Dissertations / Theses on the topic 'Genome wide screening'
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Appari, Mahesh [Verfasser]. "Genome-wide screening of biomarkers in androgen insensitivity syndrome (AIS) / Mahesh Appari." Kiel : Universitätsbibliothek Kiel, 2009. http://d-nb.info/1019866764/34.
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Christ, Ryan. "Ancestral trees as weighted networks : scalable screening for genome wide association studies." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:8f382d56-2d5d-4a4f-9b39-41700897e02e.
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Several haplotype-based methods have been developed to identify loci where multiple mutations of low to moderate frequency and effect size modulate disease susceptibility. Most such approaches either do not scale to hundreds of thousands of genomes or do not explicitly model recombination and the block-like structure of haplotypes. Using a novel checkpointing technique and a C-core, vectorized implementation of an Hidden Markov Model (HMM) based on the Li & Stephens Model, at each single nucleotide polymorphism (SNP) along the genome, we obtain a local genetic distance between all pairs of haplotypes in a phased dataset. To rapidly test this local distance matrix for association with a phenotype, we derive two finite sample central limit type theorems for quadratic forms which do not require any further assumptions on the matrix other than it is free of outliers, for which we have an easily calculable, formal condition. We combine these results with a novel concentration inequality for Gaussian quadratic forms to upper and lower bound p-values for quadratic forms while avoiding a full eigendecomposition of each matrix. Applying our HMM implementation and quadratic form screening method, we recover known loci associated with malaria susceptibility and uncover new potential associations in a pilot dataset of 6,136 haplotypes.
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Löllgen, Ruth Mari Caroline. "Genome wide screening of loss of heterozygosity in human midgut carcinoid tumors with fluorescent technique." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972556702.
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Kaemena, Daniel Fraser. "CRISPR/Cas9 genome-wide loss of function screening identifies novel regulators of reprogramming to pluripotency." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31184.
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In 2006, Kazutoshi Takahashi and Shinya Yamanaka demonstrated the ability of four transcription factors; Oct4, Sox2, Klf4 and c-Myc to 'reprogram' differentiated somatic cells to a pluripotent state. This technology holds huge potential in the field of regenerative medicine, but reprogramming also a model system by which to the common regulators of all forced cell identity changes, for example, transdifferentiation. Despite this, the mechanism underlying reprogramming remains poorly understood and the efficiency of induced pluripotent stem cell (iPSC) generation, inefficient. One powerful method for elucidating the gene components influencing a biological process, such as reprogramming, is screening for a phenotype of interest using genome-wide mutant libraries. Historically, large-scale knockout screens have been challenging to perform in diploid mammalian genomes, while other screening technologies such as RNAi can be disadvantaged by variable knockdown of target transcripts and off-target effects. Components of clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) prokaryote adaptive immunity systems have recently been adapted to edit genomic sequences at high efficiency in mammalian systems. Furthermore, the application of CRISPR-Cas components to perform proofof- principle genome-wide KO screens has been successfully demonstrated. I have utilised the CRISPR-Cas9 system to perform genome-wide loss-of-function screening in the context of murine iPSC reprogramming, identifying 18 novel inhibitors of reprogramming, in addition to four known inhibitors, Trp53, Cdkn1a, Jun, Dot1l and Gtf2i. Understanding how these novel reprogramming roadblocks function to inhibit the reprogramming process will provide insight into the molecular mechanisms underpinning forced cell identity changes.
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Löllgen, Ruth Mari Caroline. "Genome-wide screening of loss of heterozygosity in human midgut carcinoid tumors with fluorescent technique." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2004. http://dx.doi.org/10.18452/15047.
