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DNA-Assisted Immunoassays for High-Performance Protein AnalysesYan, Junhong January 2014 (has links)
Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages. In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.
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Proximity Ligation Assays for Disease Biomarkers AnalysisNong, Rachel Yuan January 2011 (has links)
One of the pressing needs in the field of disease biomarker discovery is new technologies that could allow high performance protein analysis in different types of clinical material, such as blood and solid tissues. This thesis includes four approaches that address important limitations of current technologies, thus enabling highly sensitive, specific and parallel protein measurements. Paper I describes a method for sensitive singleplex protein detection in complex biological samples, namely solid phase proximity ligation assay (SP-PLA). SP-PLA exhibited improved sensitivity compared to conventional sandwich immunoassays. We applied SP-PLA to validate the potential of GDF-15 as a biomarker for cardiovascular disease. Paper II describes ProteinSeq, a multiplexed immunoassay based on the principle of SP-PLA, for parallel detection of 36 proteins using next-generation sequencing as readout. ProteinSeq exhibited improved sensitivity compared to multiplexed sandwich immunoassays, and the potential to achieve even higher levels of multiplexing while preserving a high sensitivity and specificity. We applied ProteinSeq to analyze 36 proteins, including one internal control, in 5 μl of plasma samples in a cohort of patients with cardiovascular disease and healthy controls. Paper III describes PLA-DTM, a strategy for recording all possible interactions between sets of proteins in clinical samples. Individual proteins and their interactions are first encoded to dual barcoded DNA by PLA, and the barcodes are interrogated by a method named dual tag microarray (DTM). We applied the method for studying interactions among protein members of the NFκB signaling pathway. Paper IV describes a novel probing strategy for analyzing individual biomolecules in solution or in situ. The technique employs a new class of probes for unfolding proximity ligation assays - uPLA probes. The probes are designed so that each probe set is sufficient in forming and replicating circular DNA reporter, without interactions among themselves when incubated with the sample. The uPLA probing strategy provides ease in the design of multiple probe sets in parallelized assays while enhancing the specificity of detection. We used the uPLA probes to detect various targets, including synthetic DNA and cancer-related transcripts in situ.
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Development and Application of Polyclonal Antibody Based Proximity Ligation Assays in Detecting Antigenic Variants of Influenza A VirusesMartin, Brigitte Elizabeth 06 May 2017 (has links)
Influenza A virus (IAV) is a zoonotic pathogen which consists of a large genetically and antigenically diverse viral population. Swine IAVs not only cause disease outbreaks among swine, but can also be transmitted to humans, causing sporadic infections and even pandemic outbreaks apart from human seasonal IAV. Antigenic variant identification is fundamental for an effective vaccination program. Red blood cell based immunological tests have been used to identify antigenic variants among circulating IAV strains. Because these assays require viral isolation, they are time consuming and labor intensive. Thus only limited numbers of virus isolates are subjected to antigenic characterization in influenza surveillance studies and much of this important information is lost. In this project, a novel polyclonal antibody based proximity ligation assays (polyPLA) was developed and validated to characterize IAV antigenic variants directly using clinical samples. The application of this method with clinical samples from influenza surveillance had aided in the understanding of the antigenic evolution of IAV in human and swine populations.
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Automation of a solid-phase proximity ligation assay for biodefense applicationsBarkenäs, Emelie January 2013 (has links)
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.
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Validation and optimization of multiplexInSitu PLA for signalling pathway analysisSinha, Tanay Kumar January 2021 (has links)
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.
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Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian CellsNeuhaus, Jochen, Weimann, Annett, Berndt-Paetz, Mandy 17 January 2024 (has links)
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
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Proximity Ligation and Barcoding Assays : Tools for analysis of proteins and protein complexesWu, Di January 2014 (has links)
Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed. In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences. In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA). In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls. In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values < 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in the stimulated vs. non-stimulated condition using SCS. In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.
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Detection and Sequencing of Amplified Single MoleculesKe, Rongqin January 2012 (has links)
Improved analytical methods provide new opportunities for both biological research and medical applications. This thesis describes several novel molecular techniques for nucleic acid and protein analysis based on detection or sequencing of amplified single molecules (ASMs). ASMs were generated from padlock probe assay and proximity ligation assay (PLA) through a series of molecular processes. In Paper I, a simple colorimetric readout strategy for detection of ASMs generated from padlock probe assay was used for highly sensitive detection of RNA virus, showing the potential of using padlock probes in the point-of-care diagnostics. In Paper II, digital quantification of ASMs, which were generated from padlock probe assay and PLA through circle-to-circle amplification (C2CA), was used for rapid and sensitive detection of nucleic acids and proteins, aiming for applications in biodefense. In Paper III, digital quantification of ASMs that were generated from PLA without C2CA was shown to be able to improve the precision and sensitivity of PLA when compared to the conventional real-time PCR readout. In Paper IV, a non-optical approach for detection of ASMs generated from PLA was used for sensitive detection of bacterial spores. ASMs were detected through sensing oligonucleotide-functionalized magnetic nanobeads that were trapped within them. Finally, based on in situ sequencing of ASMs generated via padlock probe assay, a novel method that enabled sequencing of individual mRNA molecules in their natural context was established and presented in Paper V. Highly multiplex detection of mRNA molecules was also achieved based on in situ sequencing. In situ sequencing allows studies of mRNA molecules from different aspects that cannot be accessed by current in situ hybridization techniques, providing possibilities for discovery of new information from the complexity of transcriptome. Therefore, it has a great potential to become a useful tool for gene expression research and disease diagnostics.
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Le récepteur de l’acide rétinoïque alpha (RAR-α) : nouveau rôle dans l’adhésion des fibroblastes / The retinoic acid receptor alpha (RARα) : new role in fibroblasts adhesionAndriamoratsiresy, Dina 08 December 2016 (has links)
Les récepteurs de l’acide rétinoïque, RARα, β et γ sont des facteurs de transcription dépendants du ligand qui contrôlent l’expression de gènes spécifiques. Cependant, il s’avère depuis peu que les RAR ont aussi des effets non-transcriptionnels extranucléaires. Durant ma thèse, j’ai observé que (1) les fibroblastes invalidés pour tous les RAR ont un cytosquelette d’actine perturbé et ont perdu leurs propriétés d’adhésion (2) RARα interagit via son motif riche en proline N-terminal avec la profiline 2a (PFN2a) qui est un régulateur critique de l’élongation des filaments d’actine du cytosquelette. J’ai montré que : (1) Les RAR contrôlent la morphologie, l’adhésion et la migration des MEF via la régulation transcriptionnelle de l’expression de gènes codant pour des protéines d’adhésion (2) Dans le cytoplasme, RARα forme avec PFN2a des complexes dont le nombre contrôle le réseau d’actine et l’adhésion des MEF via un mécanisme non transcriptionnel. Ces observations mettent en exergue l’importance de la combinaison des effets génomiques et non-génomiques des RAR dans l’adhésion des cellules et ouvrent de nouvelles possibilités de dérégulation du fonctionnement des RAR dans certaines pathologies. / Retinoic acid receptors, RARα, β and γ are ligand-dependent transcription factors that control the expression of specific genes. However, growing evidence indicates that RARs also have extranuclear and non transcriptional effects. During my thesis, I observed that (1) fibroblasts invalidated for all RARs depict a disrupted actin cytoskeleton and have lost their adhesion properties (2) RARα interacts through its N-terminal proline rich motif with profilin2a (PFN2a) a critical regulator of actin filaments elongation. I have shown that: (1) RARs control the morphology, adhesion and migration of MEFs via controlling at the transcriptional level the expression of adhesion genes (2) In the cytosol, RARα forms complexes with PFN2a. The number of these complexes controls the actin network and the adhesion of MEFs via a non-transcriptional mechanism. These observations highlight the importance of the combined genomic and non-genomic effects of RARs in cell adhesion, and open new avenues for RARs deregulations in certain pathology.
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Identification of the modulators of and the molecular pathways involved in the BIN1-Tau interaction / Identification des modulateurs et des voies moléculaires impliqués dans l'interaction BIN1-TauMendes, Tiago 13 December 2018 (has links)
Les principales caractéristiques neuropathologiques de la maladie d’Alzheimer (MA) sont les plaques séniles extracellulaires composées de peptide amyloïde β (Aβ) et les enchevêtrements neurofibrillaires intracellulaires composés de Tau hyperphosphorylé. Les mécanismes conduisant à la formation de ces lésions sont encore peu connus et le laboratoire a récemment caractériser le gène “bridging integrator 1” (BIN1), deuxième facteur de risque génétique le plus associé au risque de MA, comme facteur de risque potentiellement associé à la pathologie Tau. Une interaction entre les deux protéines a été décrite in vitro et in vivo suggérant que BIN1 pourrait être impliqué dans le développement de la pathologie associée à Tau dans le cadre de la MA. Cependant, ce rôle de l'interaction BIN1-Tau dans le processus pathophysiologique de la MA n'est pas connu et il reste ainsi à déterminer si cette interaction constitue une cible thérapeutique potentielle. Ce projet a visé alors à mieux comprendre les acteurs de cette interaction en identifiant les modulateurs et les voies moléculaires impliquées dans le contrôle de l'interaction BIN1-Tau, puis de déterminer comment cette interaction est modulée dans le contexte de la MA. Nous avons utilisé pour cela des approches complémentaires de biochimie, de résonance magnétique nucléaire et de microscopie confocale. Comme modèle cellulaire, des cultures primaires de neurones de rat ont été utilisées, et la méthode “proximity ligation assay” (PLA) a été développée comme approche principale pour observer l'interaction BIN1-Tau dans ces cellules. Nous avons déterminé que l'interaction se produit entre les domaines SH3 de BIN1 et le PRD de Tau et nous avons démontré que l’interaction est modulée par la phosphorylation de Tau et BIN1: la phosphorylation de la Thréonine 231 de Tau diminue son interaction avec BIN1, tandis que la phosphorylation de BIN1 à la Thréonine 348 (T348) augmente son interaction avec Tau. Nous avons mis au point une approche de criblage d’haut contenu semi-automatisée et basé sur une bibliothèque de composés commerciaux. Ce criblage s’est basé sur des cultures primaires de neurones comme modèle cellulaire et le PLA pour détecter l'interaction BIN1-Tau. Nous