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1
Werne, Solnestam Beata. "Interpreting the human transcriptome." Doctoral thesis, KTH, Genteknologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-158320.
Повний текст джерелаАнотація:
The human body is made of billions of cells and nearly all have the same genome. However, there is a high diversity of cells, resulted from what part of the genome the cells use, i.e. which RNA molecules are expressed. Rapid advances within the field of sequencing allow us to determine the RNA molecules expressed in a specific cell at a certain time. The use of the new technologies has expanded our view of the human transcriptome and increased our understanding of when, where, and how each RNA molecule is expressed. The work presented in this thesis focuses on analysis of the human transcriptome. In Paper I, we describe an automated approach for sample preparation. This protocol was compared with the standard manual protocol, and we demonstrated that the automated version outperformed the manual process in terms of sample throughput while maintaining high reproducibility. Paper II addresses the impact of nuclear transcripts on gene expression. We compared total RNA from whole cells and from cytoplasm, showing that transcripts with long, structured 3’- and 5’-untranslated regions and transcripts with long protein coding sequences tended to be retained in the nucleus. This resulted in increased complexity of the total RNA fraction and fewer reads per unique transcript. Papers III and IV describe dynamics of the human muscle transcriptome. For Paper III, we systematically investigated the transcriptome and found remarkably high tissue homogeneity, however a large number of genes and isoforms were differentially expressed between genders. Paper IV describes transcriptome differences in response to repeated training. No transcriptome-based memory was observed, however a large number of isoforms and genes were affected by training. Paper V describes a transcript profiling protocol based on the method Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification. We designed the method for a few selected transcripts whose expression patterns are important for detecting breast cancer cells, and optimized the method for single cell analysis. We successfully detected cells in human blood samples and applied the method to single cells, confirming the heterogeneity of a cell population.Människokroppen är uppbyggd av miljarder celler och nästan alla innehåller samma arvsmassa. Trots detta finns det många olika celler med olika funktioner vilket är en följd av vilken del av arvsmassan som cellerna använder, dvs vilka RNA-molekyler som finns i varje cell. Den snabba utvecklingen av sekvenseringstekniker har gjort det möjligt att studera när, var och hur varje RNA-molekyl är uttryckt och att få en djupare förståelse för hur människans celler fungerar. Arbetet som presenteras i denna avhandling fokuserar på analys av RNA-molekyler i människans celler. I artikel I beskriver vi en automatiserad metod för att förbereda cellprov för RNA-sekvensering. Det automatiserade protokollet jämfördes med det manuella protokollet, och vi visade att det automatiserade protokollet överträffade det manuella när det gällde provkapacitet samtidigt som en höga reproducerbarheten behölls. I artikel II undersökte vi effekterna som RNA-molekyler från en del av cellen (cellkärnan) har på den totala mängden uttryckta RNA-molekyler. Vi jämförde RNA från hela cellen och från en del av cellen (cytoplasman) och visade att RNA-molekyler med långa och strukturerade 3'- och 5'-otranslaterade regioner och RNA-molekyler med långa proteinkodande sekvenser tenderade att hållas kvar i cellkärnan till en högre grad. Detta resulterade i en ökad komplexitet av RNA-molekylerna i hela cellen, medan vi i cytoplasma-fraktionen lättare kunde hitta de korta och svagt uttryckta RNA-molekyler. I Artikel III och IV studerar vi RNA-molekyler i människans skelettmuskler. I artikel III visar vi att andelen RNA-molekyler uttryckta i skelettmuskler är väldigt lika mellan muskler och mellan olika personer, men att ett stort antal RNA-molekyler var uttryckta i olika nivåer hos kvinnor och män. Artikel IV beskriver RNA-nivåer som svar på upprepade perioder av uthållighetsträning. Artikel V beskriver en metod för att studera ett fåtal utvalda RNA-molekyler. Vi valde RNA-molekyler vars uttryck är viktigt vid analys av bröstcancerceller, och optimerade metoden för analys av enskilda celler. Vi analyserade cancerceller från blodprov och använde metoden för att titta på RNA-nivåer i enskilda celler från en grupp av celler och visade på skillnader i RNA-nivåer inom gruppen.
QC 20150115
2
Natarajan, Sripriya 1978. "Defining the human endothelial transcriptome." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33082.
Повний текст джерелаАнотація:
Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2005.Includes bibliographical references (leaves 91-100).
Advances in microarray technology facilitate the study of biological systems at a genome-wide level. Meaningful analysis of these transcriptional profiling studies, however, demands the concomitant development of novel computational techniques that take into account the size and complexity of the data. We have devised statistical algorithms that use replicate microarrays to define a genome-wide expression profile of a given cell type and to determine a list of genes that are significantly differentially expressed between experimental conditions. Applying these algorithms to the study of cultured human umbilical vein endothelial cells (HUVEC), we have found approximately 54% of all genes to be expressed at a detectable level in HUVEC under basal conditions. The set of highest expressed genes is enriched in nucleic acid binding proteins, cytoskeletal proteins and isomerases as well as certain known markers of endothelium, and the complete list of genes can be found at ... We have also studied the effect of a 4-hour exposure of HUVEC to 10 U/mL of IL-1, and detected 491 upregulated and 259 downregulated statistically significant genes, including several chemokines and cytokines, as well as members of the TNFAIP3 family, the KLFfamily and the Notch pathway. Applying these rigorous statistical techniques to genome-wide expression datasets underscores known patterns of endothelial inflammatory gene regulation and unveils new pathways as well.
(cont.) Finally, we performed a direct comparison of direct-labeled microarrays with amplified RNA microarrays for an initial assessment of the effect of the additional noise of amplification on the outputs of the statistical algorithms. These techniques can be applied to additional genome-wide profiling studies of endothelium and other cell types to refine our understanding of transcriptomes and the gene regulatory network governing cellular function and pathophysiology.
by Sripriya Natarajan.
S.M.
3
Oldham, Michael Clark. "Transcriptome organization in human and chimpanzee brains." Diss., Restricted to subscribing institutions, 2009. http://proquest.umi.com/pqdweb?did=1872073991&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
Повний текст джерела
4
Wetterbom, Anna. "Genome and Transcriptome Comparisons between Human and Chimpanzee." Doctoral thesis, Uppsala universitet, Genomik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-112893.
Повний текст джерелаАнотація:
The chimpanzee is humankind’s closest living relative and the two species diverged ~6 million years ago. Comparative studies of the human and chimpanzee genomes and transcriptomes are of great interest to understand the molecular mechanisms of speciation and the development of species-specific traits. The aim of this thesis is to characterize differences between the two species with regard to their genome sequences and the resulting transcript profiles. The first two papers focus on indel divergence and in particular, indels causing premature termination codons (PTCs) in 8% of the chimpanzee genes. The density of PTC genes is correlated with both the distance to the telomere and the indel divergence. Many PTC genes have several associated transcripts and since not all are affected by the PTC we propose that PTCs may affect the pattern of expressed isoforms. In the third paper, we investigate the transcriptome divergence in cerebellum, heart and liver, using high-density exon arrays. The results show that gene expression differs more between tissues than between species. Approximately 15% of the genes are differentially expressed between species, and half of the genes show different splicing patterns. We identify 28 cassette exons which are only included in one of the species, often in a tissue-specific manner. In the fourth paper, we use massive parallel sequencing to study the chimpanzee transcriptome in frontal cortex and liver. We estimate gene expression and search for novel transcribed regions (TRs). The majority of TRs are located close to genes and possibly extend the annotations. A subset of TRs are not found in the human genome. The brain transcriptome differs substantially from that of the liver and we identify a subset of genes enriched with TRs in frontal cortex. In conclusion, this thesis provides evidence of extensive genomic and transcriptomic variability between human and chimpanzee. The findings provide a basis for further studies of the underlying differences affecting phenotypic divergence between human and chimpanzee.
5
Symes, A. J. "Epithelial specific transcriptome map of the human prostate." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302555/.
Повний текст джерелаАнотація:
The prostate has a zonal anatomy, with differing susceptibilities to disease (benign prostatic hyperplasia originates from the transition zone, prostate cancer largely arises in the peripheral zone). The molecular reasons for this are not understood. Previous prostate cancer microarray studies have used whole benign, diseased or tissue adjacent to the carcinoma as normal controls, for what is an epithelial disease. This study provides a gene expression profile of normal, non-diseased prostate, or a ‘reference prostate gene expression profile’. This has been compared to prostate cancer to identify novel biomarkers of disease. This study also investigates zonal differences in gene expression between different anatomical zones of the prostate. I used normal, human donor prostate tissue, laser capture microdissection (LCM), and Affymetrix gene expression arrays to achieve these aims. Eight LCM prostate epithelial samples from 3 donor prostates were used. The gene expression data was validated by low density real-time PCR and immunohistochemistry on a prostate tissue microarray. Major differences in gene expression were discovered between whole tissue and LCM epithelium only prostate using homology tables. Novel prostate adenocarcinoma genes were identified using a publicly available LCM prostate cancer gene expression array dataset. 9318 genes showed significant differential expression in normal vs. cancer datasets. Three targets, MCM2, NR1D1 and ABCA1 were validated at the protein level. Expression of NR1D1 and ABCA1 were increased in cancer, suggesting they are novel epithelial biomarkers of prostate cancer. An analysis of zonal differences in gene expression found significant differences between zones. Zonal specific markers included TGM4 (central zone), LPL (peripheral zone), and COL9A1 (transition zone). This study provides: (i) a gene expression profile of the normal prostate epithelium (ii) novel, prostate adenocarcinoma specific gene and protein markers and (iii) the first gene expression profile of normal epithelium on the basis of zonal anatomy of the prostate.
6
Corral, Vázquez Celia. "Human sperm transcriptome: characterization, biological relevance, and biomarker functionality." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/669365.
Повний текст джерелаАнотація:
Se ha demostrado que la contribución del espermatozoide al embrión va más allá de la transmisión del genoma paterno. Diversos estudios han mostrado que el espermatozoide humano contiene una compleja población de RNAs implicados en funciones relacionadas con la fertilidad. Por tanto, la visión de estas moléculas como meros restos de eventos previos ha quedado atrás. Este nuevo paradigma abre las puertas a nuevas aplicaciones del RNA en el ámbito de los biomarcadores de fertilidad. Sin embargo, los análisis transcriptómicos en espermatozoides presentan diversas limitaciones debidas a la heterogeneidad y delicada naturaleza de estas moléculas, además de la poca cantidad de RNA contenida en dichas células.
En este contexto, el objetivo de esta Tesis Doctoral es caracterizar el transcriptoma del espermatozoide humano y establecer las bases para desarrollar nuevos biomarcadores de fertilidad masculina. Dentro de este objetivo, se plantearon las siguientes metas: 1) optimizar metodologías específicas para analizar el RNA espermático mediante qRT-PCR y RNA-seq; 2) proporcionar un perfil integrado y una caracterización funcional de los mRNAs y lncRNAs espermáticos mediante RNA-seq; y 3) establecer nuevos biomarcadores de fertilidad a partir de la carga transcriptómica del espermatozoide.
Con este propósito, se adaptaron tanto el protocolo experimental como el análisis de datos a las limitaciones propias del RNA espermático y a la tecnología transcriptómica usada. Por tanto, se implementaron métodos de eliminación de células no espermáticas de las muestras seminales, así como controles de calidad para asegurar la ausencia de DNA y RNA no espermático. Además, se usó un método basado en solventes orgánicos para los estudios qRT-PCR, y kits de solventes no orgánicos para RNA-seq.
Los datos obtenidos se normalizaron usando métodos específicos de la técnica empleada. En concreto, para la normalización de los datos de expresión de miRNAs espermáticos en estudios singleplex qRT-PCR era necesario establecer miRNAs normalizadores. Esto se consiguió comparando los resultados derivados de unos datos que se normalizaron mediante: i) el método Mean-Centering Restricted (MCR); y ii) el nivel de expresión de diferentes miRNAs. Los miRNAs hsa-miR-100-5p y hsa-miR-30a-5p mostraron una expresión estable y ubicua, y su uso derivó en resultados con una calidad semejante a los conseguidos mediante la normalización por MCR. Por tanto, se sugirió esta combinación de miRNAs como la mejor opción para la normalización de futuros estudios singleplex qRT-PCR de miRNAs espermáticos.
Por otro lado, se empleó RNA-seq, para caracterizar el transcriptoma espermático de individuos fértiles. Los resultados revelaron una red de mRNAs y lncRNAs en alto estado de fragmentación, pero que contenían un grupo de transcritos ubicuos. Los análisis de Ontología Génica de todos los mRNAs expresados mostraron una implicación en procesos de espermatogénesis y reproducción, la cual era más significativa en los análisis de los mRNAs altamente expresados, ubicuos y altamente estables. Aparte, los potenciales genes dianas en cis de los lncRNAs mostraron relación con procesos de desarrollo embrionario y adhesión celular, la cual prevalecía en los genes dianas que no estaban expresados en espermatozoides.
