol components such as sulfuric acid and secondary organic aerosol (SOA). Unlike soot, sulfuric acid and SOA are thought to induce cooling effects. However, soot – sulfuric acid – SOA interactions are postulated to amplify soot’s warming effect. Condensed materials such as sulfuric acid and SOA can modify soot’s morphology, i.e. the distribution of soot aggregates and condensates in space, thereby altering its properties and atmospheric life time. The overall aim of this thesis is to characterize freshly emitted soot and the transformations in morphology and optical properties it undergoes upon condensation of sulfuric acid and SOA. Two frameworks were developed for quantifying the in-situ morphological properties of BC mixed with either primary organic aerosol (POA) during evaporation process or sulfuric acid and/or SOA during condensation process. The morphological transformation of soot particles was quantified with these frameworks in terms of void fractions, effective densities, and in-situ dynamic shape factors. Soot morphological transformation during condensation process was shown to occur via two complementary and sequential processes: the filling of voids within particles and mobility diameter growth. In addition, the light absorption of soot from two flame types (an industrial flame and three lab-scale flames) was studied. Significant quantities of light absorbing organics (referred to as brown carbon, BrC) were observed in lab-scale flames, but the mature soot in the industrial flame did not contain BrC. The mass absorption cross section (MAC) of BC and BrC from lab-scale flames was quantified, and the values for BrC proved to be comparable to those for BC at a short wavelength (405 nm). The most widely used model for quantifying the optical properties of coated soot at present is core-shell Mie theory, in which the key parameter is the refractive index. This study identified and evaluated alternatives to Mie theory. It was found that the nature of the condensed material can significantly influence the light absorption of coated soot particles, and that reaction between soot and sulfuric acid can have particularly important effects. The absorption cross section of soot was significantly reduced (by up to 26%) upon interaction of the soot surface with sulfuric acid, whereas the absorption cross section increased significantly when soot was coated with SOA or acidity-mediated SOA. Field studies of soot coated with other aerosol components, i.e. organics, sulfate and nitrate, were conducted in Beijing, and the results were compared to the lab studies. The findings suggested that ambient BC particles during summertime in Beijing was internally mixed and heavily coated since their effective density was similar to the material density of the coatings.
A tale of two songs: oligomeric and monomeric functions of the molecular chaperone CCT
(2024-11-07) Córdoba Beldad, Carmen Maria; Córdoba Beldad, Carmen Maria
The chaperonin-containing tailless complex polypeptide 1 (CCT) is a 1 MDa barrel shaped molecular chaperone present in the cytoplasm of all eukaryotes. Eight different subunits, located in a fixed position, form CCT, which is mostly studied as the folding machinery of the cytoskeletal proteins actin and tubulin. Roles beyond folding have been reported for the CCT oligomer and together with the increasing evidence of monomeric CCT subunit functions, have elevated the importance of studying the chaperone CCT. Here, we show that the transcription factor STAT3, a previously reported oligomeric binding partner of CCT, does not behave as an obligate folding substate but rather is sequestered by CCT. To expand our knowledge of the monomeric roles of the CCT subunits, we have studied the interaction between CCTδ and p150Glued, a component of the dynactin complex involved in crucial biological processes such as mitosis. We show that monomeric CCTδ is required for the correct localisation of p150Glued at spindle poles and for accurate chromosome segregation. Furthermore, we explore interactions between two other CCT subunits and two regulatory components of mitosis, the centromeric protein Mis18BP1 and the outer kinetochore component KNL1. Taken together the work in this thesis extends the understanding of both oligomeric and monomeric functions of CCT beyond folding.
A Waterborne Colloidal Model System Consisting of Fluorinated Spheres Bearing Grafted PEG: Synthesis, Characterization and Properties
(2016-01-08) Ulama, Jeanette
Model systems have expanded our knowledge of numerous phenomena in Colloid Science, such as the appearance of glasses, order-disorder transitions involving crystals and attraction induced formation of gels. So far, the existing colloidal model systems have been limited mainly to nonaqueous media. Given that water is such an important solvent, an aqueous colloidal model system is called for. Here we present such an aqueous colloidal model system with core-shell particle morphology, where the interior is composed of spherical fluorinated cores and the exterior of a poly(ethylene glycol) (PEG) polymer graft. To synthesize these colloids, we have adopted a semi-batch emulsion polymerization, in which the initiator is slowly fed into the reaction mixture. Using this approach not only can monodisperse, low refractive index and sterically stabilized colloids be produced, but also various lengths of the PEG polymer could be successfully grafted onto the particles. Throughout this thesis, several different instrumental techniques have been used to gain an insight into the collective phenomena of these particles and how particle interactions contribute to the observed phase behavior. Although steric stabilization is very robust way of stabilizing colloidal particles against aggrega- tion, attractions between particles can nevertheless appear, e.g. through the addition of certain salts or addition of a non-solvent. The origin of these attractions is not fully understood. Our results show that colloidal stability increases with decreasing length of the steric stabilizer and that the polymer graft contracts as the solvent quality is worsened. The contraction is accompanied by moderately strong attractions even though the van der Waals force due to core-core interactions is essentially absent. It follows that the attractions are caused by purely polymer-mediated interactions.
