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.

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.

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.

Submitted by Rosana Amâncio (rosana.amancio@ufcg.edu.br) on 2018-07-19T20:53:36Z No. of bitstreams: 1 LARISSA CAVALCANTE COSTA - DISSERTAÇÃO PPGCNBio 2015..pdf: 1987711 bytes, checksum: 9d66a83ab8d2cec764aeaa785de117d4 (MD5) / Made available in DSpace on 2018-07-19T20:53:36Z (GMT). No. of bitstreams: 1 LARISSA CAVALCANTE COSTA - DISSERTAÇÃO PPGCNBio 2015..pdf: 1987711 bytes, checksum: 9d66a83ab8d2cec764aeaa785de117d4 (MD5) Previous issue date: 2015-07-31 / CNPq / O gênero Begomovirus pertence à família Geminiviridae de vírus que infectam plantas e são transmitidos pelo vetor mosca branca (Bemisia tabaci), também considerado umas das maiores pragas da agricultura. O presente estudo investigou a diversidade de begomovírus presente no vetor mosca branca (B. tabaci) em áreas da Paraíba, Minas Gerais e Distrito Federal. Um total de 17 amostras de moscas brancas foi coletado a partir de 12 espécies vegetaisem áreas da Paraíba, Minas Gerais e Distrito Federal e e usado para detecção do DNA-A de begomovirus por PCR, por RCA e por PCR-RCA, com primers específicos. Amplicons virais foram clonados e os clones recombinantes foram selecionados sob meio de cultura seletivo, contendo X-Gal e IPTG. A presença de amplicons foi confirmada por padrões de restrição gerados pelas enzimas EcoR I e Msp I em DNAs plasmidiais isolados dos clones selecionados e, então, sequenciados. As sequencias de DNA-A de begomovírus isoladas de moscas brancas coletadas em plantas de algodoeiro EMEPA-PB, em Emília, picão preto, barba-de-falcão, jatrofa, berinjela e malvona, presentes na área do Distrito Federal, foram analisadas quanto a porcentagem de identidade de sequencias de nucleotídeos e agrupamentos filogenéticos comparando com outras sequencias de isolados de begomovírus disponíveis no banco de dados GenBank. Como resultado, 5 espécies de begomovírus (SiYBV, CoMoV, SiMMV, BGMV e SiGLSV) evidenciaram a diversidade de begomovírus presente em moscas brancas nas áreas estudadas, mas que não necessariamente está presente nas respectivas plantas onde as moscas foram coletadas. Os primers PAL1v 1978 e PAR1c 496 descritos para detecção de DNA-A de begomovírus a partir de plantas foram eficientes para detecção específica desse componente genômico viral a partir do vetor mosca branca. A detecção específica de DNA-A do componente de begomovírus por PCR a partir do DNA total extraído de moscas brancas foi enriquecida pela amplificação por RCA. O begomovírus BGMV foi predominantemente encontrado em moscas brancas coletadas em plantas na área do Distrito Federal. A diversidade presente nos isolados dos 5 vírus identificados neste trabalho foi agrupada em 4 ramos filogenéticos suportados pela porcentagem de identidade de sequencias de nucleotídeos comparadas com sequencias dos isolados de begomovírus disponíveis no banco de dados GenBank / The Begomovirus genus belongs to Geminiviridae family of viruses that infect plants and is transmitted by whitefly (Bemisia tabaci) vector, which is also considered one of the major pests of agriculture. The present study investigated the diversity of begomovirusesin the whitefly vector (B. tabaci) in areas of Paraíba, Minas Gerais and the Federal District. A total of 17 samples of whiteflies were collected from 12 plant species in areas of Paraíba, Minas Gerais and the Federal District and used for the detection of begomovirus DNA-A by PCR, RCA and RCA-PCR with specific primers. Viral amplicons were cloned and the recombinant clones were selected on selective medium containing X-Gal and IPTG. The presence of amplicons was confirmed by restriction enzyme patterns generated by EcoR I and Msp I on plasmid DNAs of selected clones isolated and then sequenced. The begomovirus DNA-A sequences isolated from whiteflies collected from cotton plants EMEPA-PB, in Emilia, black prick, beard-of-hawk, jatropha, eggplant and malvona present in the Federal District, were analyzed bynucleotide sequence identity percentage and phylogenetic groups compared to other begomovirus strain sequences available in GenBank database. As a result, five species of begomovirus (SiYBV, CoMoV, SiMMV, BGMV e SiGLSV) revealed the diversity of begomovirus present in whiteflies in the studied areas, but that is not necessarily present in the respective plants where the flies were collected. The PAL1v 1978 PAR1c 496 primers described for begomovírus DNA-A detection from plants were also efficient for detection of this specific viral genomic component from whitefly vector. The specific detection of begomovirus DNA-A component by PCR from total DNA extracted from whiteflies was enriched by amplification by RCA. The BGMV begomovirus was found predominantly in whiteflies on plants collected in the Federal District. The diversity present in strains of thefive virus identified in this work was grouped into four phylogenetic branches supported by the nucleotide sequence identity percentage compared to sequences of the begomovirus strains available in GenBank database

