| 1. |
- Granseth, Erik, 1978-
(författare)
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Structure, prediction, evolution and genome wide studies of membrane proteins
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- α-helical membrane proteins constitute 20-30% of all proteins in a cell and are involved in many essential cellular functions. The structure is only known for a few hundred of them, which makes structural models important. The most common structural model of a membrane protein is the topology which is a two-dimensional representation of the structure. This thesis is focused on three different aspects of membrane protein structure: improving structural predictions of membrane proteins, improving the level of detail of structural models and the concept of dual topology. It is possible to improve topology models of membrane proteins by including experimental information in computer predictions. This was first performed in Escherichia coli and, by using homology, it was possible to extend the results to 225 prokaryotic organisms. The improved models covered ~80% of the membrane proteins in E. coli and ~30% of other prokaryotic organisms. However, the traditional topology concept is sometimes too simple for complex membrane protein structures, which create a need for more detailed structural models. We created two new machine learning methods, one that predicts more structural features of membrane proteins and one that predicts the distance to the membrane centre for the amino acids. These methods improve the level of detail of the structural models. The final topic of this thesis is dual topology and membrane protein evolution. We have studied a class of membrane proteins that are suggested to insert either way into the membrane, i.e. have a dual topology. These protein families might explain the frequent occurrence of internal symmetry in membrane protein structures.
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| 2. |
- Qian, Xiaoyan, 1989-
(författare)
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Towards comprehensive cellular atlases : High-throughput cell mapping by in situ sequencing
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- With recent technological advancements in single-cell biology, many aspects of individual cells are characterized with unprecedented resolution and details. Cell types in human and model organisms are redefined, and multiple organ-wide atlases are proposed to integrate different types of data to provide a comprehensive view of biological systems at cellular resolution. Incorporating location information of cells in such atlases is crucial to understanding the structure and functions. Several spatially resolved transcriptomics technologies may serve this purpose, and in situ sequencing (ISS) is among the most powerful ones.ISS detects the expression of tens to hundreds of genes in situ, i.e. inside preserved cells and tissues. ISS is a targeted approach, using probes designed to identify specific transcripts. Its key advantages, as compared to other spatially resolved gene expression analysis methods, are high throughput, cellular resolution and tissue compatibility, making it a tool ideally suited for spatial cell mapping. The work included in this thesis aims to develop tools and methods for this application.In paper I, a network analysis tool was developed to analyze ISS and other spatially resolved data. The tool enables smooth visualization of large datasets and generates networks based on colocalization. It also includes functions to test statistical significance and resolve tissue heterogeneity.In paper II, we studied spatio-temporal patterns of immune response in tuberculosis granuloma by targeting immune markers with ISS. Using the tool developed in paper I together with other methods, we established an immune response time course at the granuloma sites and found histologically different granulomas based on transcriptional information. The paper demonstrated that ISS can robustly detect transcripts in formalin-fixed paraffin-embedded tissues across biological samples and reveal biologically relevant structures.In paper III, we developed probabilistic cell typing by in situ sequencing (pciSeq), a method to spatially map cell types defined by single-cell RNA-sequencing. pciSeq is an integrated pipeline that includes gene selection, image analysis, barcode calling and cell type calling. We mapped closely related interneuron cell types of the mouse hippocampal CA1 region in 14 coronal sections and validated the results against ground truth.In paper IV, we investigated the quantification bias of ISS resulting from the probe target selection. We developed a method to sequence in situ synthesized cDNA and found that the read coverage of in situ cDNA library reflected ISS counts more closely than conventional RNA sequencing, making it possible, to some extent, to predict a probe’s performance and guide the probe design.Taken together, the developments described in this thesis comprise several tools that make ISS suitable for building cellular atlases via large-scale spatial mapping.