Adaptation and Protein Quality Control Under Metalloid Stress
(2015-04-22) Ibstedt, Sebastian
Toxic metals and metalloids are emerging as major environmental pollutants, having ecological consequences as well as being linked to a broad range of degenerative conditions in animals, plants and humans. While the toxicity of several metalloids is well established, the underlying molecular mechanisms are often not clear. Several human degenerative diseases are linked to misfolding and aggregation of specific proteins. I have shown that many of these proteins have yeast homologs that are particularly prone to misfolding and aggregation during arsenite exposure. The yeast proteins are highly dependent on chaperones for proper folding, whereas arsenite is capable of inhibiting chaperone function as well as causing additional aggregation through a propagating effect. Computational analyses further revealed that aggregation-prone proteins are abundant and have a high translation rate, but are down-regulated when the cell encounters arsenite. The mechanisms behind tellurite toxicity have eluded scientists for over a century. By using a genome-wide phenotypic screen, it was found that tellurite toxicity is linked to accumulation of elemental tellurium. Sulfate metabolism and mitochondrial respiration were found to mediate toxicity. An understanding of cellular function requires knowledge of the evolutionary processes that have formed it. However, distinguishing between adaptive and non-adaptive differentiation remains an extraordinary challenge within evolutionary biology. The last part of this thesis tests a method for exposing the role of natural selection in evolution of stress tolerance. Analysis of concerted optimization of performance in distinct fitness components followed by mapping of the genetic basis for the optimizations, compellingly suggests that the method is able to detect natural selection. The results presented here are likely to be relevant in gaining a better understanding of the mechanisms behind arsenite and tellurite poisoning and cellular defense, and may form a basis for elucidating evolutionary adaptations in other environments and organisms.
Advances in Membrane Protein Structural Biology: Lipidic Sponge Phase Crystallization, Time-Resolved Laue Diffraction and Serial Femtosecond Crystallography
(2013-05-08) Johansson, Linda
Membrane proteins carry out many essential tasks in cells such as signaling and transport, or function as electron carriers in photosynthesis and cellular respiration. The aim of this thesis has been to develop new and improve existing techniques for elucidating the structure and function of membrane proteins. Membrane proteins are difficult to crystallize due to their combination of hydrophilic and hydrophobic domains. Part of this thesis was therefore dedicated to the development of a membrane protein crystallization screen based on mimicking the protein’s native environment. The screen, consisting of 48 different lipidic sponge phase (LSP) conditions, was tested on eleven different membrane proteins and gave crystal leads for eight of these. One of these leads was the photosynthetic reaction center of the purple bacterium Blastochloris viridis (RCvir). Two high-resolution structures to 1.86 Å and 1.95 Å were obtained from data collected using different radiation doses and revealed a new space group and novel crystal packing along with a number of lipid-protein interactions. Using this new crystal form the electron-transfer reaction of RCvir was studied by time-resolved Laue diffraction where data were collected on crystals illuminated with light at room temperature. This revealed a reproducible movement of the highly conserved TyrL162 residue towards the special pair upon photoactivation. These results were combined with molecular dynamics studies to propose a coupling between the conformational orientation and protonation states within a bacterial reaction center. Finally, the LSP method was extended to a batch type of crystallization approach. This provided a large volume of micron-sized crystals suitable for structure determination at the Linac Coherent Light Source, a recently commissioned X-ray free electron laser (XFEL) facility. Data from hundreds of microcrystals were collected to low resolution and revealed yet another space group and crystal packing. After the commissioning of a high-resolution beamline, the structure of RCvir was solved to 3.5 Å resolution. This represents the highest resolution membrane protein structure determined using XFEL radiation to date.
Advancing the use of serial crystallography in drug discovery
(2025-04-02) Dunge, Andreas
This thesis explores the application of serial synchrotron crystallography (SSX) in drug discovery, focusing on the structural studies of soluble epoxide hydrolase (sEH) and cytochrome P450 3A4 (CYP3A4). Utilizing SSX, a relatively novel technique that collects data from a large number of small crystals at room temperature (RT), this research aims to investigate the structural dynamics and ligand interactions of these enzymatic targets. A workflow is developed that enables the transition from macrocrystals to microcrystals, and this study incorporates ligand soaking to explore protein-ligand interactions in their near-native states. We successfully established a method for obtaining microcrystals and soaking compounds, which facilitated a comprehensive fragment study. This study led to the identification of 40 active site-binding fragments out of 384 tested for sEH, showcasing the method's effectiveness. Additionally, the RT structures revealed conformational nuances of F497 in sEH, with inward movement observed in response to potent inhibitors. For CYP3A4, an RT structure provided valuable comparisons to cryogenic analyses, emphasizing structural differences. The thesis highlights SSX's capability to capture structural information that may be obscured under traditional cryogenic conditions, demonstrating its utility alongside conventional cryogenic macromolecular crystallography to enhance understanding of protein-ligand complexes. Additionally, the work addresses the logistical and technical challenges inherent in SSX and proposes strategies to optimize experimental conditions effectively. By focusing on these targeted studies, this research highlights the capability of SSX to advance drug discovery efforts. SSX offers a novel approach for detailed structural analysis of proteins, enabling the identification of interactions which could be hidden at cryogenic temperature which can be useful for designing effective therapeutic agents.
AI-based Spectra Processing and Analysis for NMR
(2025-04-11) Jahangiri, Amir; Jahangiri, Amir
Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful analytical technique for obtaining atomic-level information across various scientific fields. However, direct interpretation of raw NMR data is impractical due to its complexity. While traditional signal processing methods are widely used, they face limitations in handling advanced tasks. Consequently, advanced computational approaches are required to optimize spectral reconstruction and improve analytical precision. Artificial Intelligence (AI), particularly deep learning, has demonstrated significant potential in addressing these challenges. This thesis explores AI-based signal processing in NMR spectroscopy, focusing on spectral reconstruction, resolution enhancement, and quality assessment. The first study introduces Low-Rank Decoupling (LRD), a method leveraging prior knowledge of J-coupling for homonuclear virtual decoupling. LRD enhances spectral resolution while maintaining sensitivity and minimizing artifacts, outperforming conventional decoupling approaches. The second study presents Magnetic Resonance processing with Artificial intelligence (MR-Ai) as an alternative to conventional nonlinear NMR processing. A 1D WaveNet-based NMR Network (1D WNN) is developed to address non-uniformly sampled (NUS) reconstruction as a pattern recognition problem, surpassing traditional methods in stability and accuracy. MR-Ai is also adapted for virtual decoupling, demonstrating robustness against variations in J-coupling values. The third study extends MR-Ai beyond traditional NMR processing. A 2D WNN architecture is designed to reconstruct Echo (or Anti-Echo) spectra, correcting phase-twist distortions in incomplete quadrature detection. Additionally, MR-Ai introduces a reference-free evaluation metric, estimating uncertainty in spectral reconstructions for direct quality assessment without external references. The fourth study introduces Peak Probability Presentation (P3), a novel AI-driven spectral visualization technique. To achieve this, an nD WNN architecture is developed for pattern recognition in nD NMR spectra. Unlike traditional intensity-based representations, P3 assigns a probability score to each spectral point, providing artifact-free, ultimate-resolution spectral interpretation. The results demonstrate P3’s superior performance in peak detection, spectral clarity, and noise differentiation. Additionally, P3 is integrated into Targeted Acquisition (TA) to develop a quantitative spectrum quality score for real-time spectral quality assessment and optimized data acquisition. Overall, this thesis demonstrates that AI-driven NMR processing not only enhances existing methods but also introduces fundamentally new approaches to spectral reconstruction, resolution enhancement, and quality assessment. As AI evolves, its integration into NMR workflows is expected to revolutionize the field, making high-quality spectral data more accessible, interpretable, and efficient.
Airborne particulate matter in a Sub-Saharan Africa city: Nairobi, Kenya, and at an Equatorial high altitude site: Mount Kenya
(2017-09-13) Gaita, Samuel Mwaniki
In Sub-Saharan Africa (SSA), air quality is gravely understudied despite the existing influential factors such a rapid urbanization and population growth that negatively affect the environment. Majority of urban areas in SSA face challenges that include lack of social services, poor infrastructure development, exponential increase of second-hand vehicles and extensive use of biomass-based fuel for energy needs. There is a systemic lack of continuous monitoring of air pollution in most SSA cities and hence it is yet to be seen if SSA will meet the set air quality targets of the sustainable development goals (SDGs) by the year 2030. Although the focus of air quality is on the urban areas, there is a need to monitor atmospheric composition at remote areas in SSA in order to build a baseline level and understand the influence of anthropogenic and natural aerosol sources on regional/global scale. This thesis work sought to study physical and chemical properties of airborne particulate matter (PM) in a typical SSA urban area, Nairobi city, and a high altitude site, Mount Kenya. Results from spatial distribution of black carbon (BC) and PM2.5 (particulate matter less than 2.5 aerodynamic diameter) showed that air quality on the road to the city and within the city is deteriorating. Factor analysis of the PM2.5 results showed that pollution sources were traffic, mineral dust, industrial, combustion, biomass burning, secondary aerosol and aged sea salt. Traffic and mineral dust contributed about 74% of the PM2.5 in Nairobi. Exposure to particulate pollutants was expressed in terms of deposition fractions from the size segregated PM data. The head airways region was found to receive the highest dose (about 86%) compared to the tracheobronchial and pulmonary regions. The reported high PM2.5 and BC concentrations measured along the main street of Nairobi city, indicated the urban population is frequently exposed to elevated pollutants concentrations and thus have high risk factor to respiratory illnesses and lung cancer. Aerosol study from Mount Kenya showed air pollutants are transported from the surrounding and far away sources by local and regional meteorology dynamics. The results from this study provides insight into the air quality issues from pollution sources, exposure to the population and dispersal to remote regions.