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Hintergrund: Karzinoid-Tumoren des embryonalen Mitteldarms sind seltene intestinale neuroendokrine Tumoren, bei denen zum Zeitpunkt der Diagnose häufig Metastasen vorliegen. Im Gegensatz zu Karzinoiden des Vorderdarms und Respirationstraktes sind sie nicht mit der Multiplen Endokrinen Neoplasie Typ 1 (MEN1) vergesellschaftet. Die Mechanismen ihrer Tumorigenesis sind weitgehend unbekannt. Methoden: Tumorgewebe acht sporadischer, maligner Dünndarm-Karzinoide war Objekt dieser Studie über Verlust der Heterozygotie ("Loss Of Heterozygosity" (LOH)) mit 131 fluoreszierenden Mikrosatelliten. DNA Sequenz-Analyse mit Oligonucleotid Primern, die Exon 8-11 des SMAD4/DPC4 Gens flankieren sowie immunhistochemische Färbung mit Smad4/DPC4 antikörpern wurde durchgeführt. Ergebnis: Chromosom 18 wies Deletionen in 88% der Tumoren auf. Alle außer einem Tumor hatten sowohl 18p als auch 18q verloren, in einem der Tumoren war eine kleine Region telomer zu den SMAD4/DPC4/DCC Genen auf 18q21 verloren. Andere Chromosomen waren nur in drei Tumoren betroffen. LOH auf Chromosom 11q13, dem MEN1 Lokus, wurde nicht gefunden.Sequenzierung der DNA und immunhistochemische Färbung für das SMAD4/DPC4 Gen zeigten keine Aberrationen. Diskussion: Die Funde der Chromosom 18 Deletionen weisen eindeutig auf ein entscheidendes Ereignis in der Tumorigenese von Karzinoiden des Mitteldarms hin. An der Entstehung dieser Tumoren könnte ein mutmaßliches Tumor Suppressor Gen beteiligt sein, welches auf Chromosom 18 lokalisiert ist. Dahingegen ist SMAD4/DPC4 wahrscheinlich nicht in die Tumorneogenese von Carcinois Tumoren involviert.Background: Midgut carcinoid tumors are rare malignant tumors with origin in the neuroendocrine cells of the small intestine. Due to secretion of a variety of peptide hormones and biogenic amines they cause the carcinoid syndrome. Metastases are often present at first diagnosis. Despite this, patients have a realistic chance to survive for a prolonged period (30% (unresectable/metastatic disease) -79% (non-metastatic disease) 5-year survival rate) if treated by a combination of surgery and medication. Unlike their foregut counterparts, midgut carcinoid tumors are not or rarely associated with the multiple endocrine neoplasia type 1 (MEN1) syndrome. The genetic back-ground to tumorigenesis of these neoplasms is unknown. In contrast, the events involved in tumorigenesis of gastroenteropancreatic adenocarcinomas are better characterized with frequent mutations e.g. of the Smad4/DPC4, Smad2/MADR2/JV18-1 and DCC genes on chromosome 18. Methods: Eight metastatic midgut carcinoids were analysed by a genome-wide screening for loss of heterozygosity using 131 PCR-amplified fluorescent-labelled microsatellite markers. DNA sequence analysis using oligonucleotide primers flanking exons 8-11 of the Smad4/DPC4 gene and immunohistochemical staining with Smad4/DPC4 antibodies was performed. Results: Chromosome 18 was deleted in seven out of eight tumors (88%). All but one of these tumors had lost both 18p and 18q, the remaining tumor had lost the long arm but retained the short arm. Several other chromosomal alleles were lost in a subset of the tumors. Loss of heterozygosity (LOH) on chromosome 11q13, the MEN 1 locus, was not found. Smad4/DPC4 wild-type sequence and normal immunohistochemical staining for Smad4/DPC4 protein was found for all analysed tumors. Conclusions: Our finding of a high frequency of chromosome 18 deletions in 88% of the tumors strongly suggests that midgut carcinoid tumorigenesis might involve inactivation of a candidate tumor suppressor gene located in that region while Smad4/DPC4 is unlikely to be involved in that process. A more detailed analysis of the genetic events in midgut carcinoid tumors is warranted to clarify their neogenetic origin.
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Riehmer, Vera [Verfasser]. "Genome-wide screening methods in tumors of the central nervous system and cancer predisposition / Vera Riehmer." Bonn : Universitäts- und Landesbibliothek Bonn, 2014. http://d-nb.info/1054691770/34.
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Rudge, Felicity. "Genome-wide cDNA and RNAi screening to identify modulators of responses to a novel Wnt signalling inhibitor." