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1

Larsson, Ludvig. "Optimization of UMI counting strategies for Spatial Transcriptomics." Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-233838.

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Анотація:
Spatial Transcriptomics (ST) is a microarray-based RNA sequencing technology that allows for genome-wide transcriptome profiling of tissue sections with spatialresolution, which was published in Science by Ståhl and Salmén et al in 2016. Polyadenylated transcripts are captured on a microarray surface through hybridizationwith a barcoded DNA oligo that carries the information necessary to infer spatialposition and transcript uniqueness from the output sequencing reads. The ST protocolutilizes Unique Molecular Identifiers (UMIs) to remove PCR duplicates and obtain reliable estimates of gene counts. However, errors gradually accumulate in the UMIpool as a consequence of enzymatic conversion steps and these errors ought to be addressed computationally in data processing steps to produce reliable gene countestimates.In this thesis, we used ERCC reference RNAs and a set of custom-made DNA oligosas spike-ins in the ST protocol to explore sources of technical variation under controlled experimental conditions. An exploratory analysis of the spike-in data gave new insights into the chemistry which could guide future improvements of the protocol. We also developed a new strategy to bin read alignments before gene quantification and showed that this strategy produces a more reliable output. Finally, we developed an in silicosimulation of the ST library preparation to provide a framework that can be used toevaluate the performance of various computational processing strategies. The simulation was then used to benchmark a set of duplicate removal algorithms used toquantify gene expression.This master thesis project was carried out at SciLifeLab at the division of GeneTechnology under supervision of José Fernandez Navarro.
Spatial Transcriptomics (ST) är en teknologi som utvecklades av Ståhl och Salmén etal (2016) och som används för att analysera RNA från vävnadssnitt. Metoden användersig av ett mikrochip som kan fånga upp polyadenylerade molekyler från vävnaden med hjälp av oligo(dT)-prober som är riggade på ytan. Varje yt-prob innehåller en positionsspecifiksekvens som kan användas för att bestämma från vilken position på ytan enRNA-molekyl fångats och ger därmed en möjlighet att analysera transkriptomet överhela vävnadssnittet. Genom att kombinera denna teknologi med högupplöst ljusfältsmikroskopiär det möjligt att skapa en tvådimensionell representation av genuttrycksom direkt kan kopplas till vävnadens morfologi. På varje DNA-oligo finns förutompositions-specifika sekvenser dessutom en kortare sekvens, en så kallad UMI somanvänds för att avlägsna PCR-duplikat. Dessa sekvenser kan signifikant förbättra estimaten av genuttryck, men är känsliga för mutationer och fel som uppstår under de flertalet enzymatiska reaktioner som utnyttjas i ST-protokollet. Fel som uppstår i UMIsekvensen hanteras med data-baserade algoritmer och kräver en noggrann strategi för att generera en precis biologisk representation.I detta projekt användes ett sett av standardiserade RNA-molekyler (ERCC) samtskräddarsydda DNA-oligos som ett substitut för biologiskt material för att utvärderakällor till teknisk variation som har en direkt inverkan på estimeringen av genuttryck.Vi utvecklade även en ny strategi för att gruppera RNA-sekvenser och visar hur denna strategi producerar mer pålitliga resultat. Slutligen presenterar vi en in silico-simuleringav hela ST-metoden som kan användas som ett ramverk för att testa nya algoritmer för att kvantifiera genuttryck. Med detta ramverk utförde vi en riktmärkning av olikaalgoritmer som används för att eliminera PCR-duplikat och selekterade därefter en robust algoritm baserat på resultaten från simuleringen.Detta projekt utfördes på SciLifeLab på avdelningen för genteknologi underhandledning av José Fernandez Navarro.
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2

Van, Leen Eric. "On the morphogenesis of the D. melanogaster pupa : a study on gene patterning and tissue folding." Electronic Thesis or Diss., Sorbonne université, 2020. http://www.theses.fr/2020SORUS387.

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Анотація:
Au cours du développement, la coordination des comportements cellulaires est essentielle à la formation d’organes complexes et fonctionnels. L’analyse de ces processus cellulaire est essentielle pour comprendre comment les tissues se forment au cours du développement. Pour ce faire, il est tout d’abord primordial d’identifier les gènes dont l’expression est corrélée avec chacun de ces processus cellulaires. Avec pour modèle la formation de l’épithélium dorsal (le notum) de la pupe de drosophile, mon travail de thèse a visé à identifier les mécanismes moléculaires qui gouvernent la régulation spatiale de la morphogenèse de l’échelle cellulaire à l’échelle de l’organisme. Dans un premier temps, j’ai mis en œuvre une analyse de transcriptomique spatiale qui m’a permis d’identifier de nouveaux gènes impliqués dans la morphogenèse du notum. Dans un second temps, j’ai développé un microscope confocal rotatif avec l’aide de la plateforme d’imagerie de l’Institut Curie. En appliquant cette nouvelle méthode au cours du développement de la pupe jusqu’au stade adulte, j’ai pu observer la morphogenèse de l’aile et du notum de manière simultanée. J’ai ainsi identifié un nouveau mouvement morphogénétique du notum entre 45-50 hAPF qui semble indépendant de la morphogenèse de l’aile dans le temps et dans l’espace. Enfin j’ai montré que ce mouvement est contrôlée par l’expression de sérine-protéases qui libèrent l’attachement de l’épithélium à la cuticule. Ce travail de thèse représente un apport important à une compréhension fine et intégrée de la régulation de la morphogenèse et de la coordination des dynamiques cellulaires au cours du développement
In order to achieve complex shapes during development, multicellular organisms need to coordinate cellular behaviors to form complex and functional organs. Identifying genes that are expressed in patterns that correlate with cellular processes is therefore primordial. Using the dorsal epithelium (the notum) of drosophila pupa as a model, my thesis aimed at uncovering the molecular mechanisms which control the spatial regulation of morphogenesis at the cell and tissue scale. First, I developed spatial transcriptomics which enabled the identification of new expression patterns involved in notum morphogenesis. Second, I developed, in collaboration with the imaging platform of Institut Curie, Rotating Sample Confocal Microscopy. Using this technique, I was able to simultaneously observe the morphogenesis of the notum, hinge and wing blade. This enabled the discovery of a new morphogenetic movement in the notum between 45-50hAPF. My results suggest that this extensive folding and elongation of the notum is independent of folding in the wing. Furthermore, I demonstrated that the expression of serine proteases regulate the attachment of the tissue to the cuticle which triggers the onset of the folding and determines the final shape of the tissue. Overall, this work increases our understanding of the spatial regulation of morphogenesis and contributes to the knowledge on how the extracellular matrix can regulate tissue shape
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3

Peiffer, Jason, Shail Kaushik, Hajime Sakai, Mario Arteaga-Vazquez, Nidia Sanchez-Leon, Hassan Ghazal, Jean Vielle-Calzada, and Blake Meyers. "A spatial dissection of the Arabidopsis floral transcriptome by MPSS." BioMed Central, 2008. http://hdl.handle.net/10150/610079.

