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.

<p>A novel technology for protein detection, proximity ligation, has been developed along with improved methods for <i>in situ</i> synthesis of DNA microarrays. Proximity ligation enables a specific and quantitative transformation of proteins present in a sample into nucleic acid sequences. As pairs of so-called proximity probes bind the individual target protein molecules at distinct sites, these reagents are brought in close proximity. The probes consist of a protein specific binding part coupled to an oligonucleotide with either a free 3’- or 5’-end capable of hybridizing to a common connector oligonucleotide. When the probes are in proximity, promoted by target binding, then the DNA strands can be joined by enzymatic ligation. The nucleic acid sequence that is formed can then be amplified and quantitatively detected in a real-time monitored polymerase chain reaction. This convenient assay is simple to perform and allows highly sensitive protein detection. Parallel analysis of multiple proteins by DNA microarray technology is anticipated for proximity ligation and enabled by the information carrying ability of nucleic acids to define the individual proteins. Assays detecting cytokines using SELEX aptamers or antibodies, monoclonal and polyclonal, are presented in the thesis.</p><p>Microarrays synthesized <i>in situ</i> using photolithographic methods generate impure products due to damaged molecules and interrupted synthesis. Through a molecular inversion mechanism presented here, these impurities may be removed. At the end of synthesis, full-length oligonucleotides receive a functional group that can then be made to react with the solid support forming an arched structure. The 3’-ends of the oligonucleotides are then cleaved, removing the impurities from the support and allowing the liberated 3’-hydroxyl to prime polymerase extension reactions from the inverted oligonucleotides. The effect of having pure oligonucleotides probes compared to ones contaminated with shorter variants was investigated in allele specific hybridization reactions. Pure probes were shown to have greater ability to discriminate between matched and singly mismatched targets at optimal hybridization temperatures.</p>

A novel technology for protein detection, proximity ligation, has been developed along with improved methods for in situ synthesis of DNA microarrays. Proximity ligation enables a specific and quantitative transformation of proteins present in a sample into nucleic acid sequences. As pairs of so-called proximity probes bind the individual target protein molecules at distinct sites, these reagents are brought in close proximity. The probes consist of a protein specific binding part coupled to an oligonucleotide with either a free 3’- or 5’-end capable of hybridizing to a common connector oligonucleotide. When the probes are in proximity, promoted by target binding, then the DNA strands can be joined by enzymatic ligation. The nucleic acid sequence that is formed can then be amplified and quantitatively detected in a real-time monitored polymerase chain reaction. This convenient assay is simple to perform and allows highly sensitive protein detection. Parallel analysis of multiple proteins by DNA microarray technology is anticipated for proximity ligation and enabled by the information carrying ability of nucleic acids to define the individual proteins. Assays detecting cytokines using SELEX aptamers or antibodies, monoclonal and polyclonal, are presented in the thesis. Microarrays synthesized in situ using photolithographic methods generate impure products due to damaged molecules and interrupted synthesis. Through a molecular inversion mechanism presented here, these impurities may be removed. At the end of synthesis, full-length oligonucleotides receive a functional group that can then be made to react with the solid support forming an arched structure. The 3’-ends of the oligonucleotides are then cleaved, removing the impurities from the support and allowing the liberated 3’-hydroxyl to prime polymerase extension reactions from the inverted oligonucleotides. The effect of having pure oligonucleotides probes compared to ones contaminated with shorter variants was investigated in allele specific hybridization reactions. Pure probes were shown to have greater ability to discriminate between matched and singly mismatched targets at optimal hybridization temperatures.