Finalmente, el hecho de hallar transcritos ubicuos y de expresión correlacionada indicó un posible uso de estas moléculas como biomarcadores de fertilidad. Por tanto, se evaluó y se validó la presencia de pares de miRNAs espermáticos con una expresión correlacionada en individuos fértiles y no correlacionada en pacientes infértiles de diferentes etiologías (astenozoospermia, teratozoospermia, oligozoospermia e infertilidad inexplicable [UMI]). El par hsa-miR-942-5p/hsa-miR-1208 permitió clasificar correctamente el 85.71% de los casos de infertilidad, alcanzando el mayor potencial de diagnóstico de pacientes con alteraciones seminales. El par hsa-miR-34b-3p/hsa-miR-93-3p destacó por su potencial para discernir pacientes UMI. Aparte, varios pares de mRNAs y lncRNAs también mostraron expresiones correlacionadas en individuos fértiles, constituyendo unos candidatos potenciales para futuros estudios.The biological relevance of sperm contribution to the embryo has been shown to go beyond a mere transmission of the paternal genome. Several findings revealed that human spermatozoa carry a complex population of coding and non-coding RNAs with potential implications in multiple fertility-related pathways. Accordingly, the consideration of these molecules as simple residual pools of earlier processes has been left behind. This new paradigm also opens the possibility for potential applications in the field of male fertility biomarkers. However, sperm transcriptomic analysis has several limitations due to the heterogeneity and delicate nature of these molecules, besides the small amount of RNA contained in spermatozoa. In this context, the objective of this Doctoral Thesis is to characterize the human sperm transcriptome to set up the basis for developing new biomarkers of male fertility. Within this goal, the following aims were undertaken: 1) To optimize specific methodologies of sperm RNA analysis using qRT-PCR and RNA-seq strategies; 2) To provide an integrative profiling and functional characterization of sperm mRNAs and lncRNAs by RNA-seq technologies; and 3) To establish new fertility biomarkers among the transcriptomic cargo of the human spermatozoa. For this purpose, the experimental protocols and data analysis were adapted to the inherent limitations of sperm RNA and to the used transcriptomic technology. Therefore, methods for the elimination of non-sperm cells from semen samples were implemented, together with strict quality controls for ensuring the absence of DNA and non-sperm RNA. Besides, an organic solvent-based method was used for qRT-PCR studies, and non-organic solvent kits were employed for RNA-seq. The obtained data were normalized by specific methods depending on the used technique. In particular, the normalization of sperm miRNA qRT-PCR singleplex studies required the determination of a suitable set of normalizing miRNAs molecules. This was achieved by comparing the results derived from a sperm miRNA expression dataset normalized by: i) the reference Mean Centering Restricted (MCR) method; and ii) the expression level of different miRNAs. The miRNAs hsa-miR-100-5p and hsa-miR-30a-5p showed ubiquitous and stable expressions, and data normalized by their mean expression led to results with an appropriate quality when compared to MCR. Therefore, this miRNA combination was suggested as the most suitable choice for data normalization in further sperm singleplex studies. RNA-seq analysis was used to characterize the sperm transcriptome cargo of fertile individuals. Results revealed a complex network of mRNAs and lncRNAs with a high fragmentation status, but containing a host of ubiquitous transcripts. Gene ontology analyses of the whole set of expressed mRNAs showed an enrichment of spermatogenesis and reproduction processes, which was more significant in the sets of highly expressed, ubiquitous, and highly stable mRNAs. Additionally, the functional profiling of potential cis-target genes of the observed lncRNAs showed a significant involvement in embryo development and cell adhesion. This implication became more evident in those cis-target genes that were not present among the sperm mRNA cargo. Finally, the detection of ubiquitous transcripts and pairs of RNAs with correlated expressions suggested a potential use of these molecules as fertility biomarkers. Accordingly, the presence of sperm miRNA pairs with a correlated expression in fertile individuals that was disrupted in infertile patients of different ethiologies (asthenozoospermia, teratozoospermia, oligozoospermia, and Unexplained Male Infertility or UMI) was evaluated and validated by qRT-PCR. The hsa-miR-942-5p/hsa-miR-1208 pair allowed correctly classifying the 85.71% of infertile individuals, thus achieving the highest potential for discerning infertility cases with seminal alterations. Additionally, the pair hsa-miR-34b-3p/hsa-miR-93-3p was highlighted due to its high potential for discerning UMI patients. Besides, several pairs of ubiquitous lncRNAs and mRNAs were also observed to display a correlated expression in fertile individuals, becoming potential candidates for further biomarker studies.
7
Khuder, Basil. "Human Genome and Transcriptome Analysis with Next-Generation Sequencing." University of Toledo Health Science Campus / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=mco1501886695490104.
Повний текст джерела
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Chen, Jenny (Jennifer). "Evolutionary signatures for unearthing functional elements in the human transcriptome." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/117792.
Повний текст джерелаАнотація:
Thesis: Ph. D. in Bioinformatics and Integrative Genomics, Harvard-MIT Program in Health Sciences and Technology, 2018.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged student-submitted from PDF version of thesis.
Includes bibliographical references (pages 141-156).
Comparative genomics is a powerful method for identifying functional genetic elements by their evolutionary patterns across species. However, current studies largely focus on analysis of genome sequences. The recent development of RNA-sequencing reveals dimensions of regulatory information previously inaccessible to us by sequence alone. The comparison of RNA-sequencing data across mammals has great potential for addressing two open problems in biology: identifying the regulatory mechanisms crucial to mammalian physiology, and deciphering how gene regulation contributes to the diversity of mammalian phenotypes. For my thesis, I developed two methodologies for interrogating comparative transcriptomic data for biological inference. First, I developed a framework for quantifying the evolutionary forces acting on gene expression and inferring evolutionarily optimal expression levels. I demonstrate how to use this framework to identify expression pathways underlying conserved, adaptive, and disease states of mammalian biology. Second, I developed novel metrics of transcriptional evolution to evaluate the conservation of long noncoding RNAs. These metrics further reveal that long noncoding RNAs harbor distinct evolutionary signatures, suggesting that they are not a homogenous class of molecules but rather a mixture of multiple functional classes with distinct biological roles. My thesis work provides fundamental quantitative tools for asking biological questions about transcriptome evolution. These tools provide a pivotal framework for interpreting transcriptional data across species and pave the way for deciphering the regulatory changes that lead to mammalian phenotypic variation.
by Jenny Chen.
Ph. D. in Bioinformatics and Integrative Genomics
9
Xu, Guorong. "Computational Pipeline for Human Transcriptome Quantification Using RNA-seq Data." ScholarWorks@UNO, 2011. http://scholarworks.uno.edu/td/343.
Повний текст джерелаАнотація:
The main theme of this thesis research is concerned with developing a computational pipeline for processing Next-generation RNA sequencing (RNA-seq) data. RNA-seq experiments generate tens of millions of short reads for each DNA/RNA sample. The alignment of a large volume of short reads to a reference genome is a key step in NGS data analysis. Although storing alignment information in the Sequence Alignment/Map (SAM) or Binary SAM (BAM) format is now standard, biomedical researchers still have difficulty accessing useful information. In order to assist biomedical researchers to conveniently access essential information from NGS data files in SAM/BAM format, we have developed a Graphical User Interface (GUI) software tool named SAMMate to pipeline human transcriptome quantification. SAMMate allows researchers to easily process NGS data files in SAM/BAM format and is compatible with both single-end and paired-end sequencing technologies. It also allows researchers to accurately calculate gene expression abundance scores.
10
Sherwood, Karen. "Preparation, characterisation and transcriptome analysis of RNA from human vCJD brains." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/4226.
Повний текст джерелаАнотація:
The pathological mechanisms of variant Creutzfeldt-Jakob disease (vCJD) in the human brain remain poorly understood. Gene expression data may provide insight into the molecular mechanisms involved. This requires analysis of human postmortem brain tissue however; the variability in RNA preparations from human brain material is a concern. A method for the isolation of RNA from vCJD brains which minimized infectivity and reduced Proteinase K resistant prion protein levels to undetectable by biochemical assay was developed. RNA preparations were made from sample of the frontal parasagital cortex, sub-frontal cortex and cerebellum of 78 human autopsy cases; 21 vCJD, 26 other neurological disease (OND) and 31 nonneurological disease (NND). Suitable RNA metrics for these human brain RNA preparations were evaluated and the intra- and inter-case variability of RNA preparations was determined. There was marked intra- and inter-case variability in RNA integrity number (RIN), A260:280 absorbance ratio and RNA yield. In particular, RIN and A260:280 showed little variation intra-patient, although RNA yield was more variable. The effects of postmortem interval, tissue pH, age at death, gender, freeze-thaw cycles (including storage method and temperature) and agonal state were investigated; none of these parameters correlated with the marked variability observed. Parameters for matching vCJD and OND/NND cases were considered and RNA from three age and gender matched comparison groups, each containing one OND, one NND and one vCJD case, were used for gene expression analysis. Data was generated using Superarray GEArray® Focused DNA Microarray and analysed using the GEArray Expression Analysis Suite and Significance Analysis of Microarray software. A comparison between matched vCJD and NND control cases identified 26 up-regulated and 16 down-regulated genes, showing >1.5-fold change with a false discovery rate of 9%. The modulated genes were involved in cell signaling, cell death, cholesterol and lipid metabolism. Involvement of these pathways is consistent with findings in other transmissible spongiform encephalopathy studies.
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Fischer, Cornelius [Verfasser]. "Transcriptome-wide Single-cell Analysis of Human Macrophage Heterogeneity / Cornelius Fischer." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1156901510/34.
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Abudayyeh, Omar O. "Discovery of novel CRISPR enzymes for transcriptome engineering and human health." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/120887.
Повний текст джерелаАнотація:
Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, September 2018.Page 399 blank. Cataloged from PDF version of thesis.
Includes bibliographical references (pages 210-229).
RNA plays important and diverse roles in biology, yet molecular tools to measure and manipulate RNA are limited. Recently, the bacterial adaptive immune system, CRISPR, has revolutionized our ability to manipulate DNA, but no known RNA-targeting versions exist. To discover parallel bacterial RNA-targeting systems that could be used for transcriptome engineering, we developed a computational pipeline to mine for novel Class 2 CRISPR systems across more than 25,000 bacterial genomes. Among the many novel CRISPR systems, we found a programmable RNA-targeting CRISPR system, CRISPR-Cas 13, that could provide immunity to E. coli against the ssRNA MS2 phage and biochemically characterized the enzyme. We adapted CRISPR-Casl3 for modulating the transcriptome in mammalian and plant cells by heterologously expressing Casl 3 and engineering the enzyme to precisely knockdown, bind, and edit RNA. Cas 13 knockdown was as efficient as RNA interference, but much more specific, across many transcripts tested. RNA editing with Cas 13 was also highly efficient, with up to 90% base editing rates, and as low as 20 off-targets with engineered specificity versions. Lastly, we combined Cas13 with isothermal amplification to develop a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with single-molecule sensitivity and singlebase mismatch specificity. We used this Casl3a-based molecular detection platform, termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), to specifically detect pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Our results establish CRISPR-Cas13 as a flexible platform for RNA targeting with wide applications in RNA biology, diagnostics, and therapeutics.
by Omar O. Abudayyeh.
Ph. D. in Medical Engineering and Medical Physics
13
Pilling, Luke C. "Human population studies of transcriptome-wide expression in age-related traits." Thesis, University of Exeter, 2015. http://hdl.handle.net/10871/17471.
Повний текст джерелаАнотація:
This thesis presents novel investigations of three common ageing phenotypes in human population studies, using microarray technology to assess ‘transcriptome-wide’ expression in whole blood to identify mechanisms and biomarkers. Muscle strength is related to frailty and is predictive of disability in older persons. I assessed the association between transcript abundance in the InCHIANTI peripheral blood samples (N=695) and muscle strength. One gene (CEBPB) passed the multiple testing criteria, and is involved in macrophage-mediated repair of damaged muscle. I extended this work with a meta-analysis of over 7,781 individuals in four collaborating cohorts; expression of over 222 genes were significantly associated with strength, less than half of which have previously been linked to muscle in the literature. CEBPB did not replicate in these younger cohorts. I then performed the first human analysis of gene expression and cognitive function (and separately with decline in cognitive ability over nine years) in the InCHIANTI cohort (N=681), and one gene was identified; CCR2, a chemokine receptor. Evidence in mice has implicated this gene in the accumulation of β-amyloid and cognitive impairment. Finally, in a collaborative project with the Framingham Heart Study I studied age-related inflammation – another hallmark of ageing - using a novel approach to ‘transcriptome-wide’ analysis; each transcript was assessed for the proportion of the association between age and interleukin-6 (IL6) that it statistically mediated. Very few of the genes associated with IL6 alone also mediated the relationship with age. Findings include; SLC4A10, the strongest mediator, not previously linked to inflammation, and interleukin-1 beta and perforin, a cytokine and cytotoxic protein, respectively. These novel analyses highlight key molecular pathways associated with age-related phenotypes in whole blood and provide links between mouse models and humans. They provide biological insight and directions for future research.
14
Dalla, Emiliano. "Analysis of the human transcriptome and identification of conserved noncoding elements." Doctoral thesis, SISSA, 2005. http://hdl.handle.net/20.500.11767/4751.
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Johnson, Kristen. "Software for Estimation of Human Transcriptome Isoform Expression Using RNA-Seq Data." ScholarWorks@UNO, 2012. http://scholarworks.uno.edu/td/1448.
Повний текст джерелаАнотація:
The goal of this thesis research was to develop software to be used with RNA-Seq data for transcriptome quantification that was capable of handling multireads and quantifying isoforms on a more global level. Current software available for these purposes uses various forms of parameter alteration in order to work with multireads. Many still analyze isoforms per gene or per researcher determined clusters as well. By doing so, the effects of multireads are diminished or possibly wrongly represented. To address this issue, two programs, GWIE and ChromIE, were developed based on a simple iterative EM-like algorithm with no parameter manipulation. These programs are used to produce accurate isoform expression levels.