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| 3. |
- Björkholm, Patrik, 1981-
(författare)
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Protein Interactions from the Molecular to the Domain Level
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The basic unit of life is the cell, from single-cell bacteria to the largest creatures on the planet. All cells have DNA, which contains the blueprint for proteins. This information is transported in the form of messenger RNA from the genome to ribosomes where proteins are produced. Proteins are the main functional constituents of the cell, they usually have one or several functions and are the main actors in almost all essential biological processes. Proteins are what make the cell alive. Proteins are found as solitary units or as part of large complexes. Proteins can be found in all parts of the cell, the most common place being the cytoplasm, a central space in all cells. They are also commonly found integrated into or attached to various membranes.Membranes define the cell architecture. Proteins integrated into the membrane have a wide number of responsibilities: they are the gatekeepers of the cell, they secrete cellular waste products, and many of them are receptors and enzymes.The main focus of this thesis is the study of protein interactions, from the molecular level up to the protein domain level.In paper I use reoccurring local protein structures to try and predict what sections of a protein interacts with another part using only sequence information. In papers II and III we use a randomization approach on a membrane protein motif that we know interacts with a sphingomyelin lipid to find other candidate proteins that interact with sphingolipids. These are then experimentally verified as sphingolipid-binding. In the last paper, paper IV, we look at how protein domain interaction networks overlap and can be evaluated.
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| 4. |
- Boekel, Carolina, 1977-
(författare)
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Integration and topology of membrane proteins
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins comprise around 20-30% of most proteomes. They play important roles in most biochemical pathways. All receptors and ion channels are membrane proteins, which make them attractive targets for drug design. Membrane proteins insert and fold co-translationally into the endoplasmic reticular membrane of eukaryotic cells. The protein-conducting channel that inserts the protein into the membrane is called Sec61 translocon, which is a hetero-oligomeric channel that allows transmembrane segments to insert laterally into the lipid bilayer. The focus of this thesis is how the translocon recognizes the transmembrane helices and integrates them into the membrane.We have investigated the sequence requirements for the translocon-mediated integration of a transmembrane α-helix into the ER by challenging the Sec61 translocon with designed polypeptide segments in an in vitro expression system that allows a quantitative assessment of membrane insertion efficiency. Our studies suggest that helices might interact with each other already during the membrane-insertion step, possibly forming helical hairpins that partition into the membrane as a single unit. Further, the insertion efficiency for Nin-Cout vs. Nout-Cin transmembrane helices and the integration efficiency of Alzheimer’s Aβ-peptide fragments has been investigated.Finally, detailed topology mapping was performed on two biologically interesting proteins with unknown topology, the human seipin protein and Drosophila melanogaster odorant receptor OR83b.
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| 5. |
- Calado Botelho, Salomé, 1984-
(författare)
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Translocation of proteins into and across the bacterial and mitochondrial inner membranes
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Translocons are dynamic protein complexes with the ability to respond to specific signals and to transport polypeptides between two distinct environments. The Sec-type translocons are examples of such machineries that can interconvert between a pore forming conformation that translocates proteins across the membrane, and a channel-like conformation that integrates proteins into the membrane by lateral opening.This thesis aims to identify the signals encoded in the amino acid sequence of the translocating polypeptides that trigger the translocon to release defined segments into the membrane. The selected systems are the SecYEG translocon and the TIM23 complex responsible for inserting proteins into the bacterial and the mitochondrial inner membrane, respectively.These two translocons have been challenged in vivo with designed polypeptide segments and their insertion efficiency into the membrane was measured. This allowed identification of the sequence requirements that govern SecYEG- and TIM23-mediated membrane integration. For these two systems, “biological” hydrophobicity scales have been determined, giving the contributions of each of the 20 amino acids to the overall free energy of insertion of a transmembrane segment into the membrane.A closer analysis of the mitochondrial system has made it possible to additionally investigate the process of membrane dislocation mediated by the m-AAA protease. The threshold hydrophobicity required for a transmembrane segment to remain in the mitochondrial inner membrane after TIM23-mediated integration depends on whether the segment will be further acted upon by the m-AAA protease.Finally, an experimental approach is presented to distinguish between different protein sorting pathways at the level of the TIM23 complex, i.e., conservative sorting vs. stop-transfer pathways. The results suggest a connection between the metabolic state of the cell and the import of proteins into the mitochondria.
