Every year, more than a million people are diagnosed with colorectal cancer (CRC) that develops in the large intestine. It is one of the most studied cancers in the world but still more knowledge about how this cancer develops and acts is needed in order to use more effective ways to treat CRC. Autophagy is a vital mechanism in cells that is also suggested to maintain cancer cell survival. In normal cells, it plays an important role by removing damaged cells and organelles as well as eliminating pathogens. Under metabolic stress this mechanism is induced to provide enough nutrients and energy for the cell to survive. Cancer cells are exposed to greater environmental stress than normal cells and therefore, cancer cells exhibit higher levels of autophagy suggesting it to be a crucial mechanism for their survival. Gaining a deeper understanding of this essential mechanism and its activation might provide new insights and improved treatments for the fight against colorectal cancer. In situ Proximity Ligation Assay (PLA) is a protein detection method that enables sensitive and specific detection of proteins and protein-protein interactions (PPIs) in cell lines and tissue samples. The method uses simultaneous recognition of two independent antigens on a protein or protein complex together with a rolling circle amplification (RCA) to form a rolling circle product (RCP) on top of the target. By using fluorescent oligonucleotides, RCP can be visualized and is seen as a bright spot that enables sensitive detection of the target at single-molecule resolution. The aim of this study was to develop assays to detect endogenous molecular events known to be biomarkers of autophagy in situ in order to study autophagy mechanism in CRC patient samples. We focused our research on two PPIs that were known to interact when autophagy is induced. The first investigated interaction was between microtubule-associated protein 1A/1B- light chain 3 (LC3) and sequestome-1 (SQSTM1), an interaction that occurs during autophagy initiation. The second interaction was between B-cell lymphoma 2 (Bcl-2) and Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) that takes place during hypoxia-induced autophagy. To study whether these PPIs can be used as a detection method to monitor autophagy, we used a well- established cell model based on serum starvation and CoCl2 - an hypoxic mimetic- treatment of the intestinal cancer cell line Caco-2 in comparison to normal culture condition. According to isPLA quantification, detection of both PPIs was distinctly higher in treated cells compared to untreated cells giving promising results and suggesting that they can be potentially used as suitable assays to monitor these biomarkers of autophagy. For development of an improved protein detection method that enables the study of several PPIs simultaneously in a tissue sample (In situ Multiplexing), we conjugated directly a short oligonucleotide strand to the primary antibodies. These formed proximity probes could later be used in in situ for multiplexing.

Many of today’s methods for the detection of biomolecules suffer from a high limit ofdetection due to poor signal generation upon recognition of target. By applying andoptimizing proximity ligation assay (PLA) in Western blotting (WB), the limit of detectionhas been lowered down to the picomolar range. In this report I have optimized the differentparameters that affect the signal generation and explored possibilities to increase the ease ofuse, by merging protocol steps and performing signal generating reactions at roomtemperature.

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).