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Towler, James Charles. "Transcriptome activity of human cytomegalovirus (strain Merlin) in fibroblasts, epithelial cells and astrocytes." Thesis, Connect to e-thesis record to view abstract. Move to record for print version, 2007. http://theses.gla.ac.uk/42/.
Повний текст джерелаАнотація:
Thesis (Ph.D.) - University of Glasgow, 2007.Ph.D. thesis submitted to the Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, 2007. Includes bibliographical references. Print version also available.
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Cruz, Pulido Diana Patricia. "Comparative transcriptome profiling of human and pig intestinal epithelial cells after Deltacoronavirus infection." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1587711071257247.
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Vollmar, Christine. "Can the Gingival Crevicular Fluid Transcriptome Predict Healing After Dental Trauma?" The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1435011386.
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Hernandez-Ferrer, Carles 1987. "Bioinformatic tools for exposome data analysis : application to human molecular signatures of ultraviolet light effects." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/572046.
Повний текст джерелаАнотація:
Las enfermedades complejas se encuentran entre las más comunes y son causadas por una combinación de factores genéticos y ambientales (contaminación ambiental, estilo de vida, etc). Entre las enfermedades complejas que se pueden destacar se encuentran la obesidad, el asma, la hipertensión o la diabetes. Diversos estudios científicos sugieren que el hecho de padecer enfermedades complejas está condicionado a la aparición o acumulación de determinados factores ambientales. Asimismo, se ha descrito que los factores ambientales son unos de los principales contribuyentes a la carga mundial de morbilidad. Todo esto nos lleva a definir el término exposoma como el conjunto de factores ambientales a los que un individuo se ve expuesto desde la concepción hasta la muerte. El estudio de la mecánica subyacente que vincula el exposoma con la salud es un campo de investigación emergente con un fuerte potencial para proporcionar nuevos conocimientos sobre la etiología de las enfermedades.
La primera parte de esta tesis se centra en la exposición a la radiación ultravioleta. La exposición a la radiación ultravioleta proviene de fuentes tanto naturales como artificiales. La radiación ultravioleta incluye tres subtipos de radiación según su longitud de onda (UVA 315-400 nm, UVB 315-295 nm y UVC 295-200 nm). Si bien la principal fuente natural de radiación ultravioleta es el Sol, la UVC no llega a la superficie de la Tierra debido a su absorción por la capa estratosférica de ozono. En consecuencia, la exposición a radiación ultravioleta a la que estamos usualmente sometidos consisten en una mezcla de UVA (95 %) y UVB (5 %). Los efectos de la radiación ultravioleta en humanos pueden ser beneficiosos o perjudiciales dependiendo de su cantidad y forma. Los efectos perjudiciales y agudos de la radiación ultravioleta incluyen eritema, oscurecimiento del pigmento, retraso en el bronceado y engrosamiento de la epidermis. Repetidas lesiones en la piel producidas por radiación ultravioleta pueden predisponer, en última instancia, a efectos crónicos de fotoenvejecimiento, inmunosupresión y fotocarcinogénesis. El mayor efecto beneficioso de la radiación ultravioleta es la síntesis cutánea de la vitamina D. La vitamina D es necesaria para mantener el calcio fisiológico y del fósforo para la mineralización ósea y para prevenir el raquitismo, la osteomalacia y la osteoporosis. El paradigma del exposoma es trabajar con múltiples exposiciones a la vez en vez centrarse en una sola exposición. Este enfoque permite tener una visión más parecida a la realidad que vivimos. Luego, la segunda parte se centra en las herramientas para explorar cómo caracterizar y analizar el exposoma y cómo probar sus efectos en múltiples capas biológicas intermedias para proporcionar información sobre los mecanismos moleculares subyacentes que vinculan las exposiciones ambientales a los resultados de salud.Most common diseases are caused by a combination of genetic, environmental and lifestyle factors. These diseases are referred to as complex diseases. Examples of this type of diseases are obesity, asthma, hypertension or diabetes. Several empirical evidence suggest that exposures are necessary determinants of complex disease operating in a causal background of genetic diversity. Moreover, environmental factors have long been implicated as major contributors to the global disease burden. This leads to the formulation of the exposome, that contains any exposure to which an individual is subjected from conception to death. The study of the underlying mechanics that links the exposome with human health is an emerging research field with a strong potential to provide new insights into disease etiology. The first part of this thesis is focused on ultraviolet radiation (UVR) exposure. UVR exposure occurs from both natural and artificial sources. UVR includes three subtypes of radiation according to its wavelength (UVA 315-400 nm, UVB 315-295 nm, and UVC 295-200 nm). While the main natural source of UVR is the Sun, UVC radiation does not reach Earth's surface because of its absorption by the stratospheric ozone layer. Then, exposures to UVR typically consist of a mixture of UVA (95%) and UVB (5%). Effects of UVR on human can be both beneficial and detrimental, depending on the amount and form of UVR. Detrimental and acute effects of UVR include erythema, pigment darkening, delayed tanning and thickening of the epidermis. Repeated UVR-induced injury to the skin, may ultimately predispose one to the chronic effects photoaging, immunosuppression, and photocarcinogenesis. The beneficial effect of UVR is the cutaneous synthesis of vitamin D. Vitamin D is necessary to maintain physiologic calcium and phosphorous for normal bone mineralization and to prevent rickets, osteomalacia, and osteoporosis. But the exposome paradigm is to work with multiple exposures at a time and with one or more health outcomes rather focus in a single exposures analysis. This approach tends to be a more accurate snapshot of the reality that we live in complex environments. Then, the second part is focused on the tools to explore how to characterize and analyze the exposome and how to test its effects in multiple intermediate biological layers to provide insights into the underlying molecular mechanisms linking environmental exposures to health outcomes.
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Michel, Margaux [Verfasser], and Patrick [Akademischer Betreuer] Cramer. "Transient transcriptome sequencing : development and applications in human cells / Margaux Michel ; Betreuer: Patrick Cramer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1119073316/34.
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Muñoz, Aguirre Manuel. "From RNA to histological images: linking the transcriptome with human phenotypes through statistical learning." Doctoral thesis, Universitat Politècnica de Catalunya, 2021. http://hdl.handle.net/10803/672123.
Повний текст джерелаАнотація:
Genomic datasets are fundamental to broaden our understanding of human biology in the context of health and disease. However, the high-dimensional nature of gene expression and other molecular traits poses a challenge when attempting to find associations of these data types with human phenotypes. To this end, this thesis relies on statistical learning tools to mitigate the curse of dimensionality and link the human transcriptome with phenotypes at different orders of complexity: from RNA, to computationally-inferred cell type enrichments, and finishing with histological images and their corresponding free-text descriptions. We make four specific contributions. First, we built computational models based on gene expression of post-mortem human tissues in order to derive estimates of post mortem interval. Second, we redefined the basic histological types of tissue classification based on five broad transcriptional programs which define major cell types: epithelial, endothelial, mesenchymal, neural, and blood. We generated computational estimates for the enrichment of these major cell types and validated them through the analysis of histological images and free-text pathology reports, finding that departures from normal cellular enrichment correlate with disease-associated histological phenotypes. Third, we characterized the landscape of human sex-differential gene expression, finding that effects are small but ubiquitous and tend to be tissue-specific, with some of these genes being involved in biological and molecular functions related to disease and clinical phenotypes. Fourth, we proposed an in-silico methodology to spatially deconvolute gene expression from matched sample pairs of whole slide histological images and bulk RNA-seq gene expression, with the goal of replicating the spatial transcriptomics experimental technology. Within this study, we also developed a software tool to effortlessly process whole slide histological images into tiles for machine learning applications.Los conjuntos de datos genómicos son fundamentales para ampliar nuestra comprensión de la biología humana en el contexto de la salud y la enfermedad. Sin embargo, la alta dimensionalidad de la expresión génica y otros rasgos moleculares constituye un desafío para vincular estos tipos de datos con fenotipos humanos. Esta tesis se apoya en herramientas de aprendizaje estadístico para mitigar el problema de la dimensionalidad y vincular el transcriptoma humano con fenotipos a diferentes niveles de complejidad: desde el ARN, pasando por enriquecimientos de tipos celulares inferidos computacionalmente, y terminando con imágenes histológicas y sus correspondientes anotaciones en formato de texto libre. Hacemos cuatro aportaciones específicas. Primero, construimos modelos computacionales basados en la expresión génica de tejidos humanos post-mortem para realizar estimaciones del intervalo post-mortem. En segundo lugar, redefinimos los tipos histológicos básicos de clasificación de tejidos con base en cinco amplios programas transcripcionales que definen tipos celulares principales: epitelial, endotelial, mesenquimal, neural y sanguíneo. Generamos estimaciones computacionales para el enriquecimiento de estos tipos celulares principales y las validamos mediante el análisis de imágenes histológicas e informes de patología en formato de texto libre, encontrando que las desviaciones respecto a la normalidad en los enriquecimientos celulares correlacionan con fenotipos histológicos asociados con enfermedades. En tercer lugar, caracterizamos el panorama de la expresión diferencial de los genes respecto al sexo en humanos, y descubrimos que los efectos son pequeños pero ubicuos y tienden a ser específicos al tejido, con algunos de estos genes involucrados en funciones biológicas y moleculares relacionadas con enfermedades y fenotipos clínicos. En cuarto lugar, hemos propuesto una metodología in-silico para deconvolucionar espacialmente la expresión génica a partir de muestras emparejadas de imágenes histológicas y expresión génica (bulk RNA-seq), con el objetivo de replicar la tecnología experimental de transcriptómica espacial. Dentro de este estudio, también desarrollamos una herramienta de software para procesar imágenes histológicas y generar mosaicos de imágenes para aplicaciones de aprendizaje automático.
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FANTINI, VALENTINA. "Functional analysis and transcriptome profile of meninges and skin fibroblasts from human aged donors." Doctoral thesis, Università degli studi di Pavia, 2021. http://hdl.handle.net/11571/1446317.
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Noël, Floriane. "Systems Level Analysis of Immune Cell Subsets and Intercellular Communication Networks in Human Breast Cancer." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS418/document.
Повний текст джерелаАнотація:
La communication intercellulaire est à la base de l'organisation d'ordre supérieur observée dans les tissus, les organes et l'organisme. Comprendre la communication intercellulaire et ses mécanismes sous-jacents qui sont impliqués dans le cancer est essentiel. Le microenvironnement des tumeurs du sein est composé d'une grande diversité cellulaire, telle que les cellules endothéliales, stromales ou immunitaires, qui peuvent influencer la progression tumorale ainsi que la réponse au traitement. Parmi les différentes populations de cellules immunitaires, les sous-populations de cellules dendritiques (DCs) intègrent les signaux du microenvironnement puis joue un rôle critique en orchestrant le développement d’une réponse immunitaire spécifique par activation des lymphocytes T. Cependant, les différentes fonctions de ces sous-populations et leurs interactions au sein du microenvironnement tumoral restent mal décrites. L’objectif principal de ma thèse a été de comprendre l'impact du microenvironnement tumorale du sein sur les sous-populations de DCs par analyse systémique. Nous avons utilisé le séquençage de l'ARN pour analyser systématiquement les transcriptomes des pré-DC plasmacytoïdes infiltrant les tumeurs (pDC), les populations cellulaires enrichies pour les DC classiques de type 1 (cDC1e), les DC classiques de type 2, les DC CD14+ et les monocytes-macrophages chez des patientes atteintes de cancer primitif du sein luminal et cancer du sein triple négatif. Nous avons constaté que la reprogrammation transcriptionnelle des cellules présentatrices d’antigène infiltrant la tumeur est spécifique à un sous-ensemble. Ces résultats suggèrent une interaction complexe entre l'ontogenèse et l'empreinte tissulaire dans le conditionnement de la diversité des DCs et de leur fonction dans le cancer.En second lieu, j'ai cherché à étudier les communications intercellulaires afin de comprendre comment les cellules intègrent les signaux de leur environnement. Nous avons développé ICELLNET, un outil pour reconstruire les réseaux de communication intercellulaires. Cette méthode quantitative originale, intégrant les interactions ligand-récepteur et l'expression génique spécifique à un type cellulaire, peut être appliquée automatiquement à tous profils transcriptomiques de population cellulaire, que ce soit dans divers contextes pathologiques ou d’autres domaines de la biologieCell-to-cell communication is at the basis of the higher order organisation observed in tissues, organs, and organism. Understanding cell-to-cell communication, and its underlying mechanisms that drive the development of cancer is essential. Breast tumor microenvironment (TME) is composed of a great cellular diversity, such as endothelial, stromal or immune cells that can influence tumor progression as well as its response to treatment. Among the different immune cell populations, dendritic cells (DCs) subsets integrate signals from their microenvironment and are subsequently essential in orchestrating specific immune response through T cell activation. However, the differential function of these subsets, and their interactions within the TME remain poorly described. My main thesis objective was to understand the impact of the breast TME on DC subsets using systems-level analysis. We used RNA sequencing to systematically analyze the transcriptomes of tumor-infiltrating plasmacytoid pre-DCs (pDCs), cell populations enriched for type 1 classical DCs (cDC1e), type 2 classical DCs (cDC2s), CD14+DCs, and monocytes-macrophages from human primary luminal breast cancer and triple-negative breast cancer. We found that transcriptional reprogramming of tumor-infiltrating antigen-presenting cells is subset-specific. These results suggest a complex interplay between ontogeny and tissue imprinting in conditioning DC diversity and function in cancer.As a second objective, I aimed at studying the cellular communications in order to understand how cells integrate signals from their environment. I developed ICELLNET, a tool to reconstruct intercellular communication networks. This original quantitative method, integrating ligand-receptor interactions and cell type specific gene expression, can be automatically applied to any cell population level transcriptomic profile opening perspectives of application in several disease contexts and biology fields
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Kelso, Janet. "The development and application of informatics-based systems for the analysis of the human transcriptome." Thesis, University of the Western Cape, 2003. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_5101_1185442672.