The Central Dogma of molecular biology describes a framework for how genetic information is transferred in cells, placing RNA as a messenger between DNA and translated proteins. During the last years, interest in RNA research has grown tremendously due to the increasing understanding and recognition of the importance of RNA in regulation of gene expression, biochemical catalysis, and genome integrity surveillance. Most importantly, RNA content, unlike DNA, changes constantly, fine-tuning the cellular response to match the environmental conditions. There is a clear potential for RNA biomarkers, reflecting both the natural and pathological conditions in vivo. Various methods have been developed to study RNA, of which the most common tools and techniques are described in this thesis. Since many of these gold standard methods are based on detecting RNA derivative (cDNA), there is a wide scope for efficient alternative tools directly targeting RNA. In Paper I, the spatiotemporal expression of human adenovirus-5 mRNA in epithelial and blood cells infected with the virus has been studied. For this, padlock probes and rolling circle amplification (RCA) were used to visualize, quantify and analyse both viral and host cell cDNAs in different infection scenarios, at single cell level. In Paper II, direct RNA detection fidelity has been evaluated using padlock probes. A novel type of probe (iLock) that is activated on RNA via invasive cleavage mechanism, prior to RCA was developed in this approach. Using iLocks, a substantial improvement of direct RNA sensing fidelity has been observed. In Paper III, RNA modifications were introduced in otherwise DNA iLock probes to enhance the probes’ efficiency on miRNAs. Using chimeric iLock probes, multiplexed differentiation of conserved miRNA family members were performed with next- generation sequencing-by-ligation readout. Efficient replication of chimeric probes used in Paper III implies that the Phi29 DNA polymerase readily accepts RNA-containing circles as amplification substrates. In Paper IV, real-time RCA monitoring for measurement of amplification rates and analysis of amplification patterns of various RNA-containing circles was achieved. Moreover, the RCA products were sequenced as a proof for the reverse-transcriptase activity of the Phi29 DNA polymerase. This thesis effectively contributes to a better understanding of mechanisms influencing RNA detection with, but not limited to, padlock probes. It expands the available RNA analyses toolkit with novel strategies and solutions, which can be potentially adapted for RNA-focused research, in general and molecular diagnostics, in particular. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Ziel dieser Arbeit war die Entwicklung neuer Methoden zur Untersuchung zirkulärer RNA. Das erste Projekt dieser Arbeit beschäftigte sich mit der Erstellung eines universell einsetzbaren Protokolls zur Generierung einer funktionalisierten zirkulären RNA. Hierbei konnte zunächst erfolgreich eine Vorschrift zur Herstellung unmodifizierter circRNA etabliert werden. Im zweiten Schritt gelang auch die Generierung einer zirkulären RNA mit Alkin-Funktionalisierung. Geringe Ausbeuten gaben Anlass zur Entwicklung eines alternativen Verfahrens, bei dem die Zyklisierung von Kopf-Schwanz modifizierter RNA durch CuAAC vorgenommen werden sollte. Dabei konnte zunächst eine 5‘-azidmodifizierte RNA durch in vitro Transkription gebildet werden, die anschließend am 3‘-Terminus mit einem 3‘ alkinmodifizierten Baustein mit Aminfunktionalität versehen wurde. Daraufhin konnte erfolgreich eine Zyklisierung mittels CuAAC vorgenommen werden. Ein grundlegendes Problem bei diesen Arbeiten war der Nachweis, dass die gebildete RNA tatsächlich in zirkulärer Form vorlag. Im Rahmen des zweiten Projektes dieser Arbeit wurde ein Assay zur direkten Unterscheidung von zirkulären und linearen Transkripten etabliert. Mittels reverser Transkription konnte ein rolling circle Mechanismus mit dem zirkulären Transkript durchgeführt werden, was in einer multimeren cDNA resultierte. Nach Amplifizierung über qPCR ermöglichteeine Gelanalyse den Nachweis eines spezifischen Bandenmusters für das circRNA-Transkript, wohingegen das lineare Transkript lediglich eine monomere Bande generierte. Anschließend erfolgte die Weiterentwicklung des Assays zu einer spezifischen Nachweismethode für zirkuläre RNA in biologischen Proben.Dabei kann eine abschließende Gelanalyse zur Identifizierung von falsch-positiven Ergebnissen genutzt werden. Die hier etablierte Methode ermöglicht künftig einen schnellen und einfachen Nachweis von circRNA beim Screeningvon biologischen Proben. / Circular RNAs belong to the group of long, non-coding RNAs and have gene regulating functions, comparable to miRNA. However, the field of circRNA research is proceeding slowly due to the lack of efficient analytical methods. That‘s the reason why the development of new analytical methods plays a keyrole within characterisation and identification of circRNAs. This thesis comprises two projects dealing on one hand with the creation of a protocol for the generation of functional circRNA on and the other hand, an assay to differentiate circular and linear RNA. For the generation of circRNA a non-modified circRNA was produced as positiv control by using T4 RNA ligase 2. After the addition of a modification step with T4 RNA ligase 1, it was possible to generate circRNA with alkyne functionalization. Due to limited yields of modified circRNA, the protocol was adapted and a protocol for chemical ligation was established. In this new procedure a RNA with 5‘-azido modification was generated by in vitro transcription, followed by incorporation of a 3‘-alkyne modified building block with additional amine funktionality at the RNA-3‘-end. Consecutively, it was possible to perform a cyclization with the double modified RNA by CuAAC. The second project comprises the establishment of an assay in order to differentiate circular and linear RNA. A rolling circle mechanism was utilized by reverse transcription of a circular RNA transcript, resulting in a multimeric cDNA. Following DNA amplification by qPCR, a specific fragmentation pattern for circRNA was verified by gel electrophoresis. In contrast to this, for linear RNA, a monomeric DNA pattern was seen. Subsequently the assay was advanced to a detection method for circular RNA in biological samples. A final gel electrophoresis allows the identification of false-positive results. In the future, the here developed method can be applied for fast and easy detection of circRNAs in biological samples.

