| 1. |
- Danielsson, Frida, 1984-
(författare)
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Integration of RNA and protein expression profiles to study human cells
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular life is highly complex. In order to expand our understanding of the workings of human cells, in particular in the context of health and disease, detailed knowledge about the underlying molecular systems is needed. The unifying theme of this thesis concerns the use of data derived from sequencing of RNA, both within the field of transcriptomics itself and as a guide for further studies at the level of protein expression. In paper I, we showed that publicly available RNA-seq datasets are consistent across different studies, requiring only light processing for the data to cluster according to biological, rather than technical characteristics. This suggests that RNA-seq has developed into a reliable and highly reproducible technology, and that the increasing amount of publicly available RNA-seq data constitutes a valuable resource for meta-analyses. In paper II, we explored the ability to extrapolate protein concentrations by the use of RNA expression levels. We showed that mRNA and corresponding steady-state protein concentrations correlate well by introducing a gene-specific RNA-to-protein conversion factor that is stable across various cell types and tissues. The results from this study indicate the utility of RNA-seq also within the field of proteomics.The second part of the thesis starts with a paper in which we used transcriptomics to guide subsequent protein studies of the molecular mechanisms underlying malignant transformation. In paper III, we applied a transcriptomics approach to a cell model for defined steps of malignant transformation, and identified several genes with interesting expression patterns whose corresponding proteins were further analyzed with subcellular spatial resolution. Several of these proteins were further studied in clinical tumor samples, confirming that this cell model provides a relevant system for studying cancer mechanisms. In paper IV, we continued to explore the transcriptional landscape in the same cell model under moderate hypoxic conditions.To conclude, this thesis demonstrates the usefulness of RNA-seq data, from a transcriptomics perspective and beyond; to guide in analyses of protein expression, with the ultimate goal to unravel the complexity of the human cell, from a holistic point of view.
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| 2. |
- Gnann, Christian, 1994-
(författare)
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Finding order in chaos : Dissecting single-cell heterogeneity in space and time
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cell is the smallest unit of life and contains DNA, RNA, proteins and a variety of other macromolecules. In recent years, technological advances in the field of single cell biology have revealed a staggering amount of phenotypic heterogeneity between cells in a population, which were previously considered homogenous. Previous work has largely been focused on studies of RNA. As proteins however are the ultimate effectors of genetic information, this thesis aims to provide a protein-centered view on cellular heterogeneity, particularly focusing on cell cycle and cellular metabolism.Most of my work has been performed within the framework of the Human Protein Atlas project. In the context of this project, we mapped the spatial distribution of more than 13.000 human proteins with subcellular resolution and found that around a quarter of all human proteins exhibit protein expression heterogeneity.In Paper I, we hypothesized that a majority of the observed cellular heterogeneity can be explained by differences in cell cycle progression. Therefore, we generated a map of proteomic and transcriptomic heterogeneity at subcellular resolution, which we precisely aligned to the cell cycle position of individual cells. This approach allowed us to identify hundreds of previously unknown cell cycle-related proteins. With sustained proliferative signaling representing a hallmark of cancer, novel cell-cycle proteins could serve as potential new drug targets against cancer. We further show that a large part of cell cycle dependent proteome variability is not established by transcriptomic cycling. This suggests that post-translational modifications are a major contributor to the regulation of cell cycle dependent protein level changes. Therefore, in Paper II, we carried out a deep phosphoproteome mass spectrometry profiling of the same cellular model as in Paper I and identified almost 5,000 cell cycle dependent phosphosites on over 2,000 proteins. The unprecedented scale of our phosphoproteomic data allows us to link cell cycle dependent protein expression dynamics to phosphorylation events. Furthermore, we identify a large set of proteins with stable expression levels and fluctuating phosphorylation patterns along cell cycle progression that likely alters protein function.Despite identifying hundreds of novel cell cycle dependent proteins in paper I, we observed that the majority of heterogeneously expressed proteins display variable expression independent of cell cycle progression, among them a large number of metabolic enzymes. Thus, we sought to describe the extent of subcellular metabolic complexity in human cells and tissues in Paper III. While we confirm metabolic compartmentalization in our dataset, we show that around 50% of metabolic enzymes localize to multiple cellular compartments. By integrating public protein-protein interaction data with our subcellular location information, we identify several enzymes with novel compartment-specific functions. Additionally, we observe a strongly elevated number of heterogeneously expressed enzymes compared to the background of the human proteome that is largely independent of cell cycle progression. We show that this heterogeneity can be manifested in the lineage of a single cell and is conserved in situ. To conclude, we suggest that the extensive metabolic heterogeneity can establish functional metabolic states in a population of human cells.Finally, in Paper IV, we assessed the heterogeneity of the mitochondrial proteome as they are metabolic powerhouses containing an elevated number of cell cycle independent variably expressed proteins. In this study, we correlated the variable expression of over 400 mitochondrial proteins to the expression of rate limiting enzymes in important mitochondrial pathways; such as the TCA cycle and ROS metabolism. We show that enzymes in the same pathways often correlate in their expression, indicating that their expression variability may contribute to the establishment of metabolic states.Altogether, the thesis illuminates the spatiotemporal complexity of the human proteome established by protein multilocalization and expression heterogeneity as fundamental non-genetic means of functional cell regulation.