Повний текст джерелаАнотація:
Despite the fact that the sequence of the human genome is now complete it has become clear that the elucidation of the transcriptome is more complicated than previously expected. There is mounting evidence for unexpected and previously underestimated phenomena such as alternative splicing in the transcriptome. As a result, the identification of novel transcripts arising from the genome continues. Furthermore, as the volume of transcript data grows it is becoming increasingly difficult to integrate expression information which is from different sources, is stored in disparate locations, and is described using differing terminologies. Determining the function of translated transcripts also remains a complex task. Information about the expression profile &ndash
the location and timing of transcript expression &ndash
provides evidence that can be used in understanding the role of the expressed transcript in the organ or tissue under study, or in developmental pathways or disease phenotype observed.
In this dissertation I present novel computational approaches with direct biological applications to two distinct but increasingly important areas of research in gene expression research. The first addresses detection and characterisation of alternatively spliced transcripts. The second is the construction of an hierarchical controlled vocabulary for gene expression data and the annotation of expression libraries with controlled terms from the hierarchies. In the final chapter the biological questions that can be approached, and the discoveries that can be made using these systems are illustrated with a view to demonstrating how the application of informatics can both enable and accelerate biological insight into the human transcriptome.
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Clark, Katherine. "Transcriptome- and proteome-wide responses to putrescine depletion in the human malaria parasite, Plasmodium falciparum." Thesis, University of Pretoria, 2013. http://hdl.handle.net/2263/30795.
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Stefani, Maurizio. "The effect of age, sex, and end-stage heart failure on the human cardiac transcriptome." Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/13144.
Повний текст джерелаАнотація:
This thesis aimed to describe the transcriptomic changes in the human left ventricle as a result of age and gender, and to relate these changes to the pathophysiology of heart failure, in order to understand why ageing and gender modifies our susceptibility to, and progression of heart failure. Many genes were discovered to be affected by age and gender, in particular, the set of genes that code for the r-proteins, i.e. ribosomal subunit coding proteins, are downregulated with age, and are expressed at a lower level in males. I hypothesised that this may compromise the ability of myocardium to engage in necessary hypertrophy/hyperplasia to recover from a myocardial insult such as a myocardial infarction, and thus increases the risk of heart failure developing. However, no evidence of changes in nucleolar abundance, the site of ribosome synthesis, was found with age and gender. In heart failure of a variety of aetiologies, the expression of r-protein mRNA and 45s rRNA was found to be reduced, and the reduction of r-protein mRNA expression was ameliorated by left ventricular assist device support. A reduction in nucleolar abundance was also demonstrated in certain aetiologies of heart failure. I hypothesised that this may be due to an energy shortage in heart failure, partly ameliorated by left ventricular assist device support, leading to a reduction in energetically costly ribosome synthesis which is pathological in the long term.
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Oey, Harald Motzfeldt. "Non-coding Regions of the Human Transcriptome; Incidence and Potential Relevance of Selected Sequence Insertions." Thesis, Griffith University, 2007. http://hdl.handle.net/10072/366366.
Повний текст джерелаАнотація:
The human genome is made up of vast and complex deoxyribonucleic acid (DNA) molecules whose function, complexity and intellectual beauty we are barely beginning to understand. It is composed of about 3 billion base pairs, each connected to the next by single covalent bonds. Of all these nucleotides, only ~2% code for the basic building-blocks of organic life, the protein molecules. Much of the remaining DNA is seemingly without any purpose. The main cause of the large size of the human genome are insertions and expansions of repetitive DNA sequences. There are many different types of such repetitive DNA but the retroposons stand out due to their ability to be transcribed, reverse transcribed and subsequently reinserted back into the genome in new and seemingly random locations. The genome therefore accumulates an ever increasing number of such retroposon copies and the most numerous of the human retroposons, the Alu repeat, has more than 1 million copies, comprising 10% of the human genome. Once thought to be “selfish” autonomous elements inserting themselves at random in areas where they cause only a minor “discomfort” to their surroundings, Alu repeats are now assigned ever more functions and potential functions. In recent years this has culminated in the realization that an, until recently, obscure ribonucleic acid (RNA) editing enzyme, the adenosine deaminase acting on RNA (ADAR), edits these elements in their thousands in precursor-mRNA (pre-mRNA) for as yet unknown reasons. Another interesting feature of the Alu repeats, and the one that makes them such attractive targets for ADARs, is the fact that most of the 1 million Alu repeats have significant base pairing potential towards other opposite-sense Alu repeats. They are also found frequently inside genes providing abundant potential for formation of secondary structures in otherwise single-stranded gene transcripts. In this report a number of approaches are described that ultimately aim to uncover potential functions and evolutionary significance of Alu repeats, and certain other types of repetitive DNA. Towards that end, a number of genes of clinical significance, which are distinguished by their high content of Alu repeats, are investigated for potential to form secondary structures that may influence their expression. These genes include the insulin receptor and the low density lipoprotein receptor. An analysis of some gene groups and gene families with respect to their Alu content is also provided in which a pattern seems to emerge whereby Alu repeats may be involved in tissue-specific expression of different subunit isoforms involved in the mitochondrial electron transport chain. The genes coding for the electron transport chain are also investigated for their ability to form pseudogenes, which is another type of repetitive DNA that may be of functional significance. A large survey of Alu repeat pairs across exons and immediately flanking exons is also presented. That survey confirms a number of features regarding Alu distribution in genes and in the human genome in general that have been documented by others in the past. Significantly, it also shows that there is a difference in the distribution of inverted and direct Alu repeat pairs when these are intervened by an exon. The difference favors the direct Alu repeat pairs over that of the inverted Alu repeat pairs. Such a difference may well be caused by the general ability of inverted Alu repeats to form secondary structures. Such structures are hypothesized to be disruptive to a gene, and thus be selected against. However, they could also represent a possible mechanism for regulation of gene expression and may therefore be of clinical importance. Significantly, the regions where these elements are found are not usually investigated in genes suspected of causing a genetic disorders, and they may therefore potentially explain some disorders where mutations in exons, splice sites or promoters can not be found. Despite the extensive bioinformatics searches performed in the course of the work it would be surprising if there were not many more potential functions still to be discovered.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Full Text
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Venkatesh, Geetha [Verfasser]. "A systematic analysis of the genetic influence on the transcriptome in human longevity / Geetha Venkatesh." Kiel : Universitätsbibliothek Kiel, 2018. http://d-nb.info/115376847X/34.
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Schulz, Heidi. "Towards a comprehensive description of the human retinal transcriptome identification and characterization of differentially expressed genes /." Doctoral thesis, [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=970411316.
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Kindermann, Birgit. "Effects of zinc on the transcriptome and proteome of human colonic cancer cells (HT-29 cells)." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974900648.
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Guauque, Sandra Milena. "The transcriptome of human epicardial, mediastinal and subcutaneous adipose tissues in men with coronary artery disease." Thesis, Université Laval, 2011. http://www.theses.ulaval.ca/2011/28083/28083.pdf.
Повний текст джерелаАнотація:
Le tissu adipeux épicardique (TAE) est localisé à la surface du cœur en contact avec les artères coronaires, ce qui suggère un rôle dans la pathogénèse de la maladie coronarienne. Les objectifs de cette étude étaient d’identifier les gènes différentiellemment régulés entre les tissus graisseux épicardique, médiastinal et sous-cutané à l’aide des biopuces d’ADN et d’étudier leurs rôles dans le développement des maladies cardiovasculaires. Les résultats ont montré une grande similarité d’expression entre les tissus adipeux épicardique et médiastinal. Toutefois, certains gènes impliqués dans les maladies cardiovasculaires étaient régulés différemment entre ces deux tissus. L’expression des gènes codant pour le récepteur A1 de l’adénosine (ADORA1) et la prostaglandine D2 synthase (PTGDS), impliqué dans les ischémies myocardiques et la progression de l’athérosclérose, respectivement, était significativement élevée dans le TAE. Cette étude est une première étape pour comprendre le rôle biologique du TAE et ses implications dans les maladies cardiovasculaires.Increased visceral adipose tissue has been associated with the development of cardiovascular diseases (CVD). Epicardial adipose tissue (EAT) is the visceral fat depot located on the surface of the heart especially around the epica rdial coronary vessels with extension into the myocardium. The proximity of EAT to the coronary arteries suggests a role in the pathogenesis of coronary artery disease (CAD). EAT thickness was significantly correlated with the severity of CAD. However, the biological functions of EAT and its relationship with the development of CVD remain largely elusive. The objectives of this study were to identify genes that were up- or down-regulated among three distinct adipose tissues, namely EAT, mediastinal and subcutaneous using whole-genome gene expression microarrays and to study the possible relationships of these genes with the development of CVD. Overall, the transcriptional profiles of EAT and mediastinal adipose tissue were similar compared to subcutaneous adipose tissue. Despite this similarity, a number of genes involved in cardiovascular diseases were up-regulated in EAT. The expression of the adenosine A1 receptor (ADORA1), involved in myocardial ischemia, was significantly up-regulated in EAT. Levels of the prostaglandin D2 synthase (PTGDS) gene, recently associated with the progression of atherosclerosis, were significantly different in the three pairwise comparisons (epicardial > mediastinal > subcutaneous). Overexpression of ADORA1 and PTGDS in EAT may confer cardioprotection against myocardial ischemia and CAD. This study is an important first step to understand the biological function of EAT and its potential implications in CVD.
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Howard, Lynsey. "Analysis of the transcriptome : investigation of human embryonic stem cells during directed differentiation to cardiovascular lineages." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4068/.
Повний текст джерелаАнотація:
To date, the need for effective treatments to tackle ischaemic diseases such as CHD and PAD remains unmet. As such, there has been a great deal of interest in developing cell therapies in order to address these important pathologies. The main goals of a cell therapy strategy for ischaemic disease remain prompt restoration of blood supply to the affected areas in order to salvage tissue and/or regeneration of tissues previously lost to ischaemia. Derived from the Inner Cell Mass (ICM) of an embryo at the blastocyst stage, hESC have been proposed as a potential source of functional, transplantable cells for a variety of cell therapy applications. Although successful differentiation of multiple cell types from hESC has been demonstrated, the molecular processes governing the cell commitment process remain poorly understood, and differentiation efficiency often fails to provide the number of cells required to see clinical benefit in the patient. As such, a more thorough transcriptional characterisation of cardiovascular cell types derived from hESC was the goal of this study. MicroRNAs (miRNA; miR) are small (~22nt), non-coding RNAs which negatively regulate mRNA. MiR-1 and miR-133 were previously shown to play a role in regulating cardiac differentiation with miR-1 potentiating cardiac differentiation and miR-133 having an inhibitory effect. Optimisation of lentiviral vectors showed generation of single pre-miR overexpression lentiviruses for miR-1 and miR-133 in a construct using the SFFV promoter to be possible. Furthermore, it was realised SA461 hESC were unsuitable for cardiac differentiation, however, using a modified version of the LaFlamme protocol in a monolayer system resulted in beating cells with a cardiomyocyte phenotype in H1 hESC. Despite successful overexpression of miR-1 and miR-133, there was very little effect on cardiac differentiation over no virus control. Previously published methods for the generation of vascular endothelial cells (EC) have reported varying efficiency and target cell population purity (~3 – 30%). This laboratory recently reported the successful generation of functional EC-like cells from hESC in a feeder-free manner. HESC-EC were analysed by LC Sciences miRNA microarray at early time points day 0, day 2, day 4 and day 10 after initiation of differentiation with time-matched pluripotent controls. An induction of miR-99b, -181a and -181b over time was observed, and validated in H1 hESC. In addition, miR-99b, -181a and -181b were also found to be expressed in other mesodermal cell types including adult human saphenous vein endothelial cells (HSVEC). No statistically significant expression of these miRNAs could be found in representative cell types of ectoderm and endoderm germ layers, therefore it was hypothesised that these miRNAs were largely mesoderm specific. Despite initial data showing a significant difference in expression between HSVEC from control patients and patients undergoing coronary artery bypass grafting (CABG), classical pathophysiological stimuli to cause endothelial cell stress did not change the expression of miR-99b, -181a and -181b in vitro. In order to understand more about gene expression in early lineage commitment, hESC-EC were analysed by Illumina microarray at early timepoints day 0, day 2, day 4 and day 10 after initiation of differentiation with time-matched pluripotent controls. In parallel, primary human saphenous vein endothelial cells (HSVEC) were analysed. Illumina technology permitted whole-genome profiling in a high throughput chip format. Due to overall expression levels being lower intensity than expected, no cut-off of fold-change was applied to the dataset. Analysis of the dataset showed a large number of significantly differentially expressed probes at each time point: Day 2 of endothelial differentiation compared to Day 0 pluripotent control showed 1040 significant differentially expressed probe changes, Day 4 of endothelial differentiation compared to Day 0 pluripotent control showed 2400 significant differentially expressed probe changes and Day 10 of endothelial differentiation compared to Day 0 pluripotent control showed 2157 significant differentially expressed probe changes (all False Discovery Rate <0.05). Although significant downregulation of pluripotency markers were observed, few endothelial associated genes were present at hESC-EC day 10. Analysis of HSVEC compared to hESC-EC Day 10 reveals 6133 significantly differentially expressed probes (FDR <0.05). This suggests that although day 10 hESC-ECs have previously been shown satisfy criteria for endothelial cells in vitro and in vivo, their transcriptional profiling demonstrates that they remain different in comparison to adult ECs. A transient induction of several transcription factors was observed at hESC-EC day 2, accounting for some 10% of gene changes at this time point. We hypothesised that these transcription factors may play key roles in the early mesoderm/EC commitment process. Of these, FOXA2, a transcription factor not previously associated with mesoderm or EC commitment, was upregulated, and this was further validated by both qRT-PCR and ICC in SA461, H1 and RC10 hESC lines. In addition to gene expression data, an in silico prediction of gene epigenetic status was made using a previously published chromatin immunoprecipitation sequencing (ChIP-SEQ) dataset performed in H9 hESC. Approximately 3000 genes are bivalently marked, meaning they have both H3K4me3 active chromatin and H3K27me3 repressive chromatin at their transcriptional start sites (TSS). This conveys a poised state, with the potential for the gene to be rapidly activated and/or repressed. Of these bivalent genes it was noted that FOXA2 was marked as being potentially bivalent. Upon further investigation using ChIP it was revealed that FOXA2 carried both H3K4me3 and H3K27me3 chromatin modifications in the TSS region, both in H9 and SA461 pluripotent hESC. It was hypothesised that epigenetic modification was responsible for the dynamic expression of FOXA2, although this hypothesis remains to be investigated further. Lastly, several of the miRNA targets for miR-99b, miR-181a and miR-181b were downregulated by hESC-EC day 10 compared to hESC-EC day 0, although whether these targets play a role in refining differentiation to EC warrants further investigation. In summary, a range of molecular biology techniques were employed to investigate the master control of hESC differentiation. These studies have contributed to existing knowledge on mesodermal and cardiovascular lineage specification. They provide evidence to support the continued in depth investigation of these processes in order to develop a clinically relevant cell therapy for ischaemic diseases.
