| 1. |
- Grundberg, Ida, 1982-
(författare)
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Genotyping and Mutation Detection In Situ : Development and application of single-molecule techniques
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human body is composed of trillions of cells closely working together to maintain a functional organism. Every cell is unique in molecular composition and can acquire genetic variations that might cause it to turn pathological. It is essential to develop improved tools to better understand the development of normal and disease tissue, ideally enabling single-cell expression studies in preserved context of complex tissue with single-nucleotide resolution. This thesis presents the development and application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The described technique utilizes padlock probes and target-primed rolling circle amplification and is highly suitable for sensitive in situ analysis. First, a new strategy for directed cleavage of single stranded DNA was investigated, e.g. nucleic acid targets with extended 3´ ends, for successful initiation of rolling circle amplification. The presented cleavage strategy is simple and applicable for subsequent enzymatic reactions, e.g. ligation and polymerization. Specific cleavage of long target overhangs was demonstrated in synthetic oligonucleotides and in genomic DNA and the detection efficiency was substantially increased. For multiplex detection and genotyping of individual transcripts in single cells, a new in situ method was developed. The technique showed a satisfactorily detection efficiency and was later applied as a general mutation analysis tool for detection of KRAS point mutations in complex tumor tissue sections, e.g. formalin-fixed, paraffin-embedded tumor tissues and cytologic tumor imprints. Mutation status was assessed in patient samples by in situ padlock probe detection and results were confirmed by DNA-sequencing. Finally, the method was adapted for simultaneous detection of individual mRNA molecules and endogenous protein modifications in single cells using padlock probes and in situ PLA. This assay will be useful for gene expression analysis and exploration of new drugs with vague effector sites. To our knowledge, no other technique exists today that offers in situ transcript detection with single-nucleotide resolution in heterogeneous tissues. The method will especially be suitable for discrimination of highly similar transcripts, e.g. splice variants, SNPs and point mutations, within gene expression studies and for cancer diagnostics.
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| 2. |
- Jarvius, Malin, 1975-
(författare)
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Visualization of Protein Activity Status in situ Using Proximity Ligation Assays
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In 2001 the human proteome organization (HUPO) was created with the ambition to identify and characterize all proteins encoded in the human genome according to several criteria; their expression levels in different tissues and under different conditions; the sub-cellular localization; post-translational modifications; interactions, and if possible also the relationship between their structure and function.When the knowledge of different proteins and their potential interactions increases, so does the need for methods able to unravel the nature of molecular processes in cells and organized tissues, and ultimately for clinical use in samples obtained from patients. The in situ proximity ligation assay (in situ PLA) was developed to provide localized detection of proteins, post-translational modifications and protein-protein interactions in fixed cells and tissues. Dual recognition of the target or interacting targets is a prerequisite for the creation of a circular reporter DNA molecule, which subsequently is locally amplified for visualization of individual protein molecules in single cells. These features offer the high sensitivity and selectivity required for detection of even rare target molecules. Herein in situ PLA was first established and then employed as a tool for detection of both interactions and post-translational modifications in cultured cells and tissue samples. In situ PLA was also adapted to high content screening (HCS) for therapeutic effects, where it was applied for cell-based drug screening of inhibitors influencing post-translational modifications. This was performed using primary cells, paving the way for evaluation of drug effects on cells from patient as a diagnostic tool in personalized medicine. In conclusion, this thesis describes the development and applications of in situ PLA as a tool to study proteins, post-translational modifications and protein-protein interactions in genetically unmodified cells and tissues, and for clinical interactomics.
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| 3. |
- Kühnemund, Malte
(författare)
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Single Molecule Detection : Microfluidic Automation and Digital Quantification
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Much of recent progress in medical research and diagnostics has been enabled through the advances in molecular analysis technologies, which now permit the detection and analysis of single molecules with high sensitivity and specificity. Assay sensitivity is fundamentally limited by the efficiency of the detection method used for read-out. Inefficient detection systems are usually compensated for by molecular amplification at the cost of elevated assay complexity.This thesis presents microfluidic automation and digital quantification of targeted nucleic acid detection methods based on padlock and selector probes and rolling circle amplification (RCA). In paper I, the highly sensitive, yet complex circle-to-circle amplification assay was automated on a digital microfluidic chip. In paper II, a new RCA product (RCP) sensing principle was developed based on resistive pulse sensing that allows label free digital RCP quantification. In paper III, a microfluidic chip for spatial RCP enrichment was developed, which enables the detection of RCPs with an unprecedented efficiency and allows for deeper analysis of enriched RCPs through next generation sequencing chemistry. In paper IV, a smart phone was converted into a multiplex fluorescent imaging device that enables imaging and quantification of RCPs on slides as well as within cells and tissues. KRAS point mutations were detected (i) in situ, directly in tumor tissue, and (ii) by targeted sequencing of extracted tumor DNA, imaged with the smart phone RCP imager. This thesis describes the building blocks required for the development of highly sensitive low-cost RCA-based nucleic acid analysis devices for utilization in research and diagnostics.
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| 4. |
- Weibrecht, Irene, 1983-
(författare)
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Visualizing Interacting Biomolecules In Situ
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication. In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously. In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes. This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures.
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| 5. |
- Al-Amin, Rasel A., Researcher, 1983-, et al.
(författare)
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Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:22, s. e129-e129
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Tidskriftsartikel (refereegranskat)abstract
- Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues.
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| 6. |
- Al-Amin, Rasel A., 1983-, et al.
(författare)
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Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in Situ
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- It is important to determine the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimens. This is necessary to evaluate drug candidates from early stages of drug development to clinical evaluation of mutations potentially causing resistance to targeted therapy. We describe a technology where oligonucleotide-conjugated drug molecules are used to visualize and measure target engagement in situ via rolling-circle amplification (RCA) of circularized oligonucleotide probes (padlock probes). We established this target engagement-mediated amplification (TEMA) technique using kinase inhibitor precursor compounds, and we applied the assay to investigate target interactions by microscopy in pathology tissue sections and using flow cytometry for blood samples from patients, as well as in commercial arrays including almost half of all human proteins. In the variant proxTEMAtechnique, in situ proximity ligation assays were performed by combining drug-DNA conjugates with antibody-DNA conjugates to specifically reveal drug binding to particular on- or off-targets in pathological tissues sections. In conclusion, the TEMA methods successfully visualize drug-target interaction by experimental and clinically approved kinase inhibitors in situ and with kinases among a large collection of arrayed proteins.
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| 7. |
- Björkesten, Johan, 1988-
(författare)
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Dried blood sampling and digital readout to advance molecular diagnostics
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A drastically increased capacity to measure large sets of molecular features in numerous patient samples in great detail will be required to fulfill the vision of precision medicine and wellness, which may characterize molecular diagnostics in the 21st century. Also sampling procedures need a renaissance to permit continuous sampling at population levels at reasonable cost.Blood sampling is typically performed via venipuncture to draw several milliliters of blood for plasma isolation. This is inconvenient, time-consuming and costly, as well as hard to standardize. The effect on plasma protein profiles by pre-centrifugation delay was investigated in Paper II, demonstrating time- and temperature-dependent release of proteins from blood cells upon delayed plasma isolation, but almost no protein degradation as analyzed by two 92-plex protein panels (Olink® Proteomics). An alternative sampling method, where blood drops from a finger stick are collected dried on paper, is relatively non-invasive, potentially home-based and cheap. Dried blood spots can also be shipped via regular mail and compactly stored. The effect of drying and long term storage stability of a large set of proteins from dried blood spots was investigated in Paper I using Olink® technology. The main findings were that drying slightly but consistently influenced the recorded levels of blood proteins, and that long-term storage decreased the detected levels of some of the proteins with half-lives of decades.Some molecular diagnostic investigations require great accuracy to be useful, arguing for digital enumeration of individual molecules. Digital PCR is the gold standard but Paper III presents an alternative approach based on rolling circle amplification of single molecules. Another instance where extreme assay performance is required is for rare mutation detection from liquid biopsies. Paper V presents a new method offering essentially error-free genotyping of individual molecules by majority-vote decisions for counting rare mutant DNA in blood. Yet other diagnostic investigations require very simple assays. Paper IV presents a novel one-step method to detect nucleic acid sequences by combining the power of rolling circle amplification and the specificity of DNA strand displacement in a format simple enough to be used at the point of care. Altogether, the thesis spans technologies for advanced molecular diagnostics, from sample collection over assay techniques to an improved readout.