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BACKGROUND:We have further characterized floral organ-localized gene expression in the inflorescence of Arabidopsis thaliana by comparison of massively parallel signature sequencing (MPSS) data. Six libraries of RNA sequence tags from immature inflorescence tissues were constructed and matched to their respective loci in the annotated Arabidopsis genome. These signature libraries survey the floral transcriptome of wild-type tissue as well as the floral homeotic mutants, apetala1, apetala3, agamous, a superman/apetala1 double mutant, and differentiated ovules dissected from the gynoecia of wild-type inflorescences. Comparing and contrasting these MPSS floral expression libraries enabled demarcation of transcripts enriched in the petals, stamens, stigma-style, gynoecia, and those with predicted enrichment within the sepal/sepal-petals, petal-stamens, or gynoecia-stamens.RESULTS:By comparison of expression libraries, a total of 572 genes were found to have organ-enriched expression within the inflorescence. The bulk of characterized organ-enriched transcript diversity was noted in the gynoecia and stamens, whereas fewer genes demonstrated sepal or petal-localized expression. Validation of the computational analyses was performed by comparison with previously published expression data, in situ hybridizations, promoter-reporter fusions, and reverse transcription PCR. A number of well-characterized genes were accurately delineated within our system of transcript filtration. Moreover, empirical validations confirm MPSS predictions for several genes with previously uncharacterized expression patterns.CONCLUSION:This extensive MPSS analysis confirms and supplements prior microarray floral expression studies and illustrates the utility of sequence survey-based expression analysis in functional genomics. Spatial floral expression data accrued by MPSS and similar methods will be advantageous in the elucidation of more comprehensive genetic regulatory networks governing floral development.
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4

Jestin, Martin. "Modifications du microenvironnement stromal après irradiation localisée du côlon : identification de voies moléculaires pour optimiser le processus de régénération épithéliale." Electronic Thesis or Diss., Sorbonne université, 2024. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2024SORUS165.pdf.

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Анотація:
Les cancers pelviens ont une prévalence élevée et sont principalement traités par radiothérapie. Si elle permet un contrôle de la tumeur, la radiothérapie peut également provoquer des lésions aux tissus sains environnants, entrainant des complications invalidantes définies comme une maladie à part entière, la «Pelvic Radiation Disease» ou PRD. Aujourd'hui, il n'existe pas de traitement curatif pour cette pathologie fibrosante. Ce projet vise à étudier le microenvironnement du côlon après irradiation afin d'identifier, à terme, des cibles thérapeutiques pour la prise en charge des séquelles coliques de la PRD. Pour ce projet, un modèle de souris développant des lésions coliques fibrosantes similaires à celles observées chez les patients atteints de PRD a été mis au point. Il consiste en une irradiation colorectale localisée en dose unique de 26Gy. Nous avons défini 2 temps d'étude post-irradiation : à 2 semaines afin d'étudier les effets aigus de l'irradiation ainsi que le processus de régénération et 12 semaines pour étudier la fibrose. Des études histologiques ont permis de caractériser les lésions muqueuses avec un ulcère profond à 2 semaines, et des remaniements fibreux à 12 semaines. Aux 2 temps étudiés, un processus de prolifération accru et désordonné a été observé ainsi qu'un déficit en protéines de jonctions épithéliales suggérant un défaut de la fonction de barrière. Nous avons démontré l'impact du microenvironnement colique irradié sur les processus de prolifération et de différenciation épithélial par un système de coculture avec des organoïdes coliques suivi par vidéo-microscopie. Nos résultats ont permis de valider les observations in vivo, à savoir une prolifération accrue des organoïdes en présence de stroma issus de souris 12 semaines post-irradiation. Afin de caractériser les cellules mésenchymateuses du stroma après irradiation, des expériences de séquençage d'ARN sur cellule unique (à partir de cellules coliques triées EpCAM-CD45- et issues de colon total) et de transcriptomique spatiale ont été réalisées. Elles ont mis en évidence un nouveau marqueur, Edil3, spécifique de la population stromale majeure du côlon. Ce nouveau marqueur nous a permis de mieux caractériser cette population cellulaire en termes de fonction et de localisation au niveau du côlon sain. Nous avons proposé de la nommer Mésitocytes. Au temps précoce, nous avons établi que cette population pouvait se différencier vers un profil pro-inflammatoire appelé « IAF » pour «Inflammation-Associated Fibroblasts». Nous avons également observé une augmentation de l'expression de transcrits impliqués dans des fonctions cruciales telles que l'homéostasie épithéliale, l'angiogenèse et l'inflammation par la majorité des cellules mésenchymateuses. Les résultats montrent l'importance des signaux moléculaires prolifératifs issus des cellules endothéliales lymphatiques et des cellules musculaires lisses, notamment Grem-1. L'analyse de la phase chronique après irradiation, confirme l'augmentation des signaux prolifératifs par les cellules du stroma. De plus, un nouveau type de cellules fibroblastique associé à la fibrose a été observé, caractérisé par profil transcriptionnel différent des IAF observés en phase précoce. L'étude de l'impact de l'irradiation sur le compartiment épithélial a mis en évidence des modifications importantes au niveau de la population de colonocytes et l'émergence de cellules épithéliales avec un phénotype « revival », déjà décrit dans la littérature. De façon intéressante, ces populations ont des localisations spécifiques au niveau des cryptes en régénération. Nous avons également établi l'importance de gènes tels que Lypd8 et Anxa1 dans la progression des cellules épithéliales proliférantes vers un phénotype «revival». Des observations intéressantes issues des analyses de transcriptomique spatiale permettent également d'émettre des hypothèses quant au rôle des cellules immunitaires dans le processus de régénération épithélial
Pelvic cancers are highly prevalent and are mainly treated with radiotherapy. While radiation therapy may control the tumor, it can also cause damage to surrounding healthy tissue, leading to disabling complications defined as a disease “pelvic radiation disease” (PRD). Currently, there is no curative treatment for this fibrosing pathology. The aims of this project are to study the colonic microenvironment after irradiation with a view to identify new therapeutic targets to improve the management of the colonic sequelae of PRD. For this project, a mouse model developing fibrosing colonic lesions similar to those observed in PRD patients was developed. It consists of localized colorectal irradiation with a single dose of 26Gy. We defined 2 post-irradiation study periods: 2 weeks to study the acute effects of irradiation and the regeneration process, and 12 weeks to study fibrosis. Histological studies characterized the mucosal lesions, with a deep ulcer at 2 weeks and fibrous remodeling at 12 weeks. At the 2 time points studied, an increased and disorganized proliferative process was observed, as well as a deficit in epithelial junction proteins, suggesting a defect in barrier function. We demonstrated the impact of the irradiated colonic microenvironment on epithelial proliferation and differentiation processes using a co-culture system with colonic organoids monitored by video microscopy. Our results validated in vivo observations of increased organoid proliferation in the presence of stroma derived from mice 12 weeks post-irradiation.To characterize stromal mesenchymal cells after irradiation, single-cell RNA sequencing experiments (using EpCAM-CD45-sorted colonic cells and from whole colon) and spatial transcriptomics were performed. They revealed a new marker, Edil3, specific for the major stromal population of the colon. This new marker allowed us to better characterize this cell population in terms of function and localization in the healthy colon. We proposed to call them mesitocytes. In the early stages, we found that this population could differentiate towards a pro-inflammatory profile called "IAF" for "Inflammation-Associated Fibroblasts". We also observed increased expression of transcripts involved in critical functions such as epithelial homeostasis, angiogenesis and inflammation by the majority of mesenchymal cells. The results demonstrate the importance of proliferative molecular signals from lymphatic endothelial cells and smooth muscle cells, particularly Grem-1. Analysis of the chronic phase after irradiation confirms the increase in proliferative signals from stromal cells. In addition, a new fibroblast cell type associated with fibrosis was observed, characterized by a transcriptional profile different from that of the IAF observed in the early phase. The study of the effects of irradiation on the epithelial compartment revealed significant changes in the colonocyte population and the appearance of epithelial cells with a "revival" phenotype, already described in the literature. Interestingly, these populations have specific localizations in regenerating crypts. We also established the importance of genes such as Lypd8 and Anxa1 in the progression of proliferating epithelial cells towards a "revival" phenotype. Interesting observations from spatial transcriptomic analyses also allow us to hypothesize the role of immune cells in the epithelial regeneration process
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5