Les mitochondries et le réticulum endoplasmique (RE) interagissent au niveau de points de contacts appelés « Mitochondria-Associated ER Membranes » (MAM), afin d'échanger du Ca2+ via le complexe TP3Rl/Grp75/VDACl et maintenir l'homéostasie énergétique. Bien que des dysfonctions mitochondriales, un stress du RE et des altérations de l'homéostasie du Ca2+ participent au développement de l'insulino-résistance, on ne sait pas si ce sont des facteurs indépendants ou s'ils sont inter-reliés par une altération des MAM. Mes travaux de thèse ont permis de mettre en évidence un nouveau rôle des MAM dans l'insulino-résistance hépatique. J'ai mis au point et validé la technique d'in situ PLA pour visualiser et quantifier les interactions mitochondrie-RE dans les cellules. J'ai montré que l'intégrité des MAM était nécessaire pour la signalisation de l'insuline dans le foie, et qu'un défaut d'intégrité des MAM était impliqué dans l'insulino-résistance hépatique. Des données préliminaires suggèrent qu'une altération des MAM est également associée à l'insulino-résistance musculaire. J'ai ensuite mis en évidence la présence de la protéine kinase B, une protéine clé de la signalisation de l'insuline, dans les MAM, et démontré que sa phosphorylation par l'insuline est altérée dans cette fraction dans le foie de souris diabétique. Enfin, j'ai participé à la mise en évidence l) de la présence de la cyclophilin D à l'interface des MAM régulant les échanges calciques entre les deux organites dans les cardiomyocytes et les hépatocytes, et 2) d'une régulation des MAM par le glucose dans le foie qui permet un contrôle de la dynamique et de la fonction mitochondriale au cours des transitions nutritionnelles. Par conséquent, mes travaux ont permis d'améliorer les connaissances actuelles sur les partenaires, la fonction et la régulation des MAM et de dévoiler les MAM comme une nouvelle cible pour moduler la signalisation de l'insuline et le métabolisme hépatique / Mitochondria-associated endoplasmic reticulum membranes (MAM) are functional domains between both organelles involved in Ca2+ exchange, through the voltage-dependent anion channel (VDAC)-1/glucose regulated protein 75 (Grp75)/inositol 1,4,5-triphosphate receptor (TP3R)-1 complex, and regulating energy metabolism. Whereas mitochondrial dysfunction, ER stress, and altered Ca2+ homeostasis are associated with altered insulin signalling, the implication of MAM dysfunctions in insulin resistance is unknown. During my PhD, my work has underlined a new role of MAM in hepatic insulin- resistance. T have developed a quantitative method called in situ Proximity Ligation Assay to visualise and quantify the interactions between ER and mitochondria. T have shown that MAM integrity is required for insulin signalling and that disruption of MAM is implicated in hepatic insulin resistance. Preliminary data also suggest that MAM alterations are also associated with muscle insulin resistance. T have also identified the presence of the protein kinase B (PKB), a key protein involved in metabolic effects of insulin, at the MAM interface, and demonstrated that its phosphorylation by insulin is altered in this fraction in liver of diabetic mice. Lastly, T have also participated to the identification of: 1) the presence of cyclophilin D (CypD) at MAM interface which regulates calcium transfer from ER to mitochondria in both cardiomyocytes and hepatocytes, and 2) a regulation of MAM by glucose in liver, which is involved in the regulation of mitochondria dynamics and function during nutritional transitions. Consequently, my work improved the knowledge on the composition, function and regulation of MAM, and highlighted MAM as a potential new target for the modulation of hepatic insulin action and metabolism

Le cancer est l'une des principales causes de décès dans le monde. D'après la société américaine contre le cancer; en 2015, un quart des décès aux Etats-Unis est du au cancer du poumon avant même les maladies cardiaques. Cette situation nous incite et bien d'autres scientifiques dans le monde à développer des moyens plus efficaces de traitement, le diagnostic et le dépistage de la maladie. Parce que près de 90% des décès par cancer sont dus à des métastases, de nombreuses études se sont concentrées sur le mécanisme de métastases et sur son impact clinique. Les cellules tumorales circulantes (CTC) sont les cellules s’échappent de tumeurs primaires ou métastatiques pour rejoindre le flux sanguin périphérique, ces cellules sont un élément de transition dans le processus métastatique et portent ainsi des informations cruciales sur ce mécanisme encore mal compris. Les CTCs ont déjà montré leur potentiel comme biomarqueur de pronostic de la progression de la maladie et de l'indicateur de l'efficacité du traitement en fonction l’augmentation ou de la diminution de leur nombre. Leur caractérisation moléculaire peut également donner des informations vis à vis de cibles thérapeutiques possibles et des mécanismes de progression de la maladie ou de la résistance aux médicaments. Leur comptage au cours du traitement combiné avec leur caractérisation moléculaire devrait améliorer la prise en charge des patients dans le cadre de la médecine personnalisée. Cependant CTCs sont extrêmement rares, 1 à 10 cellules / ml de sang parmi les 106 globules blancs et 109 globules rouges, leur capture à partir du sang reste donc un challenge analytique. Dans les dernières décennies, Une grande variété de techniques d'enrichissement et de capture a été mise au point et l'approche microfluidique est l'une des méthodes efficaces, flexibles et à haut débit. Au sein de notre équipe, un dispositif microfluidique (système Ephesia) puissant pour la capture et l'analyse des cellules tumorales circulantes a déjà été mis au point précédemment. Le principe de capture est basé sur l'auto-assemblage de billes magnétiques greffées par des anticorps, grâce aux quelles les cellules sont enrichies via l’interaction Ab- l'antigène de surface EpCAM que l'on trouve communément dans les cellules cancéreuses d'origine épithéliale. Ce système a déjà été validé avec des lignées cellulaires et des échantillons de patients. Cependant, le système n'a pas permis l'isolement / détection des sous-populations de CTCs ou d'effectuer une caractérisation moléculaire très poussée. Par conséquent, mon projet de thèse vise à améliorer encore les capacités du système sur les deux principaux aspects: le ciblage sous-populations de CTC et à l'étude des interactions des protéines à la surface des CTCs dans le Système Ephesia... / Metastasis is the advanced stage of cancer progression and is the cause of 90% of deaths in cancer disease. During metastatic cascade, it is suggested that the successful metastatic initiation depends on the survival of circulating tumor cells (CTCs). CTCs are the cells that shed from the primary or secondary tumor sites into the blood circulation. it is now widely recognized as potential biomarker for companion diagnostics in which high number of CTCs in blood can indicate association with poor survival or high risk of disease progression. Besides, following the number of CTCs during the course of treatment can help to adapt the selected therapy and predict the treatment efficacy. On the other hand molecular characterization can provide patient stratification and identifying the therapeutic targets. However they are extremely rare in the bloodstream, estimated between 1-10 CTC among 6×106 leukocytes, 2×108 platelets and 4×109 erythrocytes per one mL of blood which makes their isolation very challenging. A very attractive way of isolation of CTCs is to integrate microfluidics. Microfluidics offers great advantages such as low volume of reagent consumption and short analysis times with automation as well as isolation and detection analysis can be integrated resulting in highly efficient biomedical devices for diagnostics. As parallel to state of the art, a powerful microfluidic device for circulating tumor cells capture and analysis had already been developed previously in our laboratory. The principle of capture is based on self-assembly of antibody-coated (EpCAM) magnetic beads in which the cells are enriched by EpCAM surface antigen which is found commonly in epithelial origin cancer cells. This system was already validated with cell lines and patients samples. However, the system did not allow isolation/detection of subpopulations of CTCs or performing high content molecular characterization. Therefore, my PhD project aimed at further improving the capabilities of the system on the main two aspects: targeting subpopulations of CTC and studying of protein interactions of CTCs in Ephesia System...