avons identifié plusieurs composés capables de moduler l'interaction BIN1-Tau, notamment U0126, un inhibiteur de MEK-1/2, qui diminue cette interaction, et la cyclosporine A, un inhibiteur de la calcineurine, qui au contraire augmente celle-ci en augmentant la phosphorylation de T348 de BIN1. Par ailleurs les “Cyclin-dependent kinases” (CDK) ont été montré comme contrôlant aussi ce site de phosphorylation. Nous avons donc mis en évidence le couple Calcineurine/CDK comme contrôlant la phosphorylation T348 de Bin1 et donc l’interaction BIN1-Tau. Nous avons également développé un modèle murin de tauopathie dans lequel nous avons surexprimé BIN1 humain. Nous avons observé que la surexpression de BIN1 résorbait les déficits de mémoire à long terme et réduisait la présence d'inclusions intracellulaires de Tau phosphorylée, provoquées par la surexpression de Tau, ce qui était associé à une augmentation de l'interaction BIN1-Tau. En utilisant des échantillons de cerveau humain post-mortem, nous avons observé que les niveaux de l’isoforme BIN1 neuronal étaient diminués dans les cerveaux d’AD, alors que les niveaux relatifs de BIN1 phosphorylé à T348 étaient augmentés, suggérant un mécanisme compensatoire. Cette étude a démontré la complexité et la dynamique de l’interaction BIN1-Tau dans les neurones, a révélé des modulateurs et des voies moléculaires potentiellement impliquées dans cette interaction, et a montré que les variations de l’expression ou de l’activité de BIN1 ont des effets directs sur l’apprentissage et la mémoire, possiblement liés à la régulation de son interaction avec Tau. / The main neuropathological hallmarks of Alzheimer’s disease (AD) are the extracellular senile plaques composed of amyloid-β peptide (Aβ) and the intracellular neurofibrillary tangles composed of hyperphosphorylated Tau. The mechanisms leading to the formation of these lesions is not well understood and our lab has recently characterized the bridging integrator 1 (BIN1) gene, the second most associated genetic risk factor of AD and the first genetic risk factor to have a potential link to Tau pathology. The interaction between BIN1 and Tau proteins has been described in vitro and in vivo, which suggests that BIN1 might help us to understand Tau pathology in the context of AD. However, the role of BIN1-Tau interaction in the pathophysiological process of AD is not known, and whether this interaction is a potential therapeutic target remains to be determined. The aim of this project is to better understand the actors of BIN1-Tau interaction through the identification of the modulators and the molecular pathways involved therein, as well as to understand how BIN1-Tau interaction is modulated in the context of AD. We employed biochemistry, nuclear magnetic resonance, and confocal microscopy. We used rat primary neuronal cultures (PNC) as the cellular model and developed the proximity ligation assay (PLA) as the main readout of the BIN1-Tau interaction in cultured neurons. We determined that the interaction occurs between BIN1’s SH3 domain and Tau’s PRD domain, and demonstrated that it is modulated by Tau and BIN1 phosphorylation: phosphorylation of Tau at Threonine 231 decreases its interaction with BIN1, while phosphorylation of BIN1 at Threonine 348 (T348) increases its interaction with Tau. We developed a novel, semi-automated high content screening (HCS) assay based on a commercial compound library, also using PNC as the cellular model and PLA as the readout of BIN1-Tau interaction. We identified several compounds that are able to modulate the BIN1-Tau interaction, most notably U0126, an inhibitor of MEK-1/2, which reduced the interaction, and Cyclosporin A, an inhibitor of Calcineurin, which increased the interaction through increasing the BIN1 phosphorylation at T348. Furthermore, Cyclin-dependent kinases (CDK) were also shown as regulator of this phosphorylation site. These results suggest that the couple Calcineurin/CDK regulates BIN1 phosphorylation at T348 and consequently the BIN1-Tau interaction. We also developed a mouse model of tauopathy in which we overexpressed human BIN1. We observed that the overexpression of BIN1 rescued the long-term memory deficits and reduced the presence of intracellular inclusions of phosphorylated Tau, caused by Tau overexpression, and this was associated with an increase of BIN1-Tau interaction. Also, using post-mortem human brain samples, we observed that the levels of the neuronal BIN1 isoform were decreased in AD brains, whereas the relative levels of BIN1 phosphorylated at T348 were increased, suggesting a compensatory mechanism. Altogether, this study demonstrated the complexity and the dynamics of BIN1-Tau interaction in neurons, revealed modulators of and molecular pathways potentially involved in this interaction, and showed that variations in BIN1 expression or activity have direct effects on learning and memory, possibly linked to the regulation of its interaction with Tau.
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