Currás, alonso Sandra. "Lung responses to radiation injury at the single cell level." Electronic Thesis or Diss., Université Paris sciences et lettres, 2021. http://www.theses.fr/2021UPSLS060.

Повний текст джерела
Анотація:
La radiothérapie constitue une option thérapeutique majeure pour le traitement du cancer du poumon. Néanmoins, la radiothérapie induit chez environ 5 à 20 % des patients traités des toxicités pulmonaires irréversibles précoces et tardives, telles que la pneumonite aiguë ou la fibrose pulmonaire induite par la radiothérapie (FPRI). La FPRI est caractérisée par une destruction progressive et irréversible de l'architecture alvéolaire avec perturbation des échanges gazeux conduisant à la mort des patients. Bien que l'ordre des événements moléculaires et cellulaires dans la progression vers la FPRI soit un aspect pathogénique clé de la maladie, leur coordination dans l'espace et le temps reste largement inexplorée. L'objectif principal de ce projet est d'étudier la dynamique dans le temps et l'espace des mécanismes cellulaires et moléculaires qui conduisent à la fibrose pulmonaire après l’irradiation. La combinaison d'analyses single cell RNA sequencing (scRNAseq), pour étudier les réponses précoces et tardives à l’irradiation au niveau d'une seule cellule, et de la single molecule (sm) FISH, pour cartographier les types de cellules spécifiques dans le tissu, a fourni des informations sur la façon dont le tissu pulmonaire se réorganise et évolue progressivement vers un état de fibrose. Les résultats de ce projet mettent en lumière les processus moléculaires induits par l’irradiation, tels que la régénération pulmonaire, la transdifférenciation des cellules épithéliales alvéolaires, l’EMT, l’inflammation et la sénescence dans les compartiments pulmonaires impliqués dans la régénération du tissue, la cicatrisation et la fibrose. Comprendre quels sont les mécanismes à l'origine de la FPRI permettra de trouver de nouveaux traitements thérapeutiques pour améliorer les soins et la qualité de vie des patients
A major therapeutic option for lung cancer treatment is radiotherapy. Nevertheless, around 5-20% of the patients treated with radiation therapy suffer from early and late irreversible lung toxicities, such as acute pneumonitis or radiotherapy induced pulmonary fibrosis (RIPF). RIPF is characterized by progressive and irreversible destruction of the alveolar architecture with disruption of gas exchange and terminal failure. Although the order of molecular and cellular events in the progression towards RIPF is a key pathogenic aspect of the disease, their coordination in space and time remains largely unexplored. The overarching aim of this project is to study the dynamics in time and space of the cellular and molecular mechanisms that lead to lung fibrosis after ionizing radiation (IR). The combination of single cell RNA sequencing (scRNAseq) analyses, to study early and late responses to injury at the single cell level, and single molecule (sm) FISH, to map specific cell types in tissue, have provided information on how mouse and human lung tissues responds to radiation injury. The results of this project highlight the dynamics on specific radiation-induced processes, such as regeneration, transdifferentiation, EMT, inflammation and senescence in the main compartments of the lung that are known to play a major role in tissue repair, regeneration and fibrosis. Importantly, this study points at a senescence process affecting specifically the endothelial cell compartment over the course of fibrosis after fibrogenic doses of IR. Understanding what are the mechanisms causing this disease will pave the way to new therapeutic options that may improve patients’ treatments and their quality of life
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6

Lötstedt, Britta. "Towards spatial host-microbiome profiling." Licentiate thesis, KTH, Genteknologi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-289384.

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Анотація:
Sequencing technologies and applications have pushed the limits and enabled novel studies of biological mechanisms, evolutionary relationships and communication networks between cells. The technical developments leading to single cell RNA-sequencing have enabled detection of rare cell populations while spatial resolution added insights into larger biological environments, like tissues and organs. Massively parallel sequencing has paved the way for integrated high-throughput analyses including that of studying gene expression, protein expression and mapping of microbial communities. This thesis starts with an introduction describing the technical and biological advancements made in recent years with focus on spatially resolved approaches. Then, a summary of recent accomplishments is presented, which enabled ongoing work in a novel field of spatial hostmicrobiome profiling. Lastly, the concluding remarks include both a future perspective and a short reflection on the current developments in the spatial multi-omics field. 16S sequencing is often used for taxonomic classification of bacteria. In Paper I, this sequencing technique was used to study the aerodigestive microbiome in pediatric lung transplant recipients. Many of these patients regretfully reject the organ after transplant, but the underlying cause is, in many cases, unknown. In this paper, multiple factors influencing rejection were examined including that of the aerodigestive microbiome. Pediatric lung transplant recipients often suffer from gastrointestinal dysmotility and the focus of this study was also to analyze changes in the microbiome in relation to irregular gastric muscle movements. The results showed that lung transplant recipients had, in general, lower microbial diversity in the gastric fluid and throat and also that the microbial overlap between lung and gastric sampling sites was significantly less in transplant recipients compared to controls. In addition, gastrointestinal dysmotility was shown to influence the gastric microbiome in lung transplant recipients, but, given the small sample size available in this study, the correlation to patient outcome could not be examined. Integrated analysis of the transcriptome and the antibody-based proteome in the same tissue section was enabled using the method developed in Paper II. Spatial Multi- Omics (SM-Omics) uses a barcoded glass array to capture mRNA and antibody-based expression of selected proteins in the same section. The antibody-based profiling of the tissue section was enabled by either immunofluorescence or DNA-barcoded antibodies that were then decoded by sequencing. The protocol was scaled-up using an automated liquidhandling system. Using this method, simultaneous profiling of the transcriptome and multiplexed protein values was determined in both the mouse brain cortex and mouse spleen. Results showed a high correlation in spatial pattern between gene expression and antibody measurements, independently of the antibody labelling technique. SM-Omics generates a high-plex multi-omics characterization of the tissue in a high throughput manner while exhibiting low technical variation.
Tekniker och applikationer som använder sekvensering har flyttat fram gränsernaoch tillåtit nya undersökningar av biologiska mekanismer, evolutionära släktskap ochkommunikationsnätverk mellan celler. De tekniska utvecklingarna som har lett fram tillRNA-sekvensering av enskilda celler har möjliggjort upptäckten av sällsynta cellpopulationer medan den rumsliga upplösningen har inneburit en ökad förståelse av störrebiologiska miljöer, såsom vävnader och organ. Massively parallel sequencing har banat vägför integrerade analyser med hög kapacitet, vilket inkluderar analys av genuttryck,proteinuttryck och kartläggning av bakteriella samhällen. Den här avhandlingen börjar meden introduktion som beskriver tekniska och biologiska framsteg som gjorts de senaste åren,med fokus på den rumsliga upplösningen. Sedan följer en summering av de senasteprestationerna som har möjliggjort det pågående arbetet i ett nytt fält som avhandlarrumslig profilering av bakterien och dess värd. Slutligen innehåller slutordet både ettframtida perspektiv samt en kort reflektion av den nuvarande utvecklingen inom fälten förrumslig mång-omik. 16S-sekvensering används ofta för att taxonomiskt klassificera bakterier. Dennasekvenseringsteknik användes i artikel I för att studera mikrobiomet i luft- ochmatspjälkningskanalen hos barn med transplanterad lunga. Dessvärre är det vanligt medavstötning av lungan efter transplantationen hos många av dessa patienter, men denunderliggande orsaken till avstötningen är, i många fall, okänd. I denna studie undersöktesflertalet faktorer, inklusive mikrobiomet i luft- och matspjälkningskanalen, som kan tänkaspåverka bortstötningen. Barn med transplanterad lunga lider ofta av störningar i magtarmkanalens rörelser och artikelns fokus var därmed även att analysera förändringar imikrobiomet i relation till dessa avvikande rörelser i mag-tarmkanalen. Resultatet visade attpatienter med transplanterad lunga generellt hade lägre bakteriell mångfald i magsaft ochhals, samt att det bakteriella överlappet mellan lunga och magsaft var signifikant mindre ipatienter med transplanterad lunga jämfört med kontrollerna. För övrigt visade det sig attstörningar i mag-tarmkanalens rörelser påverkade magsaftens mikrobiom hos patientermed transplanterad lunga, men på grund av studiens storlek på urvalet, kunde det inteundersökas hur detta korrelerade till utfallet hos patienterna. Integrerad analys av transkriptomet och antikroppsbaserad analys av proteomet isamma vävnadssnitt har möjliggjorts genom metoden som utvecklats i artikel II. SpatialMulti-Omics (SM-Omics) använder ett avkodningsbart mönster av korta DNA-segment påen glasyta för att fånga mRNA och antikroppsbaserat uttryck av utvalda proteiner frånsamma vävnadssnitt. Den antikroppsbaserade profileringen av vävnadssnittet uppnåddesgenom antingen immunofluorescens eller antikroppar märkta med DNA-segment somkunde avkodas genom sekvensering. Protokollet skalades upp genom ett automatiseratsystem för att behandla vätskor. Genom användning av denna metod kunde simultanprofilering av transkriptomet och flertalet proteiner uppnås i både hjärnbarken och mjältenhos en mus. Resultaten visade en hög korrelation i det rumsliga mönstret mellangenuttrycket och de antikroppsbaserade mätningarna, oberoende av hur antikropparnahade märkts. SM-Omics genererar en storskalig karaktärisering av vävnaden av flera omikermed hög kapacitet samtidigt som den har låg teknisk variation.

QC 2021-02-02

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7

Kaewsapsak, Pornchai. "Spatially-resolved transcriptomic mapping in live cells using peroxidase-mediated proximity biotinylation." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/113972.

Повний текст джерела
Анотація:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The spatial organization of RNA within cells is crucial for the regulation of a wide range of biological functions, spanning all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we developed two methods, termed APEX-RIP and APEX-Seq. APEX-RIP combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking, while APEX-Seq utilizes peroxidase-catalyzed in situ biotinylation on RNA. We demonstrated that APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER) membrane, with higher specificity and coverage than conventional approaches. We furthermore identified candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Similarly, APEX-Seq can isolate RNAs from mitochondrial matrix, ER-associated RNAs, OMM-associated RNAs, and potentially other non-membrane bound compartments. We also revealed many non-coding RNA candidates at these sites. Since APEX-RIP and APEX-Seq are simple, versatile, and do not require special instrumentation, we envision their broad applications in a variety of biological contexts.
by Pornchai Kaewsapsak.
Ph. D.
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8

Mignardi, Marco. "In situ Sequencing : Methods for spatially-resolved transcriptome analysis." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-110057.

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Анотація:
It is well known that cells in tissues display a large heterogeneity in gene expression due to differences in cell lineage origin and variation in the local environment at different sites in the tissue, a heterogeneity that is difficult to study by analyzing bulk RNA extracts from tissue. Recently, genome-wide transcriptome analysis technologies have enabled the analysis of this variation with single-cell resolution. In order to link the heterogeneity observed at molecular level with the morphological context of tissues, new methods are needed which achieve an additional level of information, such as spatial resolution. In this thesis I describe the development and application of padlock probes and rolling circle amplification (RCA) as molecular tools for spatially-resolved transcriptome analysis. Padlock probes allow in situ detection of individual mRNA molecules with single nucleotide resolution, visualizing the molecular information directly in the cell and tissue context. Detection of clinically relevant point mutations in tumor samples is achieved by using padlock probes in situ, allowing visualization of intra-tumor heterogeneity. To resolve more complex gene expression patterns, we developed in situ sequencing of RCA products combining padlock probes and next-generation sequencing methods. We demonstrated the use of this new method by, for the first time, sequencing short stretches of transcript molecules directly in cells and tissue. By using in situ sequencing as read-out for multiplexed padlock probe assays, we measured the expression of tens of genes in hundreds of thousands of cells, including point mutations, fusions transcripts and gene expression level. These molecular tools can complement genome-wide transcriptome analyses adding spatial resolution to the molecular information. This level of resolution is important for the understanding of many biological processes and potentially relevant for the clinical management of cancer patients.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

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9

Vickovic, Sanja. "Transcriptome-wide analysis in cells and tissues." Doctoral thesis, KTH, Genteknologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-199447.

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Анотація:
High-throughput sequencing has greatly influenced the amount of data produced and biological questions asked and answered. Sequencing approaches have also enabled rapid development of related technological fields such as single-cell and spatially resolved expression profiling. The introductory parts of this thesis give an overview of the basic molecular and technological apparatus needed to analyse the transcriptome in cells and tissues. This is succeeded by a summary of present investigations that report recent advancements in RNA profiling. RNA integrity needs to be preserved for accurate gene expression analysis. A method providing a low-cost alternative for RNA preservation was reported. Namely, a low concentration of buffered formaldehyde was used for fixation of human cell lines and peripheral blood cells (Paper I). The results from bulk RNA sequencing confirmed gene expression was not negatively impacted with the preservation procedure (r2>0.88) and that long-term storage of such samples was possible (r2=0.95). However, it is important to note that a small population of cells overexpressing a limited amount of genes can skew bulk gene expression analyses making them sufficient only in carefully designed studies. Therefore, gene expression should be investigated at the single cell resolution when possible. A method for high-throughput single cell expression profiling termed microarrayed single-cell sequencing was developed (Paper II). The method incorporated fluorescence-activated cell sorting, sample deposition and profiling of thousands of barcoded single cells in one reaction. After sample attachment to a barcoded array, a high-resolution image was taken which linked the position of each array barcode sequence to each individual deposited cell. The cDNA synthesis efficiency was estimated at 17.3% while detecting 27,427 transcripts per cell on average. Additionally, spatially resolved analysis is important in cell differentiation, organ development and pathological changes. Current methods are limited in terms of throughput, cost and time. For that reason, the spatial transcriptomics method was developed (Paper III). Here, the barcoded microarray was used to obtain spatially resolved expression profiles from tissue sections using the same imaging principle. The mouse olfactory bulb was profiled on a whole-transcriptome scale and the results showed that the expression correlated well (r2=0.94-0.97) as compared to bulk RNA sequencing. The method was 6.9% efficient, reported signal diffusion at ~2 μm and accurately deconvoluted layer-specific transcripts in an unbiased manner. Lastly, the spatial transcriptomics concept was applied to profile human breast tumours in three dimensions (Paper IV). Unbiased clustering revealed previously un-annotated regions and classified them as parts of the immune system, providing a detailed view into complex interactions and crosstalk in the whole tissue volume. Spatial tumour classification divulged that certain parts of the tumour clearly classified as other subtypes as compared to bulk analysis providing useful data for current practice diagnostics. The last part of the thesis discusses a look towards the future, how the presented methods could be used, improved upon or combined in translational research.

QC 20170109

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10

Zhang, Yang. "A visualization interface for spatial pathway regulation data." Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-237741.

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Анотація:
Data visualization is an essential methodology for bioinformatics studies. Spatial Transcriptomics(ST) is a method that aims at measuring the transcriptome of tissue sections while maintaining its spacial information. Finally, the study of biological pathway focuses on a series of biochemical reactions that take place in organisms. As these studies generate a large number of datasets, this thesis attempts to combine the ST’s data with pathwayinformation and visualize it in an intuitive way to assist user comprehension and insight.In this thesis, Python was used for integrating the dataset and JavaScript libraries wereused for building the visualization. The processing of ST pathway data together with the data visualization interface are the outcomes of this thesis. The data visualization can show the regulation of pathways in the ST data and can be accessed by modern browsers. These outcomes can help users navigate the ST and pathway datasets more effectively.
Datavisualisering är en viktig del av bioinformatik. Spatial transkriptomik (ST) är en metod som mäter transkriptom, samtidigt som den behåller spatial information. Biologiskapathways å andrasidan fokuserar på biokemiska reaktioner som sker inom organismer. Dessa studier genererar mycket data, och denna avhandling försöker att kombinera ST-data med pathway information och få en intuitiv visualisering av det integrerade datat.I avhandlingen användes Python för att integrera datat och JavaScript bibliotek för attbygga visualiseringsverktyget. Avhandlingen resulterade i en metod för att integrera STdata och pathway information, samt ett visualiseringsverktyg för ovan nämnda information.Verktyget kan visa pathway regulationer i ST data och kan användas i moderna webbläsare.Forskningen resulterade i ett verktyg som kan hjälpa forskare att förstå ST och pathwaydata.
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11

Sugiyama, Michael. "Sex differences in the intestinal transcriptome and spatial patterns of lipid uptake capacity and intestinal lipid-metabolic gene expression." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107881.

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Анотація:
The mammalian small intestine expresses three members of the fatty acid binding protein family; Fabp1, Fabp2, and Fabp6 that differ in their spatial expression pattern and ligand binding preference. In vitro studies using recombinant FABPs suggest an important role in lipid uptake and intestinal lipid metabolism, although the in vivo functions remain unclear. Mice genetically altered to lack Fabp2 exhibit a sex-dimorphic response to high fat diet feeding featuring weight gain and fatty liver in males but not females, suggesting that Fabp2 is involved in a sex-dimorphic intestinal metabolic program. We developed a technique for the analysis of sex differences in transcriptome data and applied it to a study of the intestinal transcriptome of chow-fed Fabp2-/- mice. This analysis revealed lipid metabolic pathways that are differentially regulated in male and female Fabp2-/- mice. We next conducted a high fat diet feeding study for the purpose of analyzing the effects of Fabp2 loss on lipid uptake capacity and spatial expression patterns of lipid metabolic genes identified from the transcriptome experiment. Females lacking Fabp2 exhibited an increased capacity for lipid uptake and an increased abundance of Fabp1 mRNA in the proximal intestine. Loss of Fabp2 also caused an increased excretion of total lipids in females compared to males. This suggests that females might be protected from the adverse effects of Fabp2 loss by directing excess lipids towards excretion pathways through an intracellular, Fabp1-mediated mechanism.
Trois membres de la famille des protéines liantes d'acides gras sont exprimés dans le petit intestin des mammifères, soit le Fabp1, Fabp2 et Fabp6. Ils diffèrent par leur structuration expressive spatiale et leur affinité de ligand. Des études in vitro par recombinaison génétique suggèrent que les FABPs jouent un rôle important dans l'assimilation et le métabolisme intestinal de lipide mais leurs fonctions in vivo restent incertaines par contre. Des souris déficientes de Fabp2 par manipulations génétiques répondent différemment selon leur sexe lorsque soumissent à une diète riche en lipide de manière que les mâles subissent un gain de poids et acquièrent une hyperlipidémie du foie qui sont absents chez les femelles suggérant que le fabp2 est impliqué dans un programme métabolique intestinal dimorphe basé sur le sexe. Nous avons élaboré une technique d'analyse de données de transcriptome pour déterminer la différence sexuelle du transcriptome intestinal de la souris Fabp2-/- nourri de moulé. Cette analyse révéla que la régularisation de la voie métabolique de lipides des souris mâles diffère des femelles. Nous avons poursuivis avec une étude utilisant une diète à forte teneur en lipide dans le but d'analyser l'effet sur la capacité d'assimilation de lipide et la structuration expressive spatiale des gènes du métabolisme des lipides, identifiés par l'analyse de transcriptome précédente, par la perte de Fabp2. Les femelles déficientes de Fabp2 démontrent une augmentation de la capacité d'assimiler les lipides ainsi qu'une abondance d'ARNm de Fabp1 dans l'intestin proximal. La perte de Fabp2 cause aussi une augmentation de l'excrétion total de lipide chez les femelles lorsque comparées aux mâles. Ces observations suggèrent que les femelles semblent être protégées des effets négatifs de la perte de Fabp2 en utilisant une voie intracellulaire d'excrétion des lipides excédents. Ce mécanisme est médié par Fabp1.
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12

Parreau, Simon. "Etude du remodelage vasculaire de l’Artérite à Cellules Géantes par caractérisation inflammatoire et lésionnelle spatiale." Electronic Thesis or Diss., Limoges, 2024. http://www.theses.fr/2024LIMO0040.