The work described in this dissertation focuses on developing simple yet effective assays integrating nucleic acid (NA) aptamers with rolling circle amplification (RCA) for the detection of non-NA biomarkers. The first project, a comprehensive literature review, highlights the current state of the art in functional NA-based RCA applications, and identifies shortcomings in the detection of non-NA targets with RCA. Biosensor design is critically evaluated from four key perspectives: regulation, efficiency, and detection of RCA, and the integration of all three components for point of care (POC) applications. The second project investigates how target-binding to a linear aptamer can be utilized to regulate RCA in a simple and inexpensive format. Phi29 DNA polymerase (DP) exhibits difficulty processing DNA strands that are bound to non-NA materials such as proteins. The work uses this restriction of phi29 DP as a feature by utilizing protein-binding aptamers as primer strands (aptaprimers) for RCA. The simplicity is showcased by adapting the method to a cellulose paper-based device for the real-time detection and quantification of PDGF or thrombin within minutes. As the second project is a turn-off sensor, the third project exploits the inherent 3’-exonuclease activity of phi29 DP to generate a simple turn-on assay instead. As target-bound aptamers were shown to be resistant to exonuclease activity, the phi29 DP preferentially digests target-free aptaprimers instead of target-bound aptaprimers. The target-bound aptaprimer could be liberated by a circular template (CT) by incorporating toehold-mediated strand displacement (TMSD), and used for RCA. Sensitivity was improved relative to project two, though the dynamic range was narrow owing to difficulty liberating target-bound aptaprimer at high target concentrations. Project four instead used RecJ, which has 5’-exonuclease activity, to modulate aptaprimer availability. Similarly to project three, target-binding conferred protection on the aptaprimer from 5’-exonuclease digestion by RecJ. By including a free 3’ terminus on the aptaprimer, inhibition of RCA due to target binding was avoided and CT-mediated TMSD was not needed, simplifying the assay. As well, this approach was generalizable as it was demonstrated using both a protein (thrombin) and a small molecule (ochratoxin A) target. This turn-on method further improved the assay compared to project three with a 100-fold enhancement in sensitivity and a restoration of the dynamic range. In sum, this work contributed multiple simple and sensitive approaches for the real-time fluorescent detection of proteins and small molecules with the RCA of linear aptamers. / Thesis / Doctor of Science (PhD)