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Guauque-Olarte, Sandra. "The transcriptome of human epicardial, mediastinal and subcutaneous adipose tissues in men with coronary artery disease." Master's thesis, Université Laval, 2011. http://hdl.handle.net/20.500.11794/22497.
Повний текст джерелаАнотація:
Le tissu adipeux épicardique (TAE) est localisé à la surface du cœur en contact avec les artères coronaires, ce qui suggère un rôle dans la pathogénèse de la maladie coronarienne. Les objectifs de cette étude étaient d’identifier les gènes différentiellemment régulés entre les tissus graisseux épicardique, médiastinal et sous-cutané à l’aide des biopuces d’ADN et d’étudier leurs rôles dans le développement des maladies cardiovasculaires. Les résultats ont montré une grande similarité d’expression entre les tissus adipeux épicardique et médiastinal. Toutefois, certains gènes impliqués dans les maladies cardiovasculaires étaient régulés différemment entre ces deux tissus. L’expression des gènes codant pour le récepteur A1 de l’adénosine (ADORA1) et la prostaglandine D2 synthase (PTGDS), impliqué dans les ischémies myocardiques et la progression de l’athérosclérose, respectivement, était significativement élevée dans le TAE. Cette étude est une première étape pour comprendre le rôle biologique du TAE et ses implications dans les maladies cardiovasculaires.Increased visceral adipose tissue has been associated with the development of cardiovascular diseases (CVD). Epicardial adipose tissue (EAT) is the visceral fat depot located on the surface of the heart especially around the epica rdial coronary vessels with extension into the myocardium. The proximity of EAT to the coronary arteries suggests a role in the pathogenesis of coronary artery disease (CAD). EAT thickness was significantly correlated with the severity of CAD. However, the biological functions of EAT and its relationship with the development of CVD remain largely elusive. The objectives of this study were to identify genes that were up- or down-regulated among three distinct adipose tissues, namely EAT, mediastinal and subcutaneous using whole-genome gene expression microarrays and to study the possible relationships of these genes with the development of CVD. Overall, the transcriptional profiles of EAT and mediastinal adipose tissue were similar compared to subcutaneous adipose tissue. Despite this similarity, a number of genes involved in cardiovascular diseases were up-regulated in EAT. The expression of the adenosine A1 receptor (ADORA1), involved in myocardial ischemia, was significantly up-regulated in EAT. Levels of the prostaglandin D2 synthase (PTGDS) gene, recently associated with the progression of atherosclerosis, were significantly different in the three pairwise comparisons (epicardial > mediastinal > subcutaneous). Overexpression of ADORA1 and PTGDS in EAT may confer cardioprotection against myocardial ischemia and CAD. This study is an important first step to understand the biological function of EAT and its potential implications in CVD.
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BOCCHI, VITTORIA. "THE CODING AND NON-CODING TRANSCRIPTOME OF THE HUMAN FETAL STRIATUM FROM A SINGLE-CELL PERSPECTIVE." Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/631915.
Повний текст джерелаАнотація:
The human brain is a tissue of vast complexity in terms of the cell types it comprises. Understanding how this complexity arises requires a deep understanding of how neuronal patterning progresses at the single cell level. Previous studies have concentrated on cell fate decisions during cortical development but little is known about how the lateral ganglionic eminences (LGE) develops and gives rise to the different cell populations of the striatum. Conventional approaches to classify cell types in this area have been limited to exploring relatively few markers and therefore have provided a narrow characterization of any given cell type. Furthermore, most studies have bounded their inquiries to protein-coding genes and have not investigated the role of lncRNAs that have been shown to have a high cell and tissue specificity.
Taking these aspects under consideration, here we combined bulk RNA-seq and single-cell RNA-seq to decode an unambiguous gene signature of the striatum and reveal how neural progenitors of this domain are able to differentiate at the single cell level. In particular, we deeply profiled the LGE and the surrounding neocortex and medial ganglionic eminences (MGE), from 7 to 20 postconceptional weeks (pcw), and performed de novo lncRNAs analysis that enabled us to define the first dictionary of novel lincRNAs for these areas. Furthermore, this analysis led to the establishment of a unique gene signature for the three different regions. Subsequently, we performed single-cell RNA-seq of the LGE at 7pcw that unravelled a plethora of different cell populations of the LGE. Pseudotemporal ordering of these cells uncovered the first developmental trajectory of striatal neurons and how they transition from early progenitors to mature medium spiny neuron (MSNs) and their coding and non-coding transcriptional signature. This array of cells will shortly be complemented by another batch of single-cell libraries from later time points (9-11pcw) that will be used to full characterize the early steps of neural ramifications that lead to the generation of the human striatum.
The relevance of the approach relies on the availability of extremely rare human fetal samples combined with the most revolutionary RNA sequencing technologies and highly elaborate computational tools that enable the investigation of a brain region, the striatum, whose development is poorly understood and which plays a major role in human brain physiology and pathology.
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Albrecht, Marco [Verfasser], and Lauster [Akademischer Betreuer] Roland. "Global Transcriptome Analysis of the Human Pathogens Chlamydia trachomatis and Chlamydia pneumoniae / Marco Albrecht. Betreuer: Lauster Roland." Berlin : Universitätsbibliothek der Technischen Universität Berlin, 2011. http://d-nb.info/1018072705/34.
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Aledani, Tamadir Hamid Wadi. "Expression des ARNm et des microARN dans les cellules de cumulus humains : impact de l'âge maternel." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTT011/document.
Повний текст джерелаАнотація:
L'ovocyte se développe au sein d'un follicule, en contact étroit avec des cellules d'origine somatique, les cellules de cumulus (CC). Ces deux types cellulaires communiquent entre eux via des jonctions intercellulaires, permettant ainsi la régulation et la coordination du métabolisme pendant le développement et la maturation de l'ovocyte. Notre hypothèse est que l'expression et la régulation des gènes dans les CC joue un rôle crucial dans des fonctions essentielles pour la croissance de l'ovocyte et l'acquisition de sa compétence. Mes travaux de thèse comportent deux parties. Dans la première partie nous avons utilisé le séquençage haut débit pour examiner le répertoire des microARN (communément appelés miRNA) dans les cellules de cumulus et dans l'ovocyte. Les miRNA, séquences d'ARN non codantes dont la longueur varie entre 19 et 25 nucléotides, ont émergé récemment comme régulateurs majeurs de nombreux processus biologiques, dont le vieillissement. Nous avons identifié 32 miRNA spécifiquement dans les cellules de cumulus humains et seulement 3 dans l'ovocyte MII. Dans la seconde partie de nos travaux, nous avons analysé l'impact de l'âge maternel sur l'expression des gènes dans les cellules de cumulus. Alors qu'une baisse de la compétence de l'ovocyte avec l'avancement de l'âge maternel est bien établie, les bases moléculaires de ce phénomène demeurent peu connues. Dans une première étape pour aborder cette question, nous avons utilisé des puces à ADN pour analyser les profils d'expression des gènes des CC en fonction de l'âge maternel. De façon remarquable l'âge maternel impacte significativement l'expression de gènes qui sont critiques pour la maturation de l'ovocyte tels que les gènes impliqués dans l'angiogenèse, les voies de signalisation de TGF-ß et de l'insuline. Par l'utilisation d'outils bioinformatiques, nous avons aussi identifié des miRNA potentiels régulateurs de gènes impliqués dans des processus ou des voies impactés par l'âge ; ils pourraient constituer de nouveaux biomarqueurs pour prédire un vieillissement ovarien prématuré ainsi que la qualité et la compétence de l'ovocyteThe oocyte develops into a follicle where it is in close contact with cumulus cells (CCs), of somatic origin. The two cell types undergo a bidirectional communication via gap junctions, which results in the regulation and coordination of the metabolism during oocyte development and maturation. We assume that gene expression and regulation in the CCs play a crucial role in functions that are essential for oocyte growth and competence acquisition. The present study may be subdivided in two parts. In the first part we used deep sequencing to investigate the repertoire of miRNAs in the cumulus cells and the oocyte. MicroRNAs that are noncoding RNA sequences whose length is approximately 19-25 nucleotides have emerged as important regulators in many biological processes including aging. Our data showed that 32 miRNAs were specifically expressed in human cumulus cells while only 3 miRNAs were identified in MII human oocyte. The impact of maternal age on gene expression in cumulus cells was addressed in a second part of my thesis work. While the correlation of oocyte competence decline with advancing maternal age is well established, little is known on its molecular basis. In a first attempt to address this issue, we used microarrays to study gene expression profiles of human cumulus cells according to maternal age. Remarkably, maternal age greatly impacted expression of genes that are critical for oocyte maturation such as genes involved in angiogenesis, TGF-β signaling, and insulin signaling pathways. Also, using bioinformatic tools, we identified miRNAs that potentially target some of the genes involved in the aging-impacted processes and pathways; this could candidate them as new biomarkers to predict premature ovarian aging and oocyte quality and competence
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Guha, Rusha. "Integrated analysis of mRNA and miRNA in human differentiating muscle cells." Doctoral thesis, Università degli studi di Padova, 2014. http://hdl.handle.net/11577/3423841.
Повний текст джерелаАнотація:
Muscles are responsible for the movement of body and take up roughly half of the person’s body weight. Skeletal muscles are the only voluntary muscle tissue in human body, being controlled consciously.
Skeletal muscle cells form when many smaller progenitor cells lump themselves together to constitute long, straight, multinucleated fibers. The proliferating muscle cells are called myoblasts. Myoblasts fuse together to form multinucleated non-proliferating cells called myotubes. The transition from proliferative to differentiated state involves a complete shift of the cell’s transcriptome based on a network of regulation at the molecular level. To uncover the intricacies of molecular activities involved in the process of muscle differentiation it is essential to have deeper and through understanding of the transcriptome in its entirety. RNA seq, which is a high through put sequencing technology, gives us the opportunity to reach into the deepest level of the transcriptome.
By means of the RNA seq technology I obtained the in-depth view of the transcriptome of human muscle cells from the proliferative to the differentiated stage. The analysis was extended also to small RNAs, to have a full picture of the transcriptome. I performed the analysis with the specific objectives of identifying the expressed genes and finding out differential gene expression, of both mRNA and miRNA. I also investigated the crosstalk between mRNA and miRNA, employing two separate methods : miRNA over expression and Ago2 immunoprecipitation followed by RNA seq.
The over expression experiments were carried out with 5 different miRNAs and in all cases I found more genes turned up than turned down. These results suggest that miRNA might either have a role in mRNA stabilization or it could play a part in a double negative mechanism by inhibiting some negative factor. By comparing the genes down regulated after miRNA over expression with Ago2-enriched genes we have found several candidate genes which are most likely under the down regulatory control of miRNA.
Skeletal muscle has enormous plasticity and can endure a lot of stress. To study this aspect of muscle we performed mechanical stretch of differentiating muscle cells. With RNA seq we got the in-depth view of the transcriptome as a response to stretch. We performed the analysis at two time points after stretch and found that stretch triggered immune response genes soon after, but enhanced muscle structural-protein genes expression over a prolonged course of time when the immune response takes a back seat.
I believe that our thorough transcriptome analysis, including miRNA and mRNA interaction studies during myogenesis, contributes towards the better understanding of the process regulating muscle developmentI muscoli sono responsabili dei movimenti del corpo e costituiscono circa metà del peso di una persona. I muscoli scheletrici sono i soli tessuti muscolari volontari, essendo controllati coscientemente. Le cellule del muscolo scheletrico si formano quando diverse piccole cellule progenitrici si conglobano tra loro per formare lunghe affusolate fibre multinucleate. Le cellule muscolari proliferanti sono chiamate mioblasti. I mioblasti si fondono tra loro per formare cellule multinucleate e non proliferanti, chiamate miotubi. La transizione dallo stato proliferante a quello differenziato implica un completo cambiamento del trascrittoma cellulare, basato su una rete di regolazione a livello molecolare. Per scoprire il groviglio di attività molecolari implicate nel processo di differenziamento muscolare è essenziale avere una più profonda ed estesa comprensione del transcrittoma nella sua interezza. L'RNA seq è una tecnologia di sequenziamento massivo che ci offre l'opportunità di accedere ai livelli più approfonditi del trascrittoma. Con la tecnologia dell'RNA seq ho ottenuto una precisa visione del trascrittoma delle cellule muscolari umane, sia allo stadio proliferativo che a quello differenziato. Per avere una visione completa del trascrittoma, l'analisi è stata estesa anche agli small RNA. Ho svolto queste analisi con l'obiettivo specifico di identificare i geni espressi e di evidenziare in particolare quelli differenzialmente espressi, sia per quanto riguarda gli mRNA che i miRNA. Ho anche analizzato il crosstalk tra mRNA e miRNA, impiegando due diversi metodi: sovraespressione dei miRNA e immunoprecipitazione di Ago2, seguita da RNA seq. Gli esperimenti di sovraespressione sono stati condotti con 5 diversi miRNA e in tutti i casi ho trovato più geni che hanno aumentato il loro livello piuttosto di geni che l'hanno diminuito. Questi risultati suggeriscono che i miRNA potrebbero avere un ruolo nella stabilizzazione degli mRNA, oppure potrebbero avere un ruolo in un doppio meccanismo negativo, inibendo a loro volta fattori negativi. Confrontando i geni che diminuiscono di livello dopo la sovraespressione di miRNA con i geni arricchiti dall'immunoprecipitazione con Ago2, abbiamo trovato diversi geni candidati per essere sotto il controllo inibitorio dei miRNA. Il muscolo scheletrico ha una grande plasticità e può sopportare un notevole stress. Per studiare questo aspetto del muscolo abbiamo sottoposto le cellule in differenziamento a stiramento meccanico. Con l'RNA seq abbiamo ottenuto un'approfondita visione del trascrittoma in risposta allo stiramento. Abbiamo effettuato queste analisi a due diversi tempi dopo lo stiramento ed abbiamo trovato che nel periodo immediatamente successivo allo stimolo vengono sovraespressi geni implicati nella risposta immunitaria, mentre successivamente sono attivati i geni codificanti proteine muscolari strutturali, quando allo stesso tempo la risposta immunitaria viene inibita. Sono convinta che la nostra approfondita analisi del trascrittoma che include l'interazione di mRNA e miRNA durante la miogenesi, possa contribuire ad una maggiore comprensione di processi che regolano lo sviluppo muscolare
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Fagerberg, Linn. "Mapping the human proteome using bioinformatic methods." Doctoral thesis, KTH, Proteomik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-31477.
Повний текст джерелаАнотація:
The fundamental goal of proteomics is to gain an understanding of the expression and function of the proteome on the level of individual proteins, on the level of defined cell types and on the level of the entire organism. In this thesis, the human proteome is explored using membrane protein topology prediction methods to define the human membrane proteome and by global protein expression profiling, which relies on a complex study of the location and expression levels of proteins in tissues and cells. A whole-proteome analysis was performed based on the predicted protein-coding genes of humans using a selection of membrane protein topology prediction methods. The study used a majority decision-based method, which estimated that approximately 26% of the human genes encode for a membrane protein. The prediction results are displayed in a visualization tool to facilitate the selection of antigens to be used for antibody generation. Global protein expression profiles in a large number of cells and tissues in the human body were analyzed for more than 4000 protein targets, based on data from the antibody-based immunohistochemistry and immunofluorescence methods within the framework of the Human Protein Atlas project. The results revealed few cell-type specific proteins and a high fraction of human proteins expressed in most cells, suggesting that cell and tissue specificity is attained by a fine-tuned regulation of protein levels. The expression profiles were also used to analyze the relationship between 45 cell lines by hierarchical clustering and principal component analysis. The global protein expression patterns overall reflected the tumor origin of the cells, and also allowed for identification of proteins of importance for distinguishing different categories of cell lines, as defined by phenotype of progenitor cell. In addition, the protein distribution in 16 subcellular compartments in three of the human cell lines was mapped. A large fraction of proteins were localized in two or more compartments and, in line with previous results, a majority of proteins were detected in all three cell lines. Finally, mass spectrometry-based protein expression levels were compared to RNA-seq-based transcript expression levels in three cell lines. Highly ubiquitous mRNA expression was found and the changes of expression levels between the cell lines showed high correlations between proteins and transcripts. Large general differences in abundance of proteins from various functional classes were observed. A comparison between categories based on expression levels revealed that, in general, genes with varying expression levels between the cell lines or only expressed in one cell line were highly enriched for cell-surface proteins. These studies show a path for a systematic analysis to characterize the proteome in human cells, tissues and organs.QC 20110317
The Human Protein Atlas project
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ITALIANI, PAOLA. "DEFINITION OF AN IN VITRO MODEL OF HUMAN MONOCYTE ACTIVATION REPRESENTATIVE OF THE DEFENSIVE INFLAMMATORY RESPONSE." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/217442.
Повний текст джерелаАнотація:
La reazione di difesa innata/infiammatoria è attivata in risposta a patogeni esterni o a segnali provenienti dal tessuto danneggiato. I monociti/macrofagi hanno un ruolo chiave nell’inizio e risoluzione della infiammazione per mezzo di differenti programmi di attivazione. Infatti i macrofagi possono adottare in vivo una varietà di fenotipi diversi che dipendono dai cambiamenti del microambiente tissutale, esibendo un continuum di stati funzionali diversi. Inoltre i monociti del sangue periferico non sono una popolazione omogenea ma differiscono nei loro fenotipi e funzioni. Nonostante l’esplosivo aumento di informazioni sull’argomento, molte questioni sono ancora aperte riguardo la caratterizzazione fenotipica e funzionale dei monociti/macrofagi, e il loro ruolo durante l’omeostasi e l’infiammazione. La maggior parte dei dati provengono da studi sul topo e molti immunologi fanno ancora affidamento su modelli di topo malgrado la distanza evolutiva e le differenze tra i sistemi immuni murino e umano. Nel tentativo di capire le questioni di cui sopra e di dirigere gli sforzi verso una immunobiologia basata sull’uomo, il fine di questo lavoro è stato quello di costruire e validare un modello umano della risposta di difesa innata/infiammatoria in vitro che ricapitolasse le differenti fasi della reazione infiammatoria, dal reclutamento e inizio, allo sviluppo e risoluzione dell’infiammazione e conseguente ripristino della omeostasi. Il modello è basato su monociti umani primari del sangue esposti in coltura a cambiamenti sequenziali delle condizioni microambientali (chemiochine, citochine, temperatura, molecole di derivazione batterica, ecc.) per 48 h. L’analisi al citofluorimetro ha dimostrato che la popolazione monocitaria utilizzata era rappresentativa dell’eterogeneità monocitaria così come presente nella circolazione sanguigna. Tutte le fasi della risposta infiammatoria sono state definite mediante analisi trascrittomica effettuata con U133Plus 2.0 GeneChip (Affymetrix). I risultati sono stati confrontati e integrati con profili trascrizionali pubblicamente disponibili di monociti/macrofagi, raccolti e annotati in un database ad hoc. Il profilo trascrittomico di alcuni fattori trascrizionali e fattori correlati con l’infiammazione sono stati confermati e validati mediante qPCR e ELISA. La “cluster analysis” ha rivelato cluster ampi e distinti che comprendono geni con un chiaro andamento che ben descrivono le differenti fasi dell’infiammazione. Per ottenere maggiori indicazioni sul ruolo biologico dei geni differenzialmente espressi durante la risposta infammatoria, ciascun cluster è stato analizzato con la GSEA (Gene Set Enrichment Analysis). I set di geni identificati dalla GSEA correlati con il profilo di espressione dei differenti cluster ha rivelato che la fase infiammatoria era arricchita di pathway infiammatorie mentre la fase anti-infiammatoria, così come quella di risoluzione, di pathway relative al metabolismo, al ciclo cellulare e al riarrangiamento genico. Inoltre confrontando le liste dei geni differenzialmente espressi tra monociti e macrofagi M1 e tra monociti e macrofagi M2 estratte dal meta-database, è stato dimostrato che i monociti trattati in vitro secondo il modello mostrano un profilo M1 durante la fase infiammatoria e M2 durante la risoluzione. L’espressione genica dei fattori trascrizionali e di quelli relativi alla infiammazione rispecchiavano il profilo di espressione ottenuto con microarray. In conclusione i dati di microarray e l’analisi cinetica dei fattori infiammatori e anti-infiammatori validano il modello in vitro proposto, modello che consente di descrivere la sequenza tempo-dipendente e coordinata degli eventi relativi alla infiammazione.The innate/inflammatory defensive reaction is activated in response to foreign pathogens or signals from damaged tissue. Monocytes/macrophages are key players in the initiation and resolution of inflammation by different activation programmes. Indeed in vivo macrophages can adopt a variety of different phenotypes depending on changes in the tissue microenvironment displaying a continuum of diverse functional states. Moreover peripheral blood monocytes are not a homogeneous population but differ in their phenotypes and functions. In spite of the explosive growth of data, many issues are still open about the phenotypic and functional characterization of monocytes/macrophages, and their role during the homeostasis and in inflammatory conditions. The great majority of the data originates from studies in mice and many immunologists still rely on mouse models despite the evolutionary distance and the differences between the murine and human immune systems. In an attempt to understanding the above issues, and to direct efforts in human immunobiology, the aim of this work was to build and validate a human model of innate/inflammatory defence response in vitro that recapitulates the different phases of the inflammatory reaction, from recruitment and initiation, to development and resolution of inflammation, and re-establishment of homeostasis. The model is based on human primary blood monocytes exposed in culture to sequential changes of microenvironmental conditions (chemokines and cytokines, temperature, bacterial-derived molecules, etc.) for 48 h. The flow cytometrical analysis has shown that the monocyte population used is representative of the monocyte heterogeneity as present in the circulation. All phases of the inflammatory response were profiled by transcriptomic analysis carried out with U133Plus 2.0 GeneChip (Affymetrix). Results were compared and integrated with publicly available transcriptional profiles of monocyte/macrophages, collected and annotated in an ad hoc database. The transcriptomic profiling of some transcriptional and inflammatory-related factors were confirmed and validated by qPCR and by ELISA. The “cluster analysis” revealed broad distinct clusters comprising genes with a clear behaviour that well described the different phases of inflammation. To gain more insight into the biologic role of the genes that are differentially expressed during the inflammatory response, each cluster was subjected to gene set enrichment analysis (GSEA). The gene sets identified by GSEA correlated with the expression profile of different clusters revealed that the inflammatory phase was enriched in inflammatory pathways while the anti-inflammatory phase, as well as the resolution phase, in pathways related to metabolism, cell cycle, and gene rearrangement. Moreover, by comparing the lists of differentially expressed gene between monocytes vs. M1 macrophages and vs. M2 macrophages extracted from the meta-database, it was shown that monocytes treated in vitro according to model resemble M1 during the inflammatory phase and M2 during the resolution. The gene expression of transcriptional and inflammatory-related factors matched with the expression profile obtained with microarrays. In conclusion the microarray data and the kinetical analysis of inflammatory and anti-inflammatory factors validate the proposed in vitro model of the inflammatory response, and allowed describing the time-dependent and coordinated sequence of inflammation-related events.
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Raghavan, Bindu. "Analysis of the Human Cytomegalovirus Transcriptome and Identification and Characterization of a HCMV gene involved in disruption of Interferon Signaling." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1218473086.
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Montgiraud, Cécile. "Définition de puces à ADN dédiées aux rétrovirus endogènes humains : applications à l’analyse du contrôle épigénétique et transcriptionnel." Thesis, Lyon 1, 2011. http://www.theses.fr/2011LYO10195.