Binder-based assays are employed throughout the life sciences. Powerful signal amplification techniques have enabled detection of very rare molecule species diluted in simple buffers. Unspecific binding of primary binders leads to increased background in more complex samples. By requiring two recognition events, ligation-based molecular analyses provide highly specific detection of biomolecules in complex samples. We developed a highly multiplexed padlock-ligation assay targeting signature sequences in the hemagglutinin and neuraminidase genes. From a panel of 77 avian influenza isolates of all major serotypes, 97% were genotyped correctly in accordance with previous classifications by classical diagnostic methods (Paper I). Alternative splicing is an important mechanism expanding the proteome. Current analysis techniques fail to provide sequences of complete transcripts beyond the read length of sequencing instruments. We devised and implemented a strategy to compress the sequence information contained in the splicing pattern of a transcript into the presence or absence of sequence-blocks. We demonstrate that this assay yields information about the splicing patterns in thousands of transcripts from cellular cDNA (Paper II). Expression changes of mucin proteins and glycosylation structures are frequently observed from the early stages of cancer development. Expression of mucin 2 and sialyl-Tn are common features of intestinal metaplasia and gastric cancer, and are known to co-locate. Here we have developed an in situ proximity ligation assay (PLA) directed against mucin 2 and sialyl-Tn. Our study on intestinal metaplasia and gastric cancer tissue sections identified mucin 2 as a major carrier of sialyl-Tn in these conditions, and demonstrated how conveniently glycosylation of proteins can be studied by in situ PLA (Paper III). This thesis shows how the dual recognition requirement of ligation-based assays can be employed to detect target molecules with high specificity, to analyze several sequence features of nucleic acids or to study the proximity of two antigens in situ.

The extent of devastation caused by a biological warfare attack is highly correlated to the time from release to detection. As a step towards lowering the detection time the international project TWOBIAS was launched. Here, the main goal is to develop an automated, specific and sensitive combined detection and identification instrument capable of identifying a biological threat within an hour. The identification unit is comprised of a sample preparation module, an amplification module and a detection module and utilizes a proximity ligation assay in combination with circle-to-circle amplification in order to detect a biological threat. This thesis describes the automation of the sample preparation steps of the assay and the integration with the downstream units. The functionality of the sample preparation module was verified by subjecting it to biological samples in a laboratory and at a real-life location. The results showed that the sample preparation module was capable of preparing a sample collected in a complex environment with the same results as a sample prepared in a laboratory.

With the advent of Tyrosine kinase inhibitors (TKI) as a therapy for Chronic myeloid Leukemia (CML), the patients now enjoy a life expectancy close to that of the general population. But some patients do get unresponsive to the TKI treatment over time due to several mutations in the kinase domain of the BCR-ABL fusion protein, which further leads to activation of multiple signaling cascades within the leukemic cell, helping it survive and proliferate. This project validates and optimizes a new method of In situ PLA that incorporates the usage of different padlocks and template oligos. Multiple cross-reactivity tests and interaction assays in multiple cancer cell lines will further optimize this system as a robust multiplex protein-protein interaction detection tool. Proteins associated with the MAP-K, PI3-K, and Jak-STAT signaling pathways were the main detection targets.