In gene editing, it is important to identify the number of edited and unedited nucleic acids in the development of new therapies and drugs. Countagen is developing a technology for accelerating genomic research and their product is called GeneAbacus. The product is a consumable reagent kit for the quantification of nucleic acids, which can be used by CRISPR gene editing researchers. The DNA which is analyzed with the reagent kit is first extracted in an assay and then targeted with tailored padlock probes. The target region is amplified via RCA and the products collapse into a fluorescent DNA nanoball, which can be analyzed with a fluorescence microscope. Each fluorescent dot in the microscope corresponds to a single recognition event, making the quantification of the edited and unedited nucleic acids possible.  The purpose of this project was to count the number of DNA nanoballs in images from a fluorescence microscope with a focus on deep learning. To do this, the images were first preprocessed to enhance the image quality and then cropped into small patches, before the patches were manually annotated on image-level. The mean value from three annotators was used as the label and the labelled images were used to train a ResNet by using a regression- based approach. PyTorch and the API Fastai were used for training and the applied method was transfer learning. The network was trained in two stages: first, the newly added layers were trained for feature extraction, and then the pre-trained base model was unfrozen and trained for fine-tuning. To find the position of the nanoballs in the images, Class Activation Maps (CAMs) and Gradient-weighted Class Activation Mapping (Grad-CAMs) were created, and the local maxima were calculated to produce statistics.  The best-performing model was a ResNet34 trained with batch size 32 and the loss function Huber loss. The model inference showed that the deep learning model counted the nanoballs in the same interval as the observers in 40 of 50 test images. The created CAMs and Grad-CAMs had too low resolution to find the coordinates of the detected nanoballs.  During this project, the nanoballs were only counted in small patches, but the goal was to find nanoballs in a large image. This project has been limited by time and unfortunately, the step where the number of nanoballs in the different patches were to be summed was not performed. However, the study showed that it is possible to implement and train a deep learning model to count nanoballs in small patches. It also showed that the activation maps had too low resolution to be able to find the positions of the nanoballs by looking for local maxima. The results showed that the number of patches used as training samples did not greatly impact the model’s performance when comparing 300 patches and 450 patches. Manual annotation of nanoballs was a difficult task since the nanoballs are moving when the images are taken, which results in unsharp nanoballs in some patches. Therefore, the manual annotation should probably be performed by experts to get the correct labels for the training. To improve the model and be able to find the positions of the nanoballs further investigation is needed.

I detta examensarbete användes olika bildbehandlingstekniker för detektion och kvantifiering av DNA-mikrobollar, mer specifikt rolling circle amplification-produkter, på mikroskopibilder. Avsikten med detta arbete var att hjälpa Countagen AB utforska pipelines för bildbehandling för sin produkt där de analyserar utfallet av genredigeringsförsök på ett billigare och snabbare sätt än dagens konventionella sekvenseringsmetoder. Två olika metoder för objektdetektion användes i detta arbete. Big-FISH, som bygger på Laplacian of Gaussian och detektion av lokala maxima, samt LodeSTAR, en single-shot, self-supervised djupinlärningsmodell. Förbehandling av bilder var också en central del av detta projekt. DeepSpot, en djupinlärningsmodell för framhävning av punkter, användes för att framhäva mikrobollarna så att de lätt kunde upptäckas, och en top-hat-transform användes för att filtrera bort bakgrunden från bilderna. De olika metoderna utvärderades på ett dataset med manuellt annoterade bilder, en spädningsserie av prover samt prover med samma koncentration. Detta för att få värden på precision, recall och F1-score samt mäta hur robust modellen är när det gäller att detektera punkter. Den modell som presterade bäst var LodeSTAR, med en F1-score på 83% på det annoterade datasetet. / In this thesis project, different image processing techniques were utilized for the detection and quantification of DNA microballs on fluorescence microscopy images. These microballs consisted of rolling circle amplification products, of regions of interest. This was done to aid Countagen AB in exploring image processing pipelines for their product where they analyze gene editing efficiency in a cheaper and faster manner than today's conventional sequencing methods. Two different object detection methods: Big-FISH, which builds on Laplacian of Gaussian and local maxima detection, and LodeSTAR, a single-shot, self-supervised deep learning model, were evaluated for this task of detection and quantification. Image preprocessing was a central part of this project. DeepSpot, a deep learning model for spot enhancement was used to highlight the microballs, and a white top-hat transform was applied to the images for background subtraction. The different methods were evaluated on a test set of manually annotated images, a dilution series of samples, and samples with the same concentration to obtain precision, recall, and F1 scores, as well as gauge the robustness of the model in detecting spots. The best-performing model was LodeSTAR, with an F1-score of 83% on the test set.

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.