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| 3. |
- Sariyar, Sanem
(författare)
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Spatiotemporal Profiling of Human Development Using Multiplexed Imaging
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human development is complex and intricate, where the positions of cells, expression of key markers, and cell-cell interactions contribute to the development of various organs from different germ layers and the establishment of the body axis. Therefore, understanding human development within spatial and temporal aspects is crucial. Spatial and temporal aspects can be studiedthrough multiplexed imaging, which enables the assessment of multiple markers on the same tissue, offering critical insights into protein expressions in the cells and tissues. Within the scope of this thesis, we focused on the spatial and single-cell profiling of cell types during the first trimester of human development, both at the systemic and organ levels, using multiplexed imaging. Paper I of this thesis presents a spatial and single-cell map of the developing human lung in the first trimester. We used multiplexed imaging on post-conception week 6 to 13 lungs employing a 30-plex antibody panel and, as a result, analyzed nearly 1 million cells. We provide a spatially resolved cell type composition of the developing human lung, focusing on spatiotemporal changes in the cell types, such as immune cells, endothelial cells, lymphatic cells, and proliferative cell states. Key findings of the first paper are that the proliferation patterns in the epithelium reveal differences in the elongation of smaller and larger distal and proximal airways and the presence of some immune cells around arteries, highlighting location-function relationships. Additionally, this paper represents the first application of multiplexed imaging on the developing human lung. Paper II aimed to systematically investigate human development in whole embryos by focusing on cell types such as immune and endothelial cells. We analyzed human whole embryo tissues from week 3 to 5 using a 28-plex multiplexed antibody panel. A key finding of the paper is the appearance of liver immune cells as early as week 4 and differences in their marker expression profiles compared to the other immune cells. In Paper III, we proposed a simple and flexible open-source method for visualizing in situ expressions of hundreds of genes, which can be combined with other methods, such as multiplexed imaging. In Paper IV, we explored the spatial dynamics of the developing human heart at the cellular and subcellular levels. In conclusion, this thesis elucidates the spatiotemporal changes during the first trimester of human development by presenting spatial maps of developing organs and whole embryos at various stages. The objective is to illustrate the characteristics of a healthy state, contributing to a better understanding of abnormalities associated with congenital diseases.
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| 4. |
- Stenström, Lovisa
(författare)
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The spatiotemporal protein landscape of human cells
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis focuses on the spatiotemporal mapping of proteins at a subcellular level. In other words, determining the cellular location of proteins over time. From a biological point of view, knowledge about protein location is fundamental to understand protein function. In the longer run, this also means better understanding of cells in the context of health and disease, since protein malfunction and mislocalization are two important factors during disease development. Using an antibody-based imaging approach, Paper I contains a subcellular map of 12 003 protein in 30 different cellular structures, freely accessible as part of the Human Protein Atlas (www.proteinatlas.org). Apart from enabling exploration of the organellar proteomes, we conclude that half of the human proteins localize to multiple compartments, and that almost one fifth display cell-to-cell variations in terms of protein expression. Paper II aimed to decrease the cumbersome work of manual protein location annotations by leveraging the power of the crowd through citizen science. By integrating the image-classification task into a video game with a massive player base, EVE online, protein location labels could be efficiently and rapidly assessed compared to manual curation from a few experts. To compare the performance of the players, a deep learning classifier was developed. The algorithm was capable of classifying protein location in images containing several challenging localization problems, such as multilocalizing proteins, cell line variations and rare classes. Using the protein location data from Paper I and Paper II, Paper III presents an image-based characterization of the nucleolar proteome. In total, 1 318 nucleolar proteins are included, of which 157 localizes to a fourth nucleolar compartment, the nucleolar rim. Additionally, 65 proteins were detected on the chromosomal periphery during mitosis, and these could be further divided into two recruitment phenotypes with different temporal profiles. Also, the mitotic chromosome proteins are enriched for intrinsically disordered domains, suggesting liquid-like properties of the perichromosomal layer. Paper IV presents a systematic dissection of the variable proteome drafted in Paper I. We show evidence for 539 proteins being correlated to cell cycle variations, of which a minority are also cycling at a transcriptional level, suggesting protein regulation at a translational or post-translational level. Additionally, we detected hundreds of proteins with previously unknown relations to mitosis and the cell cycle, many being linked to proliferation and oncogenic functions.In conclusion, Paper I and Paper II provide a basis for further in-depth studies of proteins at a subcellular level, while Paper III and Paper IV show how this resource can be used to study proteins in space and time. The results enable system-level investigation of protein dynamics, as well as provide exciting insights into organellar organization, such as the nucleolus.