Повний текст джерелаАнотація:
Les rétrovirus endogènes (ERV) sont constitutifs du génome des eucaryotes et représentent environ 400000 loci dans le génome humain divisés en différentes familles. Ces HERV (Human ERV) sont pour la majorité silencieux en contexte physiologique excepté dans le placenta mais présentent une activité transcriptionnelle en contexte pathologique comme par exemple dans les cancers. Il est difficile de comprendre de façon systématique les mécanismes de régulation/dérégulation des HERV et leur implication en contexte physiopathologique car il n’existe à ce jour aucun critère permettant de distinguer qu’elles sont les longues terminaisons répétées (LTR) transcriptionnellement actives dans l’ensemble de ces éléments de régulation. Nous avons développé deux générations de puces à ADN haute densité afin d’appréhender quelles étaient les LTR réactivées dans les cancers et de comprendre les mécanismes sous-jacents à la transcription des HERV. Avec la première version de la puce HERV, nous avons notamment identifié six loci de la famille HERV-W différentiellement exprimés dans le cancer testiculaire dont le locus ERVWE1 qui code pour la syncytine-1 impliquée dans la morphogénèse placentaire. L’analyse de l’ADN des tumeurs et des tissus sains adjacents démontre que l’hypométhylation des régions U3 promotrices est un pré-requis à l’activation des HERV. La deuxième version de la puce HERV a été utilisée pour une recherche de biomarqueurs pronostiques dans le cancer du poumon non à petites cellules. Ceci a permis de mettre en évidence des réactivations de HERV dans certains échantillons cancéreux et illustre la difficulté d’une telle approche au regard des disparités inter-individusEndogenous Retroviruses (ERVs) are inherited part of the Eukaryotic genomes, and represent about 400,000 loci in the Human genome divided in distinct families. The majority of HERVs (Human ERV) are mainly silent in most physiological contexts excepted in placenta, whereas a significant expression is observed in pathological contexts such as cancers. It is difficult to understand HERV (de)regulation mechanisms and their implication in physio-pathological contexts, as there is no criteria defining transcriptional active promoters HERV long terminal repeats (LTRs) among all these regulatory élements. We developed two versions of highdensity DNA microarray to specifically detect LTR reactivated in cancers and try to understand transcription mechanism of HERV. With the first version of HERV-microarray, we identified six HERV-W loci over-expressed in testicular cancer, including the domesticated ERVWE1 locus which produces an envelope protein dubbed Syncytin-1 associated with placenta development. The analysis of DNA from tumoral versus normal tissue reveals that hypomethylation of U3 promoters in tumors is a prerequisite of HERV activation. The second version of HERV-microarray was used to identify prognosis biomarkers in non small cell lung cancer. This study identified HERV reactivation in some samples and highlighted difficulties of such approach due to inter-individuals disparities
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Zaitseva, Olena [Verfasser], Lutz [Akademischer Betreuer] [Gutachter] Walter, Jörg [Gutachter] Stülke, and Matthias [Gutachter] Dobbelstein. "Analysis of the transcriptome of human NK lymphocytes / Olena Zaitseva ; Gutachter: Lutz Walter, Jörg Stülke, Matthias Dobbelstein ; Betreuer: Lutz Walter." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2016. http://d-nb.info/1117908445/34.
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Annab, Karima. "Etude de l’expression génique de différents syndromes progéroïdes en utilisant le modèle des cellules souches à pluripotence induite." Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0101.
Повний текст джерелаАнотація:
Les syndromes progéroïdes regroupent un ensemble de pathologies caractérisées par un vieillissement précoce et accéléré. Le syndrome le plus connu et étudié est la progéria de Hutchinson-Gilford dont l'incidence est de 1 cas sur 8 millions ce qui en fait une maladie très rare. Nous avons étudié trois symptômes progéroïdes dont le syndrome HGPS, un syndrome HGPS-like ainsi qu'un syndrome APS. Ces pathologies ont de nombreux symptômes en commun dont une ostéolyse, une lipodystrophie, ainsi qu'une atteinte cardiovasculaire. Ces trois syndromes sont provoqués par différentes mutations du gène LMNA qui code pour les Lamines A et C. Nous avons utilisé le modèle des iPSCs afin d'étudier in vitro la physiopathologie de ces trois syndromes en les comparant à des cellules contrôles. Les cellules dérivées de la voie mésenchymateuse étant majoritairement altérées dans ces pathologies, nous avons créé des modèles in vitro d'étude de la différentiation en MSCs. De plus, ces patients présentant des altérations arterio-veineuses, nous avons analysé la différenciation en VSMCs. Le phénotype des ces cellules a été analysé et les profils transcriptomiques comparés pour les différentes lignées. Des gènes communs, impliqués dans le stress oxydatif et dans des systèmes de réparation géniques ont été retrouvés comme étant altérés. De plus, nous avons mis en évidence des altérations de voies de signalisation indispensables à la survie et à la prolifération cellulaire en comparant les cellules progéroïdes aux contrôles. Certaines de ces voies biologiques ouvrent de nouvelles perspectives dans la compréhension des symptômes observés chez ces patientsProgeroid syndromes are a group of pathologies characterized by accelerated and early aging. One of the most studied of these diseases is HGPS, with an estimated incidence of 1 in 8 millions birth making it an extremely rare disease. We focused our attention on three different progeroid syndromes including classic HGPS, a HGPS-like and an atypical progeroid syndrome. These pathologies share many symptoms, including osteolysis, lipodystrophy, and cardiovascular alterations. These 3 syndromes are caused by 3 different mutations in the LMNA gene that encodes A- and C-type lamins, inducing production of a truncated Lamin A in HGPS and HGPS-like and production of a mutated Lamin with a p.T528M substitution in APS. We produced hiPSCs to create a model of these different diseases and investigate in vitro the physiopathology of these syndromes by comparing them to control cells. Cells derived from mesenchymal stem cells being the most impaired type of tissue, we established in vitro models in order to study the differentiation of hiPSCs into MSCs. In addition given the massive cardiovascular defects in these patients, we also investigated differentiation toward the VSMCs. Cell phenotypes were carefully characterized and we compared the transcripttomic profile of the different cell types. We identified dysregulation in genes involved in oxidative stress response and in DNA repair in progeroid cells. In addition, pathways essential for cell survival and proliferation are also modified when comparing progeroid and controls cells. Altogether, these results might explain some of the symptoms observed in progeroid patients but also reveal pathways involved in ageing
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Rotival, Maxime. "Approches intégrées du génome et du transcriptome dans les maladies complexes humaines." Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00665244.
Повний текст джерелаАнотація:
Cette thèse a pour objet l'étude du lien génotype-transcriptome et de son influence sur le développement des maladies multifactorielles. Les apports de ce travail sont à la fois méthodologiques et appliqués. Nous étudions d'abord le lien génotype-transcriptome en établissant la liste des eQTL (expression Quantitative Trait Loci) dans le monocyte et nous évaluons l'apport de l'observation des eQTL pour l'interprétation des analyses d'association génome entier (GWAS). Nous proposons ensuite une méthode pour l'identification de variants génétiques affectant des modules de gènesco-régulés que nous appliquons à l'étude des données d'expression de monocytes issus d'une large étude populationnelle (GHS). Nous mettons ainsi en évidence plusieurs loci affectant l'expression de modules de gènes co-régulés, dont plusieurs sont impliqués dans la prédisposition au diabète de type I. Nous montrons également que le processus d'isolation des cellules monocytaires peut engendrer des biais liés à la contamination par des types cellulaires non désirés et nous proposons une approche pour contrôler ce type de biais dans les analyses.
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Mutez, Eugénie. "Apport du transcriptome des cellules mononucléées sanguines à l'étude de cas familiaux et sporadiques atteints de la maladie de Parkinson." Phd thesis, Université du Droit et de la Santé - Lille II, 2011. http://tel.archives-ouvertes.fr/tel-00912324.
Повний текст джерелаАнотація:
La maladie de Parkinson (MP) est caractérisée par la mort des neurones dopaminergiques de la substance noire et la présence de corps de Lewy. Son diagnostic reste sujet à des erreurs notamment aux stades précoces. Les cellules mononucléées sanguines périphériques (PBMC) jouent un rôle dans la cascade délétère et sont le reflet d'événements associés à la MP. Même si elles ne représentent qu'un faible pourcentage, les formes génétiquement déterminées permettent d'identifier des sujets à un stade précoce. Nous avons émis l'hypothèse que les PBMC pouvaient constituer un modèle d'étude reflétant certains mécanismes de la dégénérescence du vivant du patient. Nous avons réalisé des études du transcriptome chez différents groupes de sujets malades ou porteurs de mutations pour y déceler les gènes et voies de signalisation cellulaire dérégulés. Nous avons d'abord étudié le profil d'expression génique de sujets porteurs de la mutation G2019S de LRRK2. L'analyse des puces a permis d'identifier des perturbations de voies impliquées dans la MP comme l'oxydation mitochondriale, l'inflammation et la guidance axonale. Des altérations de la voie des MAPK, du cytosquelette d'actine et du transport vésiculaire ont été notées. La liste des gènes dérégulés permet de séparer les individus selon leur statut génétique. La mutation LRRK2 est associée à un profil d'expression génique dès les stades précoces identifiable dans les PBMC. Nous nous sommes ensuite intéressés à une autre forme de MP avec duplication de SNCA. Nous avons caractérisé la relation entre le génotype et le phénotype clinique des sujets de cette famille. La duplication s'étend sur 4,928 Mb, comporte 31 gènes et résulte d'une recombinaison homologue non allélique. L'analyse de l'expression des gènes présents dans la duplication dans les PBMC d'un sujet à un stade pauci-symptomatique a montré une surexpression de SNCA. Nous avons comparé nos analyses chez les porteurs des mutations LRRK2 et SNCA et chez des parkinsoniens sporadiques. Nos analyses montrent que les sujets LRRK2 et les sujets sporadiques présentent des dérégulations communes de voies de signalisation. En revanche, les voies dérégulées chez le sujet dupliqué reflètent la pathogénie de SNCA comme l'autophagie et les voies lysosomales. Nous nous sommes intéressés à l'expression des 4 isoformes de SNCA dans les PBMC de ces 3 groupes d'individus. Les patients sporadiques et LRRK2 montrent une diminution de l'expression des 4 isoformes de SNCA dans leur PBMC. Chez le sujet dupliqué, on observe uniquement une surexpression de l'isoforme 112. Nous avons ensuite identifié les voies moléculaires associée
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Ma, Ning. "Development of techniques for analysis of the human retinal ganglion cell transcriptome : application to the role of calcium in RGC death in glaucoma." Thesis, University of East Anglia, 2013. https://ueaeprints.uea.ac.uk/48120/.
Повний текст джерелаАнотація:
Purpose: Irreversible retinal ganglion cell (RGC) death is the reason for visual loss in glaucoma. However, the mechanisms of RGC death remain unclear. The aim of this research was to develop methods to study mRNA expression profiles in human RGCs, then to use the data to investigate the role of calcium in RGC death. Methods: A planar sectioning technique was developed to isolate mRNA from serial sections of the human retina. QRT-PCR of neuronal markers validated the technique. Global gene expression analysis, using Illumina arrays, compared expression in the retina ganglion cell layer (RGCL) and entire macula (Mac). Immunohistochemistry and QRT-PCR validated gene array data. RGC death was investigated using a simulated ischemia (oxygen glucose deprivation, OGD) model in human organotypic retinal cultures (HORCs). Cell survival was measured by LDH, and RGC loss by immunohistochemistry and QRT-PCR. Western blot assessed proteases. Results: The sectioning technique developed enabled isolation of relatively large quantites of high quality mRNA from 20μm retinal sections from the macular region of the human retina. Marker genes for retinal neurons verified accurate profiling of gene expression across the retina. Gene arrays provided a list of genes that were most enriched in the RGCL. AHNAK2 and HSPA1B were the two most enriched genes in the RGCL. CAPN1 (calpain 1), a calciumdependent cysteine proteases, was in the gene list. Its expression was confirmed to be mainly in the inner retina. OGD caused calpain activation and induced RGC death. Two TRP channels, TRPM-2 and TRPC-3, which mediate Ca2+ influx, were found that predominantly expressed in the RGCL. Involvement in RGC death in the OGD model using the TRP inhibitor ACA could not be confirmed. Conclusions: The technique developed has enabled determination of the human RGCL transcriptome and has allowed expression profiling of gene of interest across the retina. This could prove to be a powerful tool in the investigation of pathways involved in neurodegeneration in the retina.
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Petiti, L. "NEXT-GENERATION SEQUENCING APPROACH FOR TRANSCRIPTOME AND EPIGENOME DEFINITION OF HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS AND THEIR EARLY ERYTHROID AND MYELOID COMMITTED PROGENY." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/286531.
Повний текст джерелаАнотація:
Somatic stem cells are the basic tools of regenerative medicine and gene therapy, providing unique opportunities for the therapy of genetic and acquired disorders. The molecular mechanisms underlying fundamental characteristics of human somatic stem cells, such as self-renewal, commitment and differentiation, are still poorly understood. A better knowledge of these mechanisms is crucial to the understanding of stem cell biology and to the development of stem cell-based therapies. High-throughput approaches, such as next-generation sequencing technologies (NGS) became fundamental to study the transcriptome, the epigenome and the usage of regulatory elements in the genome. Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Here, we define the transcriptional and epigenetic profile of human hematopoietic stem/progenitor cells (HSPC) and early myeloid and erythroid progenitors through an integrative analysis of Cap Analysis of Gene Expression (CAGE) and chromatin immuno- precipitation (ChIP-seq) data, in order to identify transcription regulatory elements that act in HSPC lineage commitment. CAGE analysis enabled us to define more than 10,000 active promoters in HSPCs and in erythroid (EPP) and myeloid precursors (MPP). The different cell types shared most of the promoters, with only a small fraction (about 4%) being differentially transcribed, suggesting that the transcriptional state is largely maintained in early hematopoietic progenitors and precursors. Interestingly about 30% of the identified of cell-specific promoters was not annotated. These novel transcripts are possibly involved in HSPCs self- renewal, commitment and differentiation. To obtain a genome-wide description of the transcriptional regulatory regions in multipotent and lineage-restricted hematopoietic progenitors, we performed ChIP-seq analysis for histone methylations typical of promoters and enhancers, H3K4me3 and H3K4me1, respectively, and for H3K27ac to mark the active elements. Overall we identified more than 20,000 active enhancers that consistently changed upon erythroid and myeloid commitment: about 80 and 95% of the active enhancers mapped in HSPC disappeared in erytrhoid and myeloid progenitors, respectively, while novel enhancers are acquired during lineage commitment. These data indicate that enhancers are dramatically redefined during lineage commitment, and that differential enhancer usage is responsible for the differential regulation of promoter activity underlying lineage restriction. This study provided an overview of the differential transcriptional programs of HSPCs and committed myeloid and erythroid hematopoietic precursors and represents a unique source of genes and regulatory regions involved in self-renewal, commitment and differentiation of human hematopoietic stem/progenitor cells and their progeny.