Algorithms and Interaction Potentials: phase density, surface tension and carbon dioxide
(2012-12-14) Persson, Rasmus
This thesis is an amalgamation of articles (Papers I–V) by the author. We present a perturbation algorithm to calculate the phase density, and thus the partition function including its temperature dependence. It works for Hamiltonians that are not too dissimilar, for which an extra degree of freedom interpolating between the two is defined so that microcanonical sampling allows the calculation of the ratio between the phase densities at any energy. The method is illustrated on a number of problems of different dimensionalities. In Paper I, we consider an anharmonic Einstein crystal, the square-well tetradecamer, and liquid gold. In addition, we consider the one-dimensional rotor and the low-dimensional ideal gas in a homogeneous external field, two Hamiltonians that display a phase transition at well-defined critical energies. We consider the interaction of linear molecules and discuss two coarse-grained pair potentials for their description. In Paper III, one of these potentials has been parametrized for the vapor-liquid envelope of carbon dioxide using two adjustablesobtaining good agreement, but for a detailed description of the carbon dioxide dimer and trimer structures, such coarse-graining fails. As reported in Paper IV, conventional all-atom force field descriptions also fail in describing the experimental second and third virial coefficients but an all-atom description with coarse-grained, single-site anisotropic three-body dispersion and single-site electrostatic induction manages to reproduce them. In addition, we note that this simple anisotropic three-body dispersion correction is essential for predicting the correct relative stability of the experimental trimer conformations when combined with a literature parametrization of the dimer ab initio potential energy surface. We present a simple method for calculating the surface tension with respect to vacuum from cluster simulations, by relating the scalar pressure to the infinitesimal isothermal pressure-volume work and equating it with the expression from classical nucleation theory. We then discuss the effect of molecular polarization on the surface tension using this method, as well as study its effect on the second and third virial coefficients of the fluid of polarizable Stockmayer molecules. The surface tension increases with polarizability, but so does its rate of decrease with temperature. The Tolman length is found positive and largely insensitive to temperature but increases non-linearly with increasing molecular polarizability. We discuss the semi-empirical calculation of the crystal-water surface tension of the pharmaceutical bicalutamide as reported in Paper V and provide a slight modification of the procedure.
Alkali Metals and Tar in Biomass Thermochemical Conversion: Development and Application of Online Measurement Techniques
(2018-02-08) Gall, Dan
Biomass is a renewable resource that can substitute the fossil-based products we depend on today. However, the conversion techniques require further improvement in order to be competitive with the traditional industry. One of the limitations is associated with the absence of measurement methods with sufficient time resolution that can be used to characterize the complex systems and optimize process conditions. This thesis presents the development and application of novel measurement methods, capable of time-resolved characterization of tar and alkali metals, which are key components in biomass gasification. The measurement methods are mainly adapted from aerosol science and based on thermal analysis and surface ionization of aerosol particles. Long-term measurements are achieved by dilution and conditioning of hot product gas, which allow condensable components to form aerosol particles that are subsequently analyzed. The developed methods are used to determine alkali, tar and particle concentrations in industrial scale facilities for biomass gasification. The aerosol characterization methods are also applied in flame chemistry and radiation research. Studies performed in dual-fluidized bed (DFB) gasification systems indicate that the alkali metal content of biomass to a large extent is emitted during the gasification process, and observed concentrations are close to the levels predicted by equilibrium calculations. The high alkali concentrations have implications for catalytic processes in the fluidized beds and for downstream processes including corrosion, fouling, and upgrading to commercial products. The developed methods are employed to characterize the transient conditions when changes in operational parameters and additives are used to optimize the gasification process. A significant increase of the alkali metal concentration was observed when alkali salts were inserted directly to a gasifier, which suggests a fast volatilization in the reducing environment. Additives to the combustion side of the DFB imposed notable effects in the product gas, and the results provide information regarding the transfer mechanisms of inorganic compounds in the system. Additions of olivine and ilmenite reduced the gas-phase alkali metal concentration, indicating a fast reaction between alkali metal compounds and the minerals. The applied changes also affected the production of condensable tar, and the tar concentration was found to anti-correlate with the alkali metal concentration when a sand bed was used, while no clear trend was observed with an olivine bed. The studies confirm that several options are available to improve the alkali metal and tar behavior in biomass gasification, and suggest that online monitoring is needed to study and optimize the underlying processes.