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| 8. |
- Darmanis, Spyros, 1983-
(författare)
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Solid-phase Proximity Ligation Assays : High-performance and multiplex protein analyses
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein biomarkers circulating in blood hold the promise of improved diagnosis, prognosis and follow-up of treatment of disease via minimally invasive procedures. For the discovery and validation of such biomarkers, methods are needed that can facilitate parallel, highly specific and in-depth analysis of the blood proteome. The work presented in this thesis intends to develop and apply such assays, building on the concept of the proximity ligation assay (PLA). In paper I, I present an easy and non-expensive alternative for the conjugation of oligonucleotides to antibodies via biotin-streptavidin-biotin interaction. This approach can be used when large sets of antibodies and/or oligos need to be validated for their performance as probes in PLA reactions. In paper II, a solid-phase variant of PLA (SP-PLA) for the detection and quantification of proteins in blood is presented. SP-PLA exhibited an improved limit of detection compared to commercial ELISA assays by two orders of magnitude. In addition SP-PLA exhibited a broader dynamic range by at least one order of magnitude and required only 5 μl of sample, rendering the method very well suited for analyses of precious bio-banked material. Last but not least, SP-PLA was used to validate the diagnostic potential of GDF-15 as a biomarker for cardiovascular disease in a set of cardiovascular disease patients and healthy controls. Paper III discusses the development of a multiplex SP-PLA (MultiPLAy) for the simultaneous detection of 36 proteins in just 5 μl of sample. MultiPLAy exhibited an improved LOD when compared to state-of-the-art bead-based sandwich assays. Most importantly, we observed only a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that much higher levels of multiplexing will be possible. The assay was used to identify putative biomarkers in sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD). Subsequent multivariate analysis revealed previously known diagnostic biomarkers. Furthermore, we successfully applied next-generation sequencing as a readout for the protein assays, allowing for the first time digital recording of protein profiles in blood. In paper IV, we investigated the suitability of prostasomes as blood biomarkers in patients with prostate cancer using a newly developed PLA assay (4PLA) that utilizes five binders for the detection of complex target molecules. The assay successfully detected significantly elevated levels of prostasomes in blood samples from prostate cancer patients prior to radical prostatectomy, compared to controls and men with benign biopsy results.
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| 9. |
- Ebai, Tonge
(författare)
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Development of Enhanced Molecular Diagnostic Tools for Protein Detection and Analysis
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Improved diagnosis, prognosis and disease follow-up is a fundamental procedure and a constant challenge in medicine. Among the different molecular biomarkers, proteins are the essential regulatory component in blood; hence, by developing enhanced specific and sensitive molecular tools will gives great insight into the different processes in disease treatment. In this thesis, we build on the proximity ligation assay to develop and apply new adaptable methods to facilitate protein detection.In paper I, I present a variant of the proximity ligation assay (we call PLARCA) using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer. PLARCA detected femtomolar levels of these proteins in patient samples, which was considerably below the detection threshold for ELISA.In paper II, we developed and adapted a new method into the in situ PLA methods for detection and identification of extracellular vesicles (EVs) using flow cytometry as readout (a method we call ExoPLA). We identified five target proteins on the surface of the Evs and using three colors, we identified the EV using flow cytometer.In paper III, we aim to improve the efficiency of in situ PLA by creating and developing new designs and versions of the assay we called Unfold probes Through comparison of detection of protein using in situ PLA versus Unfold probes, we observed considerable decrease in non-specific signals, and also a lower detection threshold.In paper IV, we describe the development of a solid phase proximity extension (sp-PEA) assay for protein detection and quantification. We compared detection of IL-8, TNF-alpha, IL-10 and IL-6 using spPEA and PEA; spPEA demonstrations over 2 orders of magnitudes in the lower detection concentrations by decreased in background noise.
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| 10. |
- Hammond, Maria, 1984-
(författare)
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DNA-Mediated Detection and Profiling of Protein Complexes
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules.Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).
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