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| 6. |
- Hessa, Tara, 1972-
(författare)
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Integration of Transmembrane Helices into the Endoplasmic Reticulum
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins reside in cell and organelle membranes. They play significant roles in many processes vital to living cells. Receptors and ion channels are examples of membrane proteins that regulate the physiological state of the cell and are attractive targets for drug development.In eukaryotic cells most membrane proteins insert and fold cotranslationally into the endoplasmic reticular membrane. The insertion process is mediated by the Sec61 translocon which is a hetero-oligomeric protein-conducting channel that allows transmembrane segments to exit laterally into the lipid bilayer. How the translocon recognizes the molecular characteristics of transmembrane helices and integrate them into the lipid bilayer is the focus of this thesis.We have determined the sequence requirements for translocon-mediated integration of a transmembrane -helix into the ER membrane by challenging the Sec61 translocon with designed polypeptide segments in an in vitro expression system that permits quantitative assessment of membrane insertion efficiency. A biological hydrophobicity scale and a position-dependent free energy matrix have been developed, describing the contribution of each of the 20 amino acids in each position of a 19-residues long polypeptide segment to the overall free energy of a single transmembrane segment insertion. These studies suggest that the translocon provides direct contact between the nascent chain and the lipids in the membrane and that this protein-lipid interaction is the basis for the recognition of transmembrane helices in the translocon.
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| 7. |
- Krzywkowski, Tomasz, 1986-
(författare)
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iLocks: a novel tool for RNA assays with improved specificity
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Central Dogma of molecular biology describes a framework for how genetic information is transferred in cells, placing RNA as a messenger between DNA and translated proteins. During the last years, interest in RNA research has grown tremendously due to the increasing understanding and recognition of the importance of RNA in regulation of gene expression, biochemical catalysis, and genome integrity surveillance. Most importantly, RNA content, unlike DNA, changes constantly, fine-tuning the cellular response to match the environmental conditions. There is a clear potential for RNA biomarkers, reflecting both the natural and pathological conditions in vivo.Various methods have been developed to study RNA, of which the most common tools and techniques are described in this thesis. Since many of these gold standard methods are based on detecting RNA derivative (cDNA), there is a wide scope for efficient alternative tools directly targeting RNA. In Paper I, the spatiotemporal expression of human adenovirus-5 mRNA in epithelial and blood cells infected with the virus has been studied. For this, padlock probes and rolling circle amplification (RCA) were used to visualize, quantify and analyse both viral and host cell cDNAs in different infection scenarios, at single cell level. In Paper II, direct RNA detection fidelity has been evaluated using padlock probes. A novel type of probe (iLock) that is activated on RNA via invasive cleavage mechanism, prior to RCA was developed in this approach. Using iLocks, a substantial improvement of direct RNA sensing fidelity has been observed. In Paper III, RNA modifications were introduced in otherwise DNA iLock probes to enhance the probes’ efficiency on miRNAs. Using chimeric iLock probes, multiplexed differentiation of conserved miRNA family members were performed with next- generation sequencing-by-ligation readout. Efficient replication of chimeric probes used in Paper III implies that the Phi29 DNA polymerase readily accepts RNA-containing circles as amplification substrates. In Paper IV, real-time RCA monitoring for measurement of amplification rates and analysis of amplification patterns of various RNA-containing circles was achieved. Moreover, the RCA products were sequenced as a proof for the reverse-transcriptase activity of the Phi29 DNA polymerase.This thesis effectively contributes to a better understanding of mechanisms influencing RNA detection with, but not limited to, padlock probes. It expands the available RNA analyses toolkit with novel strategies and solutions, which can be potentially adapted for RNA-focused research, in general and molecular diagnostics, in particular.
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| 8. |
- Lloris-Garcerá, Pilar, 1985-
(författare)
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EmrE, a puzzling transporter : Assembly, biogenesis and evolution of a dual-topology membrane protein
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biological membranes are the key to cell existence, as they are able to both isolate and connect their interior with the environment. Membranes are composed of lipids and proteins that create a semi-permeable barrier; because the lipid bilayer stops free diffusion of most molecules and ions, membrane proteins play an important role in connecting the interior of the cell with its environment. They function as receptors, sensing signals to trigger a response; cell adhesion molecules, holding neighboring cells together, or transporters and channels importing nutrients and extruding waste, among other chemical compounds, in a controlled manner.In order for membrane proteins to function correctly, proper insertion, folding and oligomerization in the bilayer is essential. While most membrane proteins adopt a unique orientation in the membrane, some proteins adopt multiple topologies. A well-known case is the dual-topology membrane proteins that adopt two opposite orientations in the membrane. The best-studied dual-topology protein is EmrE, a dimeric multidrug transporter found in Escherichia coli, and other bacteria.The existence of dual-topology proteins raises many questions regarding oligomerization, biogenesis and evolution of membrane proteins. In this thesis, EmrE has been used as a model protein to study some of these issues. Our goals were (i) to settle the controversy regarding whether the arrangement of the monomers within the EmrE dimer is parallel or antiparallel, (ii) to test the validity of the published X-ray structure by in vivo experiments and, (iii) to elucidate the mechanism of membrane insertion (iv) and the evolution of dual-topology membrane proteins.