Le métabolisme des cellules immunitaires est assujetti à un contrôle étroit. L’inflammation, la présentation antigénique et l’expansion clonale sont des évènements qui demandent un changement rapide dans le métabolisme des cellules. Notamment, la glutamine est grandement sollicitée lors de la maturation des cellules dendritiques, les macrophages et les lymphocytes B et T pour son rôle dans la synthèse des protéines et son implication dans la phosphorylation oxydative. Au repos, les cellules présentatrices d’antigènes (CPAs) expriment l’ubiquitine ligase E3 MARCH1. MARCH1 est une protéine membranaire qui régule la réponse immunitaire en ubiquitinant, entre autres, le complexe majeur d’histocompatibilité II (CMH II) et CD86. Lors de l’activation des cellules immunitaires, son expression est réprimée, ce qui permet l’accumulation du CMH II et de CD86 sur leur membrane. Nous pensons que MARCH1 régule négativement le métabolisme des cellules immunitaires. Parmi les protéines membranaires sous le contrôle de MARCH1 pourraient se trouver des transporteurs de glutamine. La baisse rapide de MARCH1 serait nécessaire pour permettre aux cellules de modifier leur métabolisme en augmentant le transport de la glutamine. Dans le mémoire présent, nous nous sommes intéressés à l’interactôme de MARCH1. Afin de découvrir de nouvelles cibles de MARCH1, nous avons utilisé la méthode du BioID dans des cellules HEK293T. Le BioID est une méthode innovatrice permettant l’identification d’interactions interprotéines. La protéine de fusion BioID2 permet la biotinylation et l’isolation des protéines adjacentes in vivo. Ces essais de proximité nous ont permis d’identifier 41 cibles potentielles de MARCH1. Nous avons analysé l’expression de 13 de ces protéines par cytométrie en flux. Nos résultats démontrent que MARCH1 induit la dégradation de NKCC1, CD147 et SNAT2. L’expression de MARCH1 dans les cellules HEK293T engendre une diminution de SNAT2 en surface. S’il avère que MARCH1 régule le métabolisme de la glutamine dans les cellules immunitaires, il s’agirait alors d’un nouveau mécanisme par lequel cette ubiquitine ligase E3 module la réponse immunitaire. SNAT2 est nécessaire dans l’adaptation des cellules pour leurs besoins en glutamine. Nous discuterons du rôle que joue cette protéine dans l’adaptation du métabolisme et la glutaminolyse. / Immune cell metabolism is subjected to a tight control. Inflammation, antigen presentation and clonal expansion are all events that comes with a rapid change in metabolism. Glutamine is highly solicited during dendritic cells, macrophages and B and T lymphocytes maturation, due to its role in protein synthesis and oxidative phosphorylation. At steady-state, antigen presenting cells express the ubiquitin ligase E3 MARCH1. MARCH1 is a membrane protein involved in the immune response through major histocompatibility II and CD86 ubiquitination and degradation. During their activation, MARCH1 expression is repressed. This allows for accumulation of MHC II and CD86 on the cell surface, but other membrane-bound receptors and transporters are also increased during that time. Among those, proteins involved in glutamine transport are increased and thus help immune cells to adjust their intracellular nutrient pool for their new metabolic needs. We propose that MARCH1 negatively regulates immune cell metabolism through the regulation of nutrient transporters. The rapid stop in the transcription of MARCH1 induces an increase in receptors on the cytoplasmic membrane. Here, we aimed to identify the MARCH1 interactome. In order to identify new MARCH1 targets, we used the BioID proximity assays in HEK293T cells. BioID is an innovative method for the identification of protein interactions. BioID2 protein fusion can be used for in vivo biotinylation and isolation of promiscuous proteins. These proximity assays allowed us to identify 41 potential MARCH1 targets. We analyzed the expression of 13 of these proteins and found that 3 were affected by MARCH1 expression. Our results show that MARCH1 induces the degradation of NKCC1, CD147 and SNAT2. More specifically, MARCH1 expression in HEK293T induces the internalisation of the glutamine transporter SNAT2. If MARCH1 proves to regulate glutamine transport in immune cells, this would be a novel mechanism by which this ubiquitin ligase regulates adaptive immune system. Indeed, SNAT2 is required for the cellular adaption of amino acids during maturation, including glutamine. We will discuss the implications of MARCH1 as a metabolic switch and the role this would have in glutaminolysis and antigen presentation.