Alkali Release and Effects on Biomass Thermal Conversion Processes
(2022-09-19) Ge, Yaxin
High alkali content is an important feature of biomass, and it has a series of implications for thermal conversion processes. This thesis focuses on the release of alkali during various biomass thermal conversion processes, including biomass pyrolysis, char gasification, co-conversion of different types of biomass, and thermal conversion of biomass mixed with fresh and used bed materials. Alkali release is also studied in combination with fuel conversion, to elucidate the underlying relationships. In addition, particle release during steam gasification of char is investigated. The studies were carried out on different reactor scales, ranging from the micro scale to pilot scale. A reliable methodology for simultaneous monitoring of alkali release and sample mass was developed based on the application of a thermogravimetric analyzer in combination with a surface ionization detector (TGA-SID). Using TGA-SID, a significant level of alkali release was observed when wood char conversion approaches completion during CO2 gasification, while the level of alkali release from straw char decreased continuously throughout the process. Alkali migration from straw to wood was observed at temperatures above 600°C during co-pyrolysis, based on online alkali measurements. Positive and negative synergistic effects were observed during the co-gasification at low and high conversions of char, respectively. This is attributed to alkali and silicon migration from the straw to the wood. Fresh bed materials affect wood and straw char gasification reactivities and rates of alkali release, and different bed materials may play different roles. For example, an alkali-containing bed material can enhance char gasification in the initial stage, a Si-containing bed material inhibits char gasification and alkali release at high conversions of char, an Al-containing bed material can inhibit char conversion when the char has a high silicon content, and Mg- and Ca-containing bed materials ensure that alkali persists in releasable form, thus favoring substantial alkali release from the char. Used silicon bed material has a coating layer that is abundant in Ca, Si, K, and Mg. These elements can migrate to the char surface during thermal conversion processes and affect char gasification. In addition, a comprehensive system based on diluters, particle sizers, and SID has been successfully used for particle and alkali measurements in laboratory- and pilot-scale reactors. The laboratory-scale steam gasification system shows that the released levels of alkali and particles significantly increase when char conversion approaches completion. Using steam as the gasifying agent instead of CO2 results in a higher level of alkali release during most of the gasification stage. Aerosol particles are also released during steam gasification, at rates that vary by more than one order of magnitude depending on the char composition. The present study improves our understanding of alkali release, migration, and reaction during biomass thermal conversion processes. The acquired fundamental knowledge can be used for reactor design, co-gasification optimization, and selection of bed materials.
Alkali Uptake and Release from Oxygen Carriers in Chemical Looping Applications: Development and Application of Reactor Systems and Measurement Techniques
(2023-10-31) Andersson, Viktor; Andersson, Viktor
Chemical looping combustion (CLC) of biomass is a heat and power generation technology with minimal associated costs for carbon capture, potentially resulting in negative CO2 emissions. The CLC technology utilizes fluidized beds of oxygen carrier (OC) particles to separate CO2 from the combustion air. The high content of potassium and sodium compounds in biomass fuels may cause detrimental problems during thermal conversion, including agglomeration, fouling and corrosion, while also enhancing conversion processes due to their catalytic abilities. Further knowledge about processes involving these alkali metals, including their uptake and release from OC materials and the control of alkali emission, is critical for the upscaling and commercialization of biomass CLC. The aim of this thesis is to improve the understanding of interactions between alkali compounds and OCs under conditions representative of biomass CLC. A novel technique based on temperature modulated surface ionization was developed to determine the contributions of alkali chlorides, hydroxides, and sulfates to the flux from different reactors. A novel laboratory-scale reactor was developed, facilitating continuous alkali vapor injection to a fluidized bed while monitoring the concentrations of alkali and gas in the reactor exhaust. An additional method was developed to monitor the real-time alkali release and mass loss from small, fixed bed samples, including OC particles and solid biomass. The type of OC material is observed to play a crucial role in alkali uptake, where fluidized beds of the promising CLC materials: calcium manganite, manganese oxide, and ilmenite, exhibiting varying levels of efficiency depending on the specific gas conditions present. Ilmenite showed near complete absorption of the injected alkali, especially during reducing conditions, making it a promising option to limit alkali emissions. The alkali speciation analysis revealed that NaCl and KCl were the predominant alkali species emitted during NaCl and KCl injection, and a similar pattern was observed for alkali sulfates. Alkali hydroxide injections resulted in highly efficient alkali uptake with emissions dominated by alkali hydroxides and chlorides. The study highlights the balance between alkali absorption efficiency and fuel conversion and oxidizing efficiency of the OC materials. While ilmenite demonstrated excellent alkali uptake, manganese oxide and calcium manganite exhibited superior fuel conversion and oxidizing efficiency. In addition, ilmenite previously used in an industrial process releases alkali in both inert and oxidizing environments at high temperatures. The described development and application of new methods are concluded to open new possibilities to understand and optimize biomass CLC.