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Cruz, Luciana Oliveira. "Identificação e seleção de novos genes humanos associados a tumores a partir de dados obtidos no projeto Transcript Finishing Initiative (TFI)." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-16112007-111539/.
Повний текст джерелаАнотація:
Após o seqüenciamento completo do genoma humano, a busca e caracterização do conjunto completo de genes humanos constitui-se no principal desafio nesta área de investigação, sendo o passo limitante para o progresso na exploração dos dados contidos no seqüenciamento deste genoma. O projeto de transcriptoma denominado \"Transcript Finishing Initiative\" (TFI) surgiu neste contexto, com o objetivo principal de gerar fragmentos parciais de transcritos humanos, que não haviam sido descritos previamente e determinar sua seqüência, para iniciar a caracterização de novos genes humanos. A estratégia utilizada foi q alinhamento de todas as seqüências ORESTES e ESTs disponíveis com a seqüência pública do genoma humano e o agrupamento j c1usterização destas com base nas coordenadas deste genoma. Algumas das regiões que não eram cobertas por estas seqüências foram, então, completadas, por RT-PCR, utilizando-se primers ancorados nos clusters vizinhos. Cada par de clusters de ESTs selecionado para validação experimental foi designado como uma Unidade do \"Transcript Finishing\" (TFU), tendo sido validadas experimentalmente, pelo grupo TFI, Um total de 211 TFUs foram validadas, sendo que 197 seqüências consenso foram submetidas ao Genbank (CF272536-CF272733). Atualmente, apenas um pequeno número destas seqüências ainda são considerados genes novos, sem que haja um cDNA depositado em banco de dados; contudo para um número considerável destas TFUs não existe qualquer caracterização sobre sua função. Na tentativa de contribuir para melhor caracterização dos genes identificados no projeto TFI, e tendo, como base, a linha de pesquisa do laboratório, que busca genes diferencialmente expressos envolvidos em transformação maligna/tumorigênese, o presente trabalho propôs a utilização das seqüências TFUs para estudar sua possível associação com tumores de glia humanos e outros tipos de tumores (de próstata e de mama). Para tanto, as TFUs foram analisadas \"in silico\" para estabelecer seu grau de ineditismo como um novo gene ou um gene sem função conhecida, e, para análise de sua expressão diferencial entre tecidos normais e tumorais de cérebro, próstata e mama. Para validar estas análises computacionais na bancada, foram gerados macro- e microarranjos de DNA utilizando-se as TFUs disponíveis como clones físicos ou amplicons, para o rastreamento com sondas das linhagens celulares A172 e T98G de glioblastomas. Os resultados das análises destes dados foram confirmados por PCR quantitativo tanto nas linhagens como em amostras clínicas de astrocitomas que apresentam diversos graus de malignidade. Como resultado, foi possível organizar um Banco de Clones Físicos de TFUs, além de identificar e selecionar uma TFU (168), cuja expressão correlaciona diretamente com o grau de malignidade dos tumores de glia. Esta seqüência corresponde a um novo gene, já que não existe a seqüência de cDNA completo nos bancos de dados. Em vista disto, a TFU168 foi selecionada para estudos funcionais posteriores, que já estão em andamento, através da obtenção de suaseqüência completa de cDNA para ensaios de superexpressão e do silenciamento gênico através de RNAi.Upon complete sequencing of the human genome, identification and characterization of the complete set of human genes constitutes the major challenge in this research field, constituting the limiting step for progress in exploration of the informations contained in the genome sequencing data. The Transcript Finishing Initiative (TFI) transcriptome project arose in this context, aiming at the generation, sequencing and characterization of partial new human transcripts and genes. The strategy adopted was the alignment of the alI the available ORESTES and EST sequences data with the public human genome sequence and their clusterization based on the coordinates of this genome. Thus, some of the regions which were not cover by ESTs and ORESTES (gaps) were then completed by RT-PCR using primers anchored in the neighboring clusters. Each pair of EST clusters selected for experimental validation was named Transcript Finishing Unit (TFU). A large number (211) of TFU s were validated and 197 -consensus sequences were submitted to the Genbank (CF272536-CF272733). At present, only a few of these sequences are considered as new genes without a full-Iength cDNA sequence deposited in the data bank, however, no functional characterization is yet available for a large number of these sequences. In an attempt to contribute to further characterization of these genes identified in the TFI project and keeping in mind the main interest of our laboratory, which is the identification of differentially expressed genes in tumor versus normal tissue, the present work aims at utilizing these TFUs to find differentially expressed genes associated with human glial tumors and with other kinds of tumors. To this end, these sequences were first subjected to in silico analysis in order to establish their degree of ineditism (new sequences and/or sequences with unknown function) and their expression profile between normal and tumoral tissues of brain, mammary gland and prostate. To validate this computational analysis, DNA macro- and microarrays were generated with the TFU sequences and screened with cDNA probes obtained from the A172 and T98G glioblastomas cell lines. The results of these screenings were confirmed by quantitative PCR both in cell lines and in tumor samples of different degrees of malignancy. The results obtained in this work allowed the organization of a TFUs Physical Clones Bank and the identification and selection of one sequence (TFU 168), whose expression is directly re1ated to the degree of tumor malignancy. This sequence constitutes a new gene, since no complete cDNA sequence is available in the data banks. Therefore, TFU168 was selected for further functional studies by obtaining its full-Iength cDNA sequence to be used for over expression by silencing this gene using RNAi.
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Perot, Philippe. "Étude du transcriptome des rétrovirus endogènes humains et implications fonctionnelles : applications à la recherche de marqueurs diagnostiques de cancers." Thesis, Lyon 1, 2012. http://www.theses.fr/2012LYO10228/document.
Повний текст джерелаАнотація:
Le génome humain contient environ 200 000 séquences d'origine rétrovirale (HERV), intégrées au fil de l'évolution et organisées aujourd'hui en familles multicopies complexes globalement réprimées par un contrôle épigénétique. L'étude du transcriptome HERV au niveau locus est compliquée par les similarités phylogénétiques au sein d'une famille et par la profusion des sites d'intégration, deux propriétés inhérentes aux éléments transposables. Dans ce travail, nous avons utilisé une méthode de conception de sondes de détection de 25 mer afin d'adresser la question de l'expression individuelle des HERV. Une puce à ADN haute densité intégrant plus de 5 500 séquences HERV et permettant une lecture fonctionnelle de l'activité de leurs LTRs a été utilisée sur un panel de tissus sains et cancéreux. Cela a permis d'identifier 1 718 séquences HERV actives, dont 326 LTRs promotrices et 209 LTRs polyA. L’étude de l’environnement génomique a mis en évidence une fenêtre d’environ 8 kb en amont des LTRs promotrices, caractérisée par une sous-représentation en gènes cellulaires en orientation sens. Nous avons également montré que le transcriptome des rétrovirus endogènes humains suit des règles de tropisme d’expression, qu’il est sensible aux états de différenciation cellulaire et qu’il ne semble pas être corrélé à l’âge des familles. Une première tentative d’exploitation de ce répertoire HERV dans un contexte clinique a visé à rechercher de nouveaux marqueurs diagnostiques du cancer de la prostate à partir de prélèvements urinaires, par la réalisation d’une étude pilote sur 45 patientsThe human genome contains around 200,000 endogenous retroviral sequences (HERV) integrated during the evolution and which are nowadays organized into complex multicopy families, globally repressed by epigenetic control. The study of the HERV transcriptome at the locus level is complicated by phylogenetic similarities within one family and by the profusion of integration sites, two inherent characteristics of transposable elements. In this work, we used a method aiming to optimally characterize individual loci associated with 25 mer probes. A custom microarray dedicated to more than 5,500 HERV sequences and allowing a functional interpretation of the LTRs expression was used on a panel of normal and tumor tissues. We therefore identified 1,718 active HERV sequences, including 326 promoter LTRs and 209 polyA LTRs. The study of the genomic environment has highlighted an approximately 8 kb zone upstream of promoter LTRs characterized by a drastic reduction in sense cellular genes. We also showed that the HERV transcriptome follows tropism rules, is sensitive to the state of cell differentiation and, unexpectedly, seems not to correlate with the age of the families. In a first attempt to use the HERV repertoire in clinical, we sought to identify new markers of prostate cancer from urine samples. This goal was pursued by conducting a pilot study on 45 patients
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Massé-Deragon, Nicolas. "Dynamique des réponses immunitaires humaines dans un modèle 3D de foie : un autre regard sur la pathogénèse hépatique du virus de la fièvre jaune." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1270/document.
Повний текст джерелаАнотація:
La fièvre jaune est une pathologie virale humaine causée par un flavivirus, le virus de la fièvre jaune et transmise par des vecteurs arthropodes. Les formes sévères, parfois mortelles, sont caractérisées par une atteinte systémique aigüe qui affecte le foie. Bien que la vaccination existe depuis près de 80 ans, des recensements réguliers d'épidémies sont encore faits. Les vaccins à base d'une souche vivante atténuée YF 17D présentent d'excellents taux de séroconversion et sont notamment caractérisés par une forte diminution de l'hépatotropisme. Néanmoins les mécanismes associés à la pathogénèse hépatique sont encore mal compris et pourraient être une aide aux développements vaccinaux contre d'autres flavivirus ou virus hépatiques. L'étude développée ici s'est inscrite dans la problématique de la représentativité des modèles cellulaires hépatiques utilisés. Afin de répondre aux pertes métaboliques et immunitaires reportées dans plusieurs modèles, nous nous sommes orientés vers des modèles organotypiques associant plusieurs populations cellulaires hépatiques et un microenvironnement caractéristique. Les modulations induites par les souches vaccinales ou sauvages du virus de la fièvre jaune ont été évaluées par une approche transcriptomique globale utilisant la technologie RNASeq et des méthodes d'analyse définies. Nos résultats montrent une plus forte permissivité des modèles cellulaires à la souche atténuée YF 17D par rapport à la souche sauvage YF Asibi. Cette observation est associée pour la souche atténuée à l'établissement précoce d'une réponse antivirale complète impliquant une détection rapide des formes réplicatives du virus, la mise en place des réponses aux IFNs de type I et de type III, la clairance virale et un contrôle des métabolismes cellulaires et hépatiques. De son côté la souche sauvage présente un délai important dans l'établissement de ces réponses amenant à de potentiels mécanismes alternatifs de la clairance virale et de dérégulations métaboliques. Ces données mettent en exergue les interactions étroites qui existent entre les systèmes immunitaires et métaboliques au niveau du foie. Nous suggérons que la forte réponse antivirale induite par la souche atténuée pourrait contribuer à la rupture de la tolérance hépatique et à l'efficacité in vivo de la souche vaccinale. En outre, la cinétique des réponses immunitaires, en combinaison avec la charge virale, peuvent déterminer l'équilibre entre la récupération et l'immunopathologie après l'infection par le virus sauvageYellow fever is a human disease caused by a flavivirus, the yellow fever virus, transmitted by arthropod vectors. Severe forms, sometimes fatal, are characterized by acute systemic disease that affects the liver. Despite an effective vaccine being available for nearly 80 years, epizootic circulation occurs and results in periodic outbreaks in endemic regions and among travelers. Vaccines based on a live attenuated strain YF 17D exhibit excellent seroconversion rate and are characterized by a strong decrease in hepatotropism. However the mechanisms involved in liver pathogenesis are poorly understood and could be helpful for future vaccine development against other flaviviruses or hepatitis viruses.There is a need to develop liver cellular model better reflecting the in vivo liver microenvironment. In this work, we used new 3D models combining several liver cell populations to evaluate immune and metabolic responses induced by yellow fever viruses. Modulations induced by both vaccine and wild-type strains were evaluated by a global transcriptomic approach using RNA-Seq technology and well-defined analysis methods. Our results show a greater permissivity of cellular models to YF 17D strain compared to the wild type YF Asibi. In addition, YF 17D infection leads to an early establishment of a complete antiviral response involving rapid detection of replicating forms of the virus, development of a strong type I and type III IFN responses, initiation of viral clearance and modulation of cellular and liver metabolism. Wild-type strain presents a significant delay in the establishment of these responses leading to potential alternative mechanisms for viral clearance and metabolic dysregulation. These data highlight the close interactions between the immune and metabolic systems in the liver.We suggest that the strong antiviral response induced by attenuated strain could contribute to the breakdown of liver tolerance and in vivo efficacy of the vaccine strain. In addition, the kinetics of immune responses, in combination with viral load, can determine the balance between the recovery and immunopathology after infection with wild type virus