The differential activation of Wnt pathways (canonical: Wnt/-catenin; non-canonical: planar cell polarity (PCP), Wnt/Ca2+) depends on the cell-specific availability and regulation of Wnt receptors, called Frizzled (FZD). FZDs selectively recruit co-receptors to activate various downstream effectors. We established a proximity ligation assay (PLA) for the detection of endogenous FZD–coreceptor interactions and analyzed time-dependent Wnt pathway activation in cultured cells. Prostate cancer cells (PC-3) stimulated by Wnt ligands (Wnt5A, Wnt10B) were analyzed by Cy3-PLA for the co-localization of FZD6 and co-receptors (canonical: LRP6, non-canonical: ROR1) at the single-cell level. Downstream effector activation was assayed by immunocytochemistry. PLA allowed the specific (siRNA-verified) detection of FZD6–LRP6 and FZD6–ROR1 complexes as highly fluorescent spots. Incubation with Wnt10B led to increased FZD6–LRP6 interactions after 2 to 4 min and resulted in nuclear accumulation of -catenin within 5 min. Wnt5A stimulation resulted in a higher number of FZD6–ROR1 complexes after 2 min. Elevated levels of phosphorylated myosin phosphatase target 1 suggested subsequent Wnt/PCP activation in PC-3. This is the first study demonstrating time-dependent interactions of endogenous Wnt (co-)receptors followed by rapid Wnt/-catenin and Wnt/PCP activation in PC-3. In conclusion, the PLA could uncover novel signatures of Wnt receptor activation in mammalian cells and may provide new insights into involved signaling routes

<p>Among the great challenges in biology are the precise quantification of specific sets of proteins and analyses of their patterns of interaction on a much larger scale than is possible today. </p><p>This thesis presents a novel protein detection technique - proximity ligation - and reports the development and application of a nucleic acid amplification technique, RCA. Proximity ligation converts information about the presence or co-localization of specific proteins to unique sets of nucleic acid sequences. For detection of target proteins or protein complexes the coincident binding by pairs or triplets of specific protein-binding reagents are required. Oligonucleotide-extensions attached to those binding reagents are joined by a DNA ligase and subsequently analyzed by standard molecular genetic techniques. The technique is shown to sensitively detect an assortment of proteins using different types of binders converted to proximity probes, including SELEX aptamers and mono- and polyclonal antibodies. I discuss factors important for using the technique to analyze many proteins simultaneously.</p><p>Quantification of target molecules requires precise amplification and detection. I show how rolling circle amplification, RCA, can be used for precise quantification of circular templates using modified molecular beacons with real-time detection. The combination of proximity-probe templated circularization and RCA results in a sensitive method with high selectivity, capable of visualizing individual immobilized proteins. This technique is used for localized detection of a set of individual proteins and protein complexes at sub-cellular resolution.</p>

Fundamental to all biological processes is the interplay between biomolecules such as proteins and nucleic acids. Studies of interactions should therefore be more informative than mere detection of expressed proteins. Preferably, such studies should be performed in material that is as biologically and clinically relevant as possible, i.e. in primary cells and tissues. In addition, to be able to take into account the heterogeneity of such samples, the analyses should be performed in situ to retain information on the sub-cellular localization where the interactions occur, enabling determination of the activity status of individual cells and allowing discrimination between e.g. tumor cells and surrounding stroma. This requires assays with an utmost level of sensitivity and selectivity. Taking these issues into consideration, the in situ proximity-ligation assay (in situ PLA) was developed, providing localized detection of proteins, protein-protein interactions and post-translational modifications in fixed cells and tissues. The high sensitivity and selectivity afforded by the assay's requirement for dual target recognition in combination with powerful signal amplification enables visualization of single protein molecules in intact single cells and tissue sections. To further increase the usefulness and application of in situ PLA, the assay was adapted to high content analysis techniques such as flow cytometry and high content screening. The use of in situ PLA in flow cytometry offers the possibility for high-throughput analysis of cells in solution with the unique characteristics offered by the assay. For high content screening, it was demonstrated that in situ PLA can enable cell-based drug screening of compounds affecting post-translational modifications and protein-protein interactions in primary cells, offering superior abilities over current assays. The methods presented in this thesis provide powerful new tools to study proteins in genetically unmodified cells and tissues, and should offer exciting new possibilities for molecular biology, diagnostics and drug discovery.