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Анотація:
Rationnel : L’artérite à cellules géantes (ACG) est une vascularite affectant l’aorte et ses principales branches, caractérisée par un infiltrat inflammatoire et un remodelage vasculaire. Les complications ischémiques de la maladie sont liées à une hyperplasie de l’intima par prolifération de myofibroblastes. Les fibroblastes adventitiels ont été peu étudiés au cours de l’ACG et pourraient participer au phénomène de remodelage vasculaire.Objectifs : Nous émettons l’hypothèse que les fibroblastes adventitiels sont impliqués dans le remodelage vasculaire de l’ACG.Méthodes : Nous avons inclus des patients ayant eu une biopsie d’artère temporale (BAT) pour suspicion d’ACG. Nous avons tout d’abord étudié la répartition intratissulaire des fibroblastes sur coupe d’artère temporale et leurs marqueurs d’activation par méthode immunohistochimique. A partir de l’isolement de fibroblastes adventitiels de BAT de sujets atteints d’ACG et de contrôles, nous avons effectué des tests fonctionnels de ces fibroblastes. Nous avons réalisé une étude transcriptomique spatiale d’artères temporales de sujets sains et de patients atteints ACG à l’aide de l’outil Digital Spatial Profiler (Nanostring) afin de mettre en évidence des signatures d’expression génomique selon les différentes couches artérielles.Résultats : Les fibroblastes de l’adventice des patients atteints d’ACG expriment des marqueurs d’activation. Ils présentent des capacités de prolifération, migration et sécrétion. L’analyse d’enrichissement montre que les fonctions liées à l’inflammation/réponse immunitaire et le remodelage vasculaire étaient en revanche significativement limitées aux couches internes (intima et media). Les fonctions immunitaires activées concernaient la différenciation des macrophages, les activations des lymphocytes T et B et du complément. Concernant les voies de remodelage vasculaire, nous avons constaté une activation : du processus métabolique du collagène, de la prolifération des fibroblastes, de l’angiogenèse, et de la prolifération des cellules musculaires lisses dans l’intima et la média. Notre analyse du réseau pharmacogénomique a permis d’identifier les gènes qui pourraient potentiellement être ciblés par des traitements actuellement approuvés ou par de nouvelles immunothérapies.Conclusion : Les fibroblastes semblent impliqués dans le processus de l’ACG. Nos résultats fournissent la première caractérisation par profilage spatial in situ des acteurs moléculaires impliqués dans l’ACG, essentielle pour la découverte de nouvelles cibles thérapeutiques potentielles pour traiter cette vascularite
Rational: Giant cell arteritis (GCA) is a vasculitis affecting the aorta and its main branches, characterized by an inflammatory infiltration and vascular remodeling. Ischemic complications of the disease are linked to intimal hyperplasia due to proliferation of myofibroblasts. Adventitial fibroblasts have been little studied in GCA and may play a role in vascular remodeling.Objectives: We hypothesize that adventitial fibroblasts are involved in GCA vascular remodeling.Methods: We included patients who had undergone temporal artery biopsy (TAB) for suspected GCA. We first studied in situ distribution of fibroblasts on temporal artery sections and their activation markers by immunohistochemistry. Using adventitial fibroblasts isolated from GCA patients and controls, functional tests on these fibroblasts were done. We performed a spatial transcriptomic study of temporal arteries from healthy subjects and GCA patients, using the Digital Spatial Profiler (Nanostring) to identify genomic expression signatures in the different arterial layers.Results: Adventitial fibroblasts from GCA patients express activation markers. They display proliferation, migration and secretion capacities. Enrichment analysis showed that functions related to inflammation/immune response and vascular remodeling were significantly restricted to the inner layers (intima and media). Activated immune functions included macrophage differentiation, T and B lymphocyte and complement activation. Regarding vascular remodeling pathways, we observed activation of collagen metabolic processes, fibroblast proliferation, angiogenesis, and smooth muscle cell proliferation in the intima and media. Our pharmacogenomic network analysis identified genes that could potentially be targeted by currently approved treatments or new immunotherapies.Conclusion: Fibroblasts appear to be involved in the GCA process. Our results provide the first characterization by in situ spatial profiling of the molecular players involved in GCA, essential for the discovery of potential new therapeutic targets to treat this vasculitis
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13

Wahle, Philipp. "Transcriptome dynamics in early Drosophila development at tissue and single-cell resolution." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/19976.

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Анотація:
Das Nervensystem von Drosophila melanogaster entwickelt sich aus dem Neuroectoderm entlang der anterior-posterioren Axe des Embryos. Dieses Gewebe unterteilt sich in drei Expressionsdomänen, die von ventral nach dorsal durch Expression der drei Homöobox-Transkriptionsfaktoren vnd, ind und msh charakterisiert sind. Vnd und Ind wurden als notwendig und ausreichend beschrieben, um die Zellidentitäten ihrer jeweiligen Gewebe zu determinieren. Um zu untersuchen, wie diese Transkriptionsfaktoren agieren, um ihre jeweiligen Zelltypen hervorzubringen, habe ich die genomweiten Genexpressionsmuster dieser verwandten Gewebe in Wildtyp-Embryos und in einer vnd-Mutante bestimmt. Ich fand heraus, dass anstelle eines Gewebes, das der IC-Domäne ähnelt, die Vnd-Mutante ein Gewebe hervorbringt, das neuronale Charakteristika zum grossen Teil verloren hat. Ich habe gefunden, dass der Transkiptionsfaktor "eyeless" bei ektopischer Expression in der VC den Vnd-Nervensystemphänotyp phänokopiert und ein ähnliches DNA-Bindemuster aufweist wie Vnd. Unsere Daten lassen vermuten, das Vnd sowohl als direkter Aktivator von VC-spezifischen Genen als auch als direkter Repressor von nicht-neuronalen Genen wirkt. Um die zeitlich-örtliche Auflösung weiter zu erhöhen, habe ich mit Nikolaos Karaiskos kollaboriert, um Einzelzell-Transkriptome von Embryos einer einzigen embryonalen Stufe zu generieren und einen Genexpressions-Atlas des frühen Drosophila-Embryos mit Einzelzellauflösung zu erstellen der die nahezu genomweite Genexpression fast jeder Zelle zu rekapitulieren. Um die Nützlichkeit der Plattform zu demonstrieren, untersuchten wir Expressionsmuster von Genen, die an der Hippo-Signalkaskade beteiligt sind. Wir prognostizierten Hippo-Signalaktivität in bestimmten Bereichen des Embryos und zeigten, dass der Hippo-Signalweg die synchronisierte Zellteilung in diesen Bereichen unterbricht.
The embryonic nervous system of Drosophila melanogaster derives from the neurogenic ectoderm, which is subdivided into three distinct domains. Several key transcription factors show expression exclusive to individual columns and endow their expression domains with characteristic identities. The genes vnd and ind, for example, are homeodomain transcription factors expressed in two columns. Both have been argued to be necessary and sufficient to confer the ventral column or intermediate column fates. To address the question how these transcription factors confer identities to their expression domains I determined the transcriptomic profile of these tissues in wild type and a Vnd mutant embryos. In order to do this, I established a protocol that allowed me to sequence the transcriptomes in developing embryos with spatio-temporal resolution. I found that upon knockout of Vnd, the ventral column largely looses its neurogenic identity rather than converting its fate, as models would predict. I identified Eyeless as a novel candidate transcription factor that shapes early nerve cord identities. Furthermore the data indicates that in Vnd acts as both, activator and repressor. Excessive co-binding with the GAGA-factor GAF suggests a mechanism by which this activation might be achieved. To push spatio-temporal resolution towards the single cell level, I collaborated with Nikolaos Karaiskos to extract single cell transcriptomes of a single developmental stage. This has allowed us to establish a digital single-cell resolved transcriptomic map of a single developmental stage. We used this map to predict expression patterns of thousands of genes with striking accuracy. We identified the Hippo signaling pathway as a spatially regulated pathway in early embryos and showed that it directs the interruption of cell cycle synchronicity in specific areas of the embryo.
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14