Allergenic Oxidation Products from Fragrance Terpenes. Chemical Analysis and Determination of Sensitizing Potency
(2013-09-05) Rudbäck, Johanna
The ubiquitous presence of fragrance compounds in consumer products has resulted in a high frequency of contact allergy to fragrances in the population. For prevention purposes and according to EU regulations, cosmetics must be labeled when containing the most prominent fragrance allergens in concentrations exceeding 0.001% in “stay-on” products and 0.01% in “rinse-off” products. Previous studies have shown that the most common fragrance terpenes oxidize upon contact with air, forming hydroperoxides which are strong skin sensitizers. Thus, cosmetics may contain allergens in form of oxidation products formed by air exposure of the fragrance terpenes. In this thesis allergenic oxidation products from fragrance terpenes have been investigated. The studies have included development of analytical methods and chemical analyses for detection of hydroperoxides in complex mixtures. Furthermore, structure-activity relationships (SARs) were investigated based on studies of oxidation products and sensitizing potencies from autoxidation of the monoterpenes a-terpinene and citronellol. Monoterpene hydroperoxides are generally difficult to determine as they have weak chromophores, exhibit low thermostability, and fragment in a similar way as parent compounds and other oxidation products in mass spectrometry. For the first time, analytical methods for detection of the highly sensitizing hydroperoxides in complex mixtures are now available utilizing mass spectrometry in combination with either liquid chromatography or gas chromatography. Low detection limits were achieved using liquid chromatography/mass spectrometry, while a high peak capacity useful for separation of hydroperoxide isomers was obtained with gas chromatography/mass spectrometry. The methods presented are sensitive enough for detection of hydroperoxides at concentrations that people may come in contact with. Thus, the analytical methods open up the possibility of investigating the clinical relevance of products in the patients’ environment through chemical analysis. Fragrance compounds in essential oils are often stated to be protected from autoxidation due to the presence of natural antioxidants. This study showed that hydroperoxides were present in the investigated essential oils already upon arrival from the supplier and that the hydroperoxide concentrations steadily increased with time and air exposure. The hydroperoxides were formed to the same extent in the essential oils compared to what is seen at air exposure of the corresponding fragrance terpenes. Thus, no significant protection against autoxidation by antioxidant activity could be observed. Neither was the rate of autoxidation substantially affected by the experimental conditions; stirring and intensity of daylight did not have any considerable effects. Autoxidation studies of -terpinene and citronellol were performed to gain knowledge regarding oxidation products and sensitizing potencies, and the results were compared to previously studied fragrance terpenes. It was shown that minor structural differences have a major impact on the dominant oxidation pathway and the stability of the oxidation products. For both compounds the sensitizing potency (as examined by the murine local lymph node assay) after air exposure was largely enhanced, around ten-fold. This shows the importance of testing with oxidized material in clinical studies and not only with the pure terpenes. In the risk assessment of fragrance chemicals, the possibility of autoxidation should be considered. To summarize, the studies in this thesis have contributed to a method toolbox of complementary analytical methods with focus on mass spectrometry for detection and quantification of fragrance hydroperoxides. In addition, fragrance terpenes as constituents of essential oils form hydroperoxides, showing that the essential oils are not natural protectors for the formation of oxidation products but rather stabilize the hydroperoxides formed. Furthermore, this thesis shows that minor structural differences between fragrance terpenes can have a major impact on the dominant autoxidation pathway and oxidation products formed. Increased understanding of how fragrance terpenes can be activated to skin sensitizers by autoxidation is necessary to obtain valid SARs for this type of compounds. In the risk assessment, their susceptibility to autoxidation as well as the sensitizing potency of formed oxidation products should be considered.
Allostery at Work: Mapping, Modulating, and Monitoring PKL Function with Chemotype Diversity
(2025-08-19) Nilsson, Oscar
Understanding the processes of protein regulation and our ability to control enzyme activity in the human body lie at the core of drug development. The liver isoform of pyruvate kinase (PKL) is a metabolic enzyme crucial for energy production. Because of its metabolic role, PKL and other pyruvate kinase isoforms are interesting drug targets for metabolic diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). In this thesis, the allosteric regulation of PKL was explored using multiple approaches to expand our knowledge about this extensively regulated enzyme. Known allosteric ligands were derivatized into fluorescent reporter probes to monitor ligand engagement with the allosteric pocket of PKL. This approach centred around the direct incorporation of fluorescent dye molecules into the scaffolds of the allosteric ligands, exploiting structurally overlapping features. A set of probes containing the environment-sensitive dye 4-sulfamoyl-7-aminobenzoxadiazole (SBD) was developed and used to measure the occupancy of unlabelled ligands in indicator displacement assays. The concept was further developed by applying this design philosophy to a more potent ligand class, generating a cell-permeable fluorescent tracer. In combination with NanoBRET technology, this tracer enabled the development of a target-engagement assay capable of detecting allosteric ligand binding to PKL in living cells. This thesis also proposes a model for how the allosteric regulation of PKL works. By modulating the enzyme with compounds derived from the known drug mitapivat, minor modifications were identified that had significant effects on functional outcomes. Biophysical techniques and molecular dynamics simulations were employed to investigate a structurally similar activator/inhibitor pair, demonstrating that they differentially affect the stability of the protein-protein interfaces. Furthermore, a fragment-based drug design effort was undertaken to map the allosteric pocket and identify novel ligand scaffolds capable of allosterically inhibiting PKL. An iterative design process starting from simple fragments yielded moderate inhibitors with large structural diversity, providing valuable insights into key binding interactions and serving as a foundation for future optimization toward more potent modulators. These findings collectively deepen our understanding of PKL allosteric regulation and provide useful chemical tools for further study. They may also inform future efforts aimed at developing ligands to modulate PKL activity in a physiological context.