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| 5. |
- Wiking, Mikaela
(författare)
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Spatial proteome profiling of the compartments of the human cell using an antibody-based approach
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human cell is complex, with countless processes ongoing in parallel in specialized compartments, the organelles. Cells can be studied in vitro by using immortalized cell lines that represent cells in vivo to a varying degree. Gene expression varies between cell types and an average cell line expresses around 10,000-12,000 genes, as measured with RNA sequencing. These genes encode the cell’s proteome; the full set of proteins that perform functions in the cell. In paper I we show that RNA sequencing is a necessary tool for studying the proteome of the human cell.By studying the proteome, and proteins’ localization in the cell, information can be assembled on how the cell functions. Image-based methods allow for detailed spatial resolution of protein localization as well as enable the study of temporal events. Visualization of a protein can be accomplished by using either a cell line that is transfected to express the protein with a fluorescent tag, or by targeting the protein with an affinity reagent such as an antibody. In paper II we present subcellular data for a majority of the human proteins, showing that there is a high degree of complexity in regard to where proteins localize in the cell.Cellular energy is generated in the mitochondria, an important organelle that is also active in many other different functions. Today approximately only a third of the estimated mitochondrial proteome has been validated experimentally, indicating that there is much more to understand with regard to the functions of the mitochondria. In paper III we explore the mitochondrial proteome, based on the results of paper II. We also present a method for sublocalizing proteins to subcompartments that can be performed in a high-throughput manner.To conclude, this thesis shows that transcriptomics is a useful tool for proteome-wide subcellular localization, and presents high-resolution spatial distribution data for the human cell with a deeper analysis of the mitochondrial proteome.
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| 6. |
- Winsnes, Casper F.
(författare)
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On computational methods for spatial mapping of the human proteome
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are complex molecules that are involved in almost every task in the body. In general, the role a protein fulfills is highly dependent on where in the cell it is located, its subcellular localization. In order to understand human biology, it is therefore imperative to gain insight into the world of proteins by examining their subcellular distribution and interaction with each other. This thesis focuses on the development of computational models capable of performing large scale spatial protein analysis on a subcellular level. Within that scope, we were able to develop models that classify the localization of proteins in immunofluorescence microscopy images as well as show how such models can integrate with other methods to gain novel insights and understanding into the roles and spatially dependent functions of proteins. In Paper I, we present and combine two separate methods for large scale protein localization. The first method is an integration of a protein localization task as a mini-game within an established massively multiplayer online video game. The second method consists of the first image-based deep neural network learning model capable of multi-label subcellular localization classification. We show that both these methods enable accurate and scalable high-throughput analysis of subcellular protein localization that overcome many of the challenges associated with such a dataset. We also show that combining the two methods yield better results than either of them do on their own, resulting in a model that is nearing human performance. In Paper II, based on the success of the neural network model from Paper I, we continue the investigation into usage of deep neural networks for the purpose of subcellular protein localization. In an effort to find the best possible model for such tasks, a machine learning image competition was developed. Over 2,000 teams participated with various kinds of architectures, resulting in a predictor that far outperforms the one presented in Paper I. The winning model is analyzed thoroughly, and we show that its internal feature representation contains biologically relevant information and that it can be used for quantitative analysis of protein patterns. Paper III takes the feature representation of immunofluorescence images from the model developed in Paper II and integrates it with features extracted from affinity purification experiments to create a hierarchical map of the human cell’s architecture. This method creates a map of protein communities grouped by subcellular structures, of which approximately 54% are putatively novel. We show that the map is biologically significant by validating several of the novel findings using affinity purification experiments and in-situ fractionation. In Paper IV, we apply what was learned in Paper I and II in order to create a model that identifies proteins residing within micronuclei. We apply the model on the image data from the Human Protein Atlas to create the first extensive mapping of the micronuclear proteome. Through enrichment analysis of the identified proteins, we propose that micronuclei harbor a more diverse set of functions than previously thought. We find that the micronuclear proteome is highly interconnected and contains many proteins that show visible variations across different micronuclei, and theorize on what this means for their role in the cell.In conclusion, Paper I and II examine and establish the possibilities of using deep neural networks for systematic subcellular protein localization analysis. Paper III and IV build upon what was learned in Papers I and II and use their models to examine protein distribution patterns and provide novel biological insights.