Marques, Christine. "Dégénérescence des neurones moteurs cortico-spinaux dans un modèle murin de sclérose latérale amyotrophique : dynamique spatio-temporelle et mécanismes moléculaires." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAJ063/document.

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Анотація:
La sclérose latérale amyotrophique (SLA) se définit cliniquement par la dégénérescence combinée des neurones moteurs cortico-spinaux (NMCS) et des motoneurones bulbaires et spinaux (MnB et MnS). Quoique l’idée d’une origine corticale de la SLA soit de plus en plus considérée, la pathologie corticale, la dynamique spatio-temporelle de la dégénérescence des NMCS et les voies moléculaires impliquées restent peu connues. Ce travail de thèse a essentiellement cherché à pallier ce manque. Nous avons montré que chez les souris Sod1G86R, la perte des NMCS, qui semble se produire en l’absence de gliose réactionnelle majeure, se manifeste de manière somatotopique et précède l'apparition des symptômes moteurs et la dégénérescence des MnS. Nous avons purifié, grâce au développement d'un nouveau protocole, les NMCS adultes du cortex cérébral de souris saines ou malades à quatre stades de la maladie. L’analyse RNA-seq a permis d’identifier de nouveaux acteurs moléculaires précoces pouvant fournir une base pour le développement d'approches thérapeutiques fondées sur le maintien de NMCS sains et fonctionnels, et à long terme, à initier des stratégies thérapeutiques alternatives pour la SLA
Amyotrophic lateral sclerosis (ALS) is clinically defined as the combined degeneration of the corticospinal motor neurons (CSMN) along with the bulbar and spinal motor neurons (BMN and SMN). While a growing body of evidence points to the cerebral cortex as the potential initiation site of ALS, little is known about the cortical pathology, the spatio-temporal dynamics of CSMN degeneration, and the molecular pathways involved. This thesis work aimed at filling this knowledge gap. In Sod1G86R, we showed that CSMN loss seems to take place without major gliosis, occurs in a somatotopic manner and precedes motor symptom appearance and SMN degeneration. We purified, thanks to the development of a novel protocol, adult CSMN from the cerebral cortex of healthy or diseased mice from early presymptomatic ages to the end stage of the disease. The RNA-seq analysis has made it possible to identify new and early molecular players in ALS. This would provide a foundation for the development of therapeutic approaches based on the maintenance of healthy and functional CSMN, and, on the long run, may in turn inform the development of alternative therapeutic strategies for ALS
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15

Sünkel, Christin. "Analyzing the neural transcriptional landscape in time and space." Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/21011.

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Анотація:
Zirkuläre RNAs sind eine Klasse endogener, tierischer RNAs. Obwohl sie hoch abundant sind, ist weder ihre Funktion noch ihre Expression im Nervensystem bekannt. Es wurde ein Katalog zirkulärer RNAs in neuralen Proben erstellt. Es konnten tausende zirkuläre RNAs von Mensch und Maus entdeckt und analysiert werden. Zirkuläre RNAs sind außerordentlich angereichert im Säugetiergehirn, ihre Sequenz ist gut konserviert und sie sind häufig gemeinsam in Mensch und Maus exprimiert. Zirkuläre RNAs waren generell höher exprimiert im Verlauf der neuronalen Differenzierung, sind stark angereichert an Synapsen und oft differentiell exprimiert. circSLC45A4 ist die Hauptisoform, die in humanem präfrontalem, embryonalen Cortex von diesem genomischen Lokus exprimiert wird und eine der am höchsten exprimierten zirkulären RNAs in diesem System. Induzierte Verminderung der Expression von circSLC45A4 ist ausreichend, um die spontane neuronale Differenzierung einer humanen Neuroblastomzelllinie zu induzieren. Dies kann durch die verstärkte Expression neuronaler Markergene belegt werden. Verminderung der Expression von circSLC45A4 im embryonalen Mauscortex verursacht eine signifikante Reduktion von basalen Progenitoren. Außerdem wurde eine signifikante Reduktion von Zellen in der kortikalen Platte nach Depletion von circSLC45A4 gemessen. Weiterhin konnten die Ergebnisse im Mauscortex dekonvoliert werden. Dies zeigte die Zunahme von Cajal-Retzius Zellen. Es wird eine Methode vorgestellt, die RNA-Sequenzierung von Einzelzellen mit räumlicher Auflösung zulässt, ohne vorherige Kenntnisse des Systems zu benötigen. 3D-seq vereint die Applikation eines physischen Gitters mit kombinatorischem Indizieren, so dass Einzelzellen individuell und räumlich markiert werden können. 3D-seq wurde an koronalen Schnitten von adultem Mausgehirn etabliert. Die Daten wurden zur Reproduktion des Gewebes in silico genutzt. 3D-seq ein leicht zu adaptierendes Protokoll, das an jedem Gewebe angewendet werden kann.
Zirkuläre RNAs sind eine Klasse endogener, tierischer RNAs. Obwohl sie hoch abundant sind, ist weder ihre Funktion noch ihre Expression im Nervensystem bekannt. Es wurde ein Katalog zirkulärer RNAs in neuralen Proben erstellt. Es konnten tausende zirkuläre RNAs von Mensch und Maus entdeckt und analysiert werden. Zirkuläre RNAs sind außerordentlich angereichert im Säugetiergehirn, ihre Sequenz ist gut konserviert und sie sind häufig gemeinsam in Mensch und Maus exprimiert. Zirkuläre RNAs waren generell höher exprimiert im Verlauf der neuronalen Differenzierung, sind stark angereichert an Synapsen und oft differentiell exprimiert. circSLC45A4 ist die Hauptisoform, die in humanem präfrontalem, embryonalen Cortex von diesem genomischen Lokus exprimiert wird und eine der am höchsten exprimierten zirkulären RNAs in diesem System. Induzierte Verminderung der Expression von circSLC45A4 ist ausreichend, um die spontane neuronale Differenzierung einer humanen Neuroblastomzelllinie zu induzieren. Dies kann durch die verstärkte Expression neuronaler Markergene belegt werden. Verminderung der Expression von circSLC45A4 im embryonalen Mauscortex verursacht eine signifikante Reduktion von basalen Progenitoren. Außerdem wurde eine signifikante Reduktion von Zellen in der kortikalen Platte nach Depletion von circSLC45A4 gemessen. Weiterhin konnten die Ergebnisse im Mauscortex dekonvoliert werden. Dies zeigte die Zunahme von Cajal-Retzius Zellen. Es wird eine Methode vorgestellt, die RNA-Sequenzierung von Einzelzellen mit räumlicher Auflösung zulässt, ohne vorherige Kenntnisse des Systems zu benötigen. 3D-seq vereint die Applikation eines physischen Gitters mit kombinatorischem Indizieren, so dass Einzelzellen individuell und räumlich markiert werden können. 3D-seq wurde an koronalen Schnitten von adultem Mausgehirn etabliert. Die Daten wurden zur Reproduktion des Gewebes in silico genutzt. 3D-seq ein leicht zu adaptierendes Protokoll, das an jedem Gewebe angewendet werden kann.
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16