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| 9. |
- Melén, Karin, 1973-
(författare)
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Topology Prediction of Membrane Proteins: Why, How and When?
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins are of broad interest since they constitute a large fraction of the proteome in all organisms, up to 20-30%. They play a crucial role in many cellular processes mediating information flow and molecular transport across otherwise nearly impermeable membranes. Traditional three-dimensional structural analyses of membrane proteins are difficult to perform, which makes studies of other structural aspects important. The topology of an α-helical membrane protein is a two-dimensional description of how the protein is embedded in the membrane and gives valuable information on both structure and function.This thesis is focused on predicting the topology of α-helical membrane proteins and on assessing and improving the prediction accuracy. Reliability scores have been derived for a number of prediction methods, and have been integrated into the widely used TMHMM predictor. The reliability score makes it possible to estimate the trustworthiness of a prediction.Mapping the full topology of a membrane protein experimentally is time-consuming and cannot be done on a genome-wide scale. However, determination of the location of one part of a membrane protein relative to the membrane is feasible. We have analyzed the impact of incorporating such experimental information a priori into TMHMM predictions and show that the accuracy increases significantly. We further show that the C-terminal location of a membrane protein (inside or outside) is the optimal information to use as a constraint in the predictions.By combining experimental techniques for determining the C-terminal location of membrane proteins with topology predictions, we have produced reliable topology models for the majority of all membrane proteins in the model organisms E. coli and S. cerevisiae. The results were further expanded to ~15,000 homologous proteins in 38 fully sequenced eukaryotic genomes. This large set of reliable topology models should be useful, in particular as the structural data for eukaryotic membrane proteins is very limited.
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| 10. |
- Nicolaus, Felix, 1990-
(författare)
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Cotranslational protein biogenesis in Escherichia coli monitored by force profile analysis
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are a diverse class of biomolecules that carry out many essential functions across all organisms. They can either be found in aqueous compartments or embedded in biological membranes of the cell and are called soluble or membrane proteins, respectively. Membrane proteins are involved in many essential cellular pathways and account for about one third of both pro- and eukaryotic proteomes. They are major drug targets and are critical when cells are engineered to secrete therapeutic proteins and industrial enzymes.In order to be functional, proteins must fold into specific 3-dimensional structures, be targeted to the right destination and can undergo additional maturation steps. Most studies have focused on the characterisation of fully synthesized proteins, and much less is known about their biogenesis while still being translated by the ribosome. Here, we focus on cotranslational events studied in the well-characterised model bacterium Escherichia coli, and take advantage of a recently developed technology that uses so-called translational arrest peptides (APs). APs stall their own translation on the ribosome unless a sufficient pulling force is applied on the nascent polypeptide chain, and can therefore be used as molecular force sensors. We found that enough force to overcome AP-induced translational arrest can be generated by transmembrane helices (TMHs) as they insert into the E. coli inner membrane. By following the stepwise cotranslational insertion of three multi-spanning integral membrane proteins, we found that a TMH starts generating a force on the nascent chain when it reaches about 45 residues away from the ribosomal peptidyl transferase center (PTC). At this distance the TMH is expected to be in the vicinity of the bacterial SecYEG translocon and begin to insert into the lipid bilayer. Interestingly, this force can be affected by the presence of other membrane-interacting segments flanking the TMH. Another intriguing finding was that an N-terminal globular domain can fold well before the downstream membrane domain starts to integrate into the membrane. Furthermore, we detected forces that are generated by residue-specific intrachain as well as interchain interactions, which suggest cotranslational folding and oligomerisation of membrane proteins. Finally, we recorded the force that is generated by a recombinant soluble protein as it folds cotranslationally in E. coli. The onset of the folding was detected when the protein’s C-terminus has not yet fully emerged from the ribosome exit tunnel, and folding was delayed in the presence of the cotranslationally acting chaperone trigger factor (TF).Taken together, the work presented in this thesis has led to a better understanding of how proteins fold, assemble as well as insert into a biological membrane during their translation, and has revealed multiple factors that contribute to the complexity of cotranslational protein biogenesis.
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