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| 7. |
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| 8. |
- Lundberg, Anna, Professor, 1972-, et al.
(författare)
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Rättsosäkerheten och nämndepersonerna
- 2021
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Ingår i: Artikel 14. - 1104-1846. ; , s. 18-21:4, s. 18-21
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Tidskriftsartikel (populärvet., debatt m.m.)
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| 9. |
- Skogs, Marie, 1981-
(författare)
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Antibody-based subcellular localization of the human proteome
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the use of antibodies and immunofluorescence for subcellular localization of proteins. The key objective is the creation of an open-source atlas with information on the subcellular location of every human protein. Knowledge of the spatial distribution and the precise location of a protein within a cell is important for its functional characterization, and describing the human proteome in terms of compartment proteomes is important to decipher cellular organization and function. Immunofluorescence and confocal microscopy of cultured cells were used for high-resolution detection of proteins on a high-throughput scale. Critical to immunofluorescence results are sample preparation and specific antibodies. Antibody staining of cells requires fixation and permeabilization, both of which can result in loss or redistribution of proteins and masking of epitopes. A high-throughput approach demands a standardized protocol suitable for the majority of proteins across cellular compartments. Paper I presents an evaluation of sample preparation techniques from which such a single fixation and permeabilization protocol was optimized. Paper II describes the results from applying this protocol to 4000 human proteins in three cell lines of different origin. Paper III presents a strategy for application-specific antibody validation. Antibodies are the key reagents in immunofluorescence, but all antibodies have potential for off-target binding and should be validated thoroughly. Antibody performance varies across sample types and applications due to the competition present and the effect of the sample preparation on antigen accessibility. In this paper application-specific validation for immunofluorescence was conducted using colocalization with fluorescently tagged protein in transgenic cell lines.
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| 10. |
- Sountoulidis, Alexandros, et al.
(författare)
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A topographic atlas defines developmental origins of cell heterogeneity in the human embryonic lung
- 2023
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Ingår i: Nature Cell Biology. - : Springer Nature. - 1465-7392 .- 1476-4679.
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Tidskriftsartikel (refereegranskat)abstract
- Sountoulidis et al. provide a spatial gene expression atlas of human embryonic lung during the first trimester of gestation and identify 83 cell identities corresponding to stable cell types or transitional states. The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.
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| 11. |
- Stadler, Charlotte, 1982-
(författare)
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Towards subcellular localization of the human proteome using bioimaging
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained.A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner.Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins.Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level.
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| 12. |
- Söderman, Emma, et al.
(författare)
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”Du förstår men du förstår ingenting” [‘You understand but you don’t understand a thing’] : Communityteater, utvisningsbarhet och översättningspolitik Emma Söderman och Anna Lundberg [Community theatre, deportability and the politics of translation]
- 2020
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Ingår i: Socialvetenskaplig tidskrift. - Linköping : Förbundet för Forskning i Socialt Arbete. - 1104-1420 .- 2003-5624. ; 27:3-4, s. 291-308
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Tidskriftsartikel (refereegranskat)abstract
- In the following article, based on two years of participatory ethnographic field-work with the No-border musical, as well as interviews with 16 of the musical’s 30 participants, community theatre is investigated in a context of deportability. We analyse the working process in the theatre group, in which actors with and without resident permits participated, through the concept of politics of translation. We show how inequalities due to the constant threat of deportation for several members, were put to the forefront during the work process of creating the musical. It concerned risks of detection for the undocumented participants as well difficult living conditions related to deportability (for example insecure access to livelihood, healthcare, housing etc.). The article conceptualizes various dimensions of working together in a group where participants live in unequal conditions as a politics of translation. This concept includes the work of language translation, and also captures translations of the different experiences mentioned above, and how different positions of power can be handled and understood, within a group who has the ambition to work together, in this case with a theatrical performance. Our analysis shows how theatre in a context of asylum rights activism can challenge and create alternatives to the conditions of deportability, whilst these simultaneously condition the activism and translation. The article contributes to knowledge about mobilization in the context of vulnerability and inequality. We hope to also contribute to a development of critical social work both within and outside of academia.
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