Analytical Approaches to Study Vesicular and Exosomal Release
(2024-02-09) Le Vo, Kim Long
Vesicles, as a special type of entity nanometers in size, are crucial for survival of multicellular organisms. Intracellular vesicles mainly contain specific signaling molecules, transmitters, and modulators whereas extracellular vesicles (EVs) are bioactive organelles carrying a wide range of proteins, genetic materials, and other molecules. Secretion from vesicles is essential to manipulate many biological pathways and intercellular communication. Understanding the regulatory mechanisms of vesicular secretion is mandatory for uncovering the pathologies of neurological disorders and developing related pharmaceuticals. Several electrochemical techniques have been proposed and developed to unravel vesicular neurotransmitters at single cell and subcellular levels. These methodologies provide high spatiotemporal resolution and sensitivity while enabling direct quantification of electroactive molecules from individual vesicles. Single cell amperometry (SCA) can be employed to determine the number of signaling transmitters being released during an exocytosis event. Vesicle impact electrochemical cytometry (VIEC) and intracellular vesicle impact electrochemical cytometry (IVIEC) are two methods that allow the quantification of the number of signaling molecules stored inside single vesicles from isolated or intracellular vesicles, respectively. In this thesis work, vesicular structure as well as their content and release have been investigated. In paper I, open carbon nanopipettes (CNPs) with different radii between 50 and 600 nm were employed to quantify vesicular content in isolated vesicles of adrenal chromaffin cells by VIEC. Paper II was continuation of work from paper I, the mechanistic study of L-DOPA was carried out by using IVIEC with various sized CNPs. SCA was introduced to capture the dynamic release of single exosomes from a single living cell in paper III. In paper IV, the combination of electrochemistry and mass spectrometry was applied to investigate the effects of ketamine on dopamine storage and exocytosis, as well as the alterations of cellular lipid composition. Generally, electrochemical methods have been considered as a powerful tool to understand structures of vesicles and their biological functions.
Anti-Virulence Strategy Targeting Sortase A, A Structural Investigation of the Sortase A Enzyme, and the Identification, Synthesis, and Evaluation of Sortase A Inhibitors
(2019-09-20) Uzelac, Ivana
The emergence of multi-resistant bacteria and their continuous spread is one of the greatest challenges when treating bacterial infections. Increased understanding of bacterial pathogenesis has revealed new strategies for treating bacteria-mediated diseases. Targeting virulence factors or virulence-mediated mechanisms is one strategy which is believed to cause less selective pressure and thereby resistance development since it would not affect bacterial growth or survival. The bacterial enzyme sortase A (SrtA) anchors the majority of virulence associated proteins to the bacterial cell wall and is a promising target for development of anti-virulence drugs. This thesis describes the investigation of SrtA conformations, derived from MD simulations, and their performance in virtual screening (VS) using a diverse set of active inhibitors and their decoys. From the performance results, SrtA structures can be selected for further docking studies and VS. Further, novel SrtA inhibitors were discovered using high throughput and fragment based screening (HTS and FBS) as starting points for hit selection. Hits were synthetically modified and evaluated using several different biochemical and biophysical assays. The HTS resulted in the discovery of substituted thiadiazoles with inhibitory activities in the low micromolar range. They probably act by binding covalently to the active site cysteine of SrtA. The fragment screening resulted in the discovery of substituted pyrazoles and isoxazoles as promising starting points for further development into more potent SrtA inhibitors. A hybrid compound combining the knowledge from the HTS and FBS was developed. The hybrid is a potent non-covalent inhibitor as opposed to the HTS compounds. The flavone morin and its effects on SrtA were also investigated, showing that morin might act as both an inhibitor and an activator. Morin seems to bind to the SrtA dimer interface inducing a conformational change in the protein allowing various fragments to bind more efficiently to the active site. This sheds further light on the importance of investigating the inhibitory mechanism of already existing SrtA inhibitors as to get a better understanding of their mode of action, which will be crucial for the development of more potent SrtA inhibitors.