Aptamers are short nucleic acid ligands generated by the process of iterative selection. Nucleic acid counterparts to protein antibodies, aptamers bind their targets with relatively high affinities by assuming characteristic shapes. Highly thermostable, open to manipulations and non-immunogenic, these olignucleotides can be readily adapted to a variety of diagnostic assays and harvested for their therapeutic potential. We have particularly focused on the unique prospects that stem from their localization patterns both in vitro and in vivo. While several assays exist for protein diagnostics, many of these are limited by the amount of target they can detect. To overcome these limitations it might prove effective to couple protein detection with nucleic acid based amplification. The Proximity Ligation Assay (PLA) is an innovative technique that facilitates protein detection on a zeptomolar range by amplifying a tiny signal via the polymerase chain reaction. PLA is based on the concept that two DNA tags when co-localized adjacent to one another on a protein surface and ligated via a connector nucleotide will form a unique amplicon that can detected using real-time PCR and in turn detect the protein. We have adapted PLA to the peptide based detection of Bacillus spores as well as the RNA aptamer based detection of cancer cells. Highly sensitive and specific, nucleic acid based PLA could serve as a promising tool in diagnostics. Aptamers have also been analyzed for their localization patterns in vivo. Using two anti-prostate specific membrane antigen RNA aptamers, we have demonstrated that there is an inherent bias for some circulating oligonucleotides over others based solely on their sequence. This phenomenon has also been explored in cancer models of mice for persistence of specific aptamers over others in tumors for therapy. An in vivo “Stealth” selection scheme has also been designed and executed to hunt for stable and robust aptamer species that are naturally chosen for survival within a mouse system. Generation of such ligands could benefit several therapeutic ventures such as targeted drug delivery past complex vasculature as in the case of the blood:brain barrier. / text