Mashayamombe, Chipo. "A rat model study of the spatio-temporal effects of progesterone signalling on the transcriptome of uterine tissues during pregnancy and parturition." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/79940/.

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Анотація:
The steroid hormone progesterone is essential for the maintenance of pregnancy in mammalian species, but its role at term and during labour is not fully understood. While the effects of progesterone be either genomic or non-genomic, this thesis focuses on the genomic effects of progesterone, and its withdrawal, on rat uterine tissues. Using laser Capture Microdissection, homogeneous cell populations from the the outer myometrium, inner myometrium, and decidua basalis were isolated from uterine horns obtained from timed-pregnant Sprague Dawley rats that were randomised to receive either progesterone, mifepristone, or vehicle treatment. These rats had a 22-day gestational period, with natural systemic withdrawal of progesterone occurring on the 19th day of gestation (GD19). RNA sequencing was employed in the examination of the transcriptomes of the uterine layers, as well as gene expression profiling between GD19 and term. Analysis of the spatial expression of mRNA and proteins in the uterine tissues revealed localisation of thyrotropin-releasing hormone mRNA, as well as the Trh protein, to the decidua in late pregnancy. In addition, tenascin-n mRNA and proteins localised to the inner myometrium but not the outer myometrium during late pregnancy. Analysis of the temporal expression of mRNA transcripts showed distinct patterns of expression within each of the analysed tissues. Following the withdrawal of progesterone on GD19, changes first occur in the outer myometrium, followed by the inner myometrium, then the decidua where mRNA expression increases between GD22 before labour and GD22 during labour. In conclusion, this thesis presents a novel approach in which rat uterine tissues were studied separately. Potential tissue-specific markers for the decidua and the inner myometrium in late pregnancy were identified. The increase in gene expression prior to labour in the decidua would suggest a signal for the onset of labour. Furthermore, changes in the myometrial tissues suggest the presence of an in-built mechanism through which the myometrium knows when to prepare for labour.
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17

"Multiplexed Single-cell Spatial Proteomics and Transcriptomics." Doctoral diss., 2018. http://hdl.handle.net/2286/R.I.51771.

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Анотація:
abstract: Single-cell proteomics and transcriptomics analysis are crucial to gain insights of healthy physiology and disease pathogenesis. The comprehensive profiling of biomolecules in individual cells of a heterogeneous system can provide deep insights into many important biological questions, such as the distinct cellular compositions or regulation of inter- and intracellular signaling pathways of healthy and diseased tissues. With multidimensional molecular imaging of many different biomarkers in patient biopsies, diseases can be accurately diagnosed to guide the selection of the ideal treatment. As an urgent need to advance single-cell analysis, imaging-based technologies have been developed to detect and quantify multiple DNA, RNA and protein molecules in single cell in situ. Novel fluorescent probes have been designed and synthesized, which targets specifically either their nucleic acid counterpart or protein epitopes. These highly multiplexed imaging-based platforms have the potential to detect and quantify 100 different protein molecules and 1000 different nucleic acids in a single cell. Using novel fluorescent probes, a large number of biomolecules have been detected and quantified in formalin-fixed paraffin-embedded (FFPE) brain tissue at single-cell resolution. By studying protein expression levels, neuronal heterogeneity has been revealed in distinct subregions of human hippocampus.
Dissertation/Thesis
Doctoral Dissertation Chemistry 2018
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18

Eng, Chee Huat (Linus). "Plus Ultra: Genome-Wide Spatial Transcriptomics with RNA seqFISH+." Thesis, 2021. https://thesis.library.caltech.edu/14204/1/CheeHuat%28Linus%29Eng_thesis.pdf.

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Анотація:

Visualizing single cells and their organization in intact tissue is crucial to understanding their governing biological function. Even though single cell RNA sequencing has provided many insights into the heterogeneity and gene expression profiles across many tissue types, the dissociation process which loses the spatial information is hindering our deeper understanding of how these transcriptional distinct cell types are organized and interacting in their native tissue environment.

The thesis begins by giving a background on how single cell RNA sequencing has transformed biology and the emergence of spatial technology such as sequential fluorescence in situ hybridization (seqFISH). While spatial methods are useful for mapping the cell types identified from single cell RNA sequencing, the need for turning spatial technology such as seqFISH, which has high detection efficiency of the transcriptome with spatial information, into an in situ discovery tool is discussed as the scientific community’s goal heads towards building spatial atlases for every human tissues and organs such as the brain.

While seqFISH has high detection efficiency, it is still limited in the number of genes capable of profiling at once. The major obstacle is the optical crowding problems when more RNA species are targeted and imaged using a fluorescence microscope. In Chapter 2, we first investigated, if the RNA molecules are instead captured on a coverslip and profiled with sequential barcoding strategy, the FISH-based method will reliably characterize the transcriptome when molecular crowding is not an issue.

Finally, in Chapter 3, we demonstrate the barcoding strategy to break through the molecular crowding limit of multiplexed FISH. From being able to profile hundreds to a thousand genes by various multiplexed FISH methods at that time in the field, we succeeded in profiling 10,000 genes by RNA seqFISH+, an evolved version of seqFISH, in various intact tissue sections, turning seqFISH+ into a spatial discovery technology with its genome-wide coverage and high detection efficiency. The work described in this part of the thesis is highlighted in Nature Method’s Method of The Year 2020- Spatially-resolved Transcriptomic article.

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