Antibiotic sensitivity and horizontal gene transfer in Escherichia coli - A genome-wide perspective
(2022-11-01) Palm, Martin
Since their discovery in the early 20th century, antibiotics have truly revolutionized human medicine. They have allowed us to treat diseases that were previously untreatable and have become a staple of modern medicine. However, along with the human use of antibiotics pathogens resistant to antibiotics emerged. Over the last decades, an arms race between bacteria developing resistance and human medicine has been raging. Today, antibiotic resistance is a global problem with even the most potent antibiotics losing their efficiency. Antibiotic resistance occurs when bacteria develop mechanisms to withstand the antibacterial effects of antibiotics. It is widely known that, once developed, resistance is selected for by the concentrations of antibiotics that are used to treat infections. In addition, it is becoming increasingly evident that even very low levels, often many times lower than those used in a clinical setting, can select for resistance. These low levels of antibiotics are commonly found in the environment where they contribute to the global reservoir of resistance by maintaining a constant level of resistance. Antibiotic resistance can also spread between bacteria through horizontal gene transfer. The major driving force behind this is believed to be bacterial conjugation. Despite this, the underlying mechanisms of conjugation are not fully understood. In this thesis, antibiotic resistance and horizontal gene transfer is explored from a genome-wide perspective. The results indicate that the presence of resistance genes alone does not give the full picture when it comes to growth at sub-inhibitory levels of antibiotics. We also discuss how conjugation can be inhibited and found both genetic and environmental factors that can impair conjugation. Overall, our findings emphasize the importance of understanding the emergence, selection, and spread of antibiotic resistance at sub-inhibitory concentrations of antibiotics.
Arsenic-induced protein aggregation and toxicity in Saccharomyces cerevisiae
(2022-08-29) Hua, Sansan
Arsenic is prevalent in the environment and this toxic metalloid poses a substantial threat to human health with 100-200 million people worldwide estimated to be at risk. Chronic exposure to arsenic is associated with neurodegenerative and age-related disorders that are characterized by the accumulation of protein aggregates, including Parkinson’s and Alzheimer’s disease. Despite of the undisputed toxicity of arsenic, our understanding of the underlying mechanisms and cellular responses is limited. This thesis has focused on arsenic-induced protein aggregation and toxicity in yeast, with the aim of elucidating how these aggregates are formed in vivo, the mechanisms by which they affect cells, how cells prevent their accumulation as well as how cells regulate the protein quality-control system to protect against toxic aggregates. The impact arsenic has on protein homeostasis may contribute to its toxicity and suspected role in protein misfolding diseases. Main findings of this thesis include the identification of novel genes whose overexpression conferred arsenic resistance. We also demonstrated the importance of accurate transcriptional and translational control for mitigating protein aggregation and toxicity during arsenite stress. In addition, we showed that the ubiquitin-proteasome system (UPS) is the main pathway that clears arsenite-induced aggregates, whilst the autophagy-vacuole pathway and the chaperone-mediated disaggregation both contribute to clearance but their roles appear less prominent than the UPS. Our findings provide novel insights into the biology of arsenic and a valuable resource for further studies on the mechanistic details of arsenic toxicity and pathogenesis.
Atmospheric Chemistry of Volatile Organic Compounds: Oxidation Products, Mechanisms and Secondary Organic Aerosol Formation
(2019-01-15) Hammes, Julia
The results from this work are a piece in understanding the complex puzzle of atmospheric aerosol formation. Secondary organic aerosol (SOA) formed by the oxidation of volatile organic compounds (VOC) in the atmosphere is a key component of air pollution with a strong negative impact on human health and influence on climate, but its formation is poorly understood. Because air pollution and climate change are major challenges facing modern societies, there is a clear need to better understand atmospheric SOA formation. SOA formation can be estimated from distributions of potential oxidation products, but such estimates are only as useful as the underlying chemical mechanisms and physical properties on which they are based. The work presented in this thesis was conducted to better characterize VOC oxidation products and the chemical mechanisms governing their formation. The SOA precursor compounds a-pinene and limonene (representing biogenic VOC) and 1,3,5- trimethylbenzene (TMB) (an anthropogenic VOC) were studied in the G-FROST and Go:PAM flow reactors to characterize their oxidation and the subsequent SOA-forming processes. Previously unknown compounds including dimer esters, carboxylic acids, nitrates and highly oxygenated molecules were identified using state-of-the-art mass spectrometric methods. These oxidation products were shown to be important SOA contributors and explicit mechanisms for their formation were proposed. Some of the identified compounds were suggested to be of extremely low volatility and thus important for new particle formation. Oxidation of TMB under conditions representative of urban environments reduced particle formation potential; this effect was attributed to the disruption of RO2 auto-oxidation cycles by NOx and subsequent nitrate formation at the expense of highly oxygenated molecules. During the course of this work, an automated algorithm was developed to extract compound-specific volatility data from FIGAERO thermograms. The scientific understanding of SOA formation would be greatly improved by a detailed knowledge of the products of VOC oxidation, the mechanisms by which they are formed, and their vapour pressures, all of which this work aims to contribute to.

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