Gastrointestinal (GI) alpha-synuclein (ASN) detection may represent a clinically useful biomarker of Parkinson's disease (PD), but this has been challenged by conflicting results of recent studies employing different immunohistochemical (IHC) methods and reporting diverse morphological patterns with variable biological interpretation. To increase sensitivity and specificity, we applied three different techniques to detect different possible conformations of ASN in GI tissue derived from biopsies of the GI tract, which were obtained from a longitudinally followed, clinically well-characterized cohort of PD subjects and healthy controls (HC) (Oxford Discovery study). With IHC, we used antibodies reactive for total (T-ASN-Abs), phosphorylated (P-ASN-Abs) and oligomeric (O-ASN-Abs) ASN; with the ASN Proximity Ligation Assay (AS-PLA), we targeted oligomeric ASN species specifically; finally, with the Paraffin-Embedded Tissue Blot (PET-Blot) we aimed to detect fibrillary conformations of ASN specifically. Optimisation and validation of the PET-Blot and PLA techniques was carried out with studies on brain tissue from subjects with ASN pathology, and these experiments were used to gain insight into morphology and distribution of different conformational variants of ASN in the brain of subjects with Lewy pathology. We specified all the detected morphological staining patterns with each technique interpreting them as pathologic or non-specific. Correlation to clinical symptoms was assessed to investigate the potential predictive or diagnostic value of specific staining patterns as biomarkers. A total of 163 GI tissue blocks were collected from 51 PD patients (113 blocks) and 21 healthy controls (50 blocks). In 31 PD patients, GI biopsies had been taken before PD diagnosis (Prodromal PD group); while in 20 PD patients biopsies were obtained after PD diagnosis (Manifest PD group). The majority of these tissues blocks were from large intestine (62%), followed by small intestine (21%), stomach (10%) and oesophagus (7%). With IHC, four ASN staining patterns were detected in GI tissue (Neuritic, Ganglionic, Epithelial, and Cellular), while two distinct staining patterns were detected with AS-PLA (cellular and diffuse signal) and with AS-PET-Blot (ASN-localised and peri-crypt signal). The level of agreement between different techniques was generally low, and no single technique or staining pattern was able to reliably distinguish PD patients (Prodromal or Manifest) from HC. Overall, our study suggests that even specific detection of ASN conformational variants currently considered pathologic was not adequate for the prediction of PD. Future studies with these or other novel techniques focusing on the upper part of the GI tract could overcome current limitations in sensitivity and specificity.

<p>Proximity ligation is a recently established technique that can provide answers to questions about the concentration, localization, interactions, modifications and functions of proteins. The method enables sensitive protein measurements with a detection limit in the low femtomolar range in complex biological samples. In proximity ligation, the challenge of detecting specific proteins is converted to the analysis of specific DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins, and to form amplifiable tag sequences upon ligation when brought in proximity. Protocols for the conversion of monoclonal or polyclonal antibodies into proximity probes through the attachment of oligonucleotide sequences are described in the thesis. In addition, the thesis describes the adaptation of the proximity ligation technology for detection of microbial pathogens, analysis of interactions between proteins and nucleic acids, and of inhibition of receptor-ligand interactions. </p><p>Nucleic acid amplification allows specific detection of pathogens with single-copy sensitivity. There are many circumstances, however, when analysis of pathogen surface antigens or the antibody response can provide increased diagnostic value. Proximity ligation reactions were used to measure numbers of virus and bacteria by detection of viral or bacterial surface proteins. Detection sensitivities similar to those of nuclear acid-based detection reactions were achieved directly in infected samples for a parvovirus and for an intracellular bacterium. </p><p>Biological processes are orchestrated by interactions of proteins with molecules in their environment, and investigations of interactions between proteins and other biomolecules are thus of great importance. Protocols were established for very specific and sensitive homogeneous-phase analysis of interactions between proteins and specific nucleic acid sequences. Finally, the proximity ligation mechanism was used to monitor interactions between VEGF-A and two of its receptors, VEGFR-1 and VEGFR-2, and to characterize the effects of agents disrupting this interaction.</p>

<p>Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences.</p><p>Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected. </p><p>A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels.</p><p>A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels. </p><p>I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction. </p><p>Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes</p>

<p>Padlock probes and proximity ligation are two powerful molecular tools for detection of nucleic acids and proteins, respectively. Both methods result in the formation of DNA reporter molecules upon recognition of specific target molecules. These reporter molecules can be designed to include tag sequences that can be analyzed by techniques for nucleic acid analysis. Herein, I present a dual-tag microarray (DTM) platform that is suitable for high-performance analyses of DNA reporter molecule libraries, generated by padlock and proximity probing reactions. The DTM platform was applied for analysis of mRNA transcripts using padlock probes, and of cytokines using proximity ligation. The platform drastically improved specificity of detection, and it allowed precise measurements of proteins and nucleic acids over wide dynamic ranges.</p><p>The thesis also presents two techniques for multi-probe analyses of biomolecules: the triple-specific proximity ligation assay (3PLA) for protein analyses, and the spliceotyping assay for mRNA analyses. 3PLA allows highly specific measurements of as little as hundreds of target protein molecules by interrogating three target epitopes simultaneously. In spliceotyping the exon composition of individual transcripts are represented as a series of tag sequences in DNA reporter molecules, via a series of target-dependent ligation reactions. Next, the splicing patterns along individual transcripts can be revealed by amplified single molecule detection and step-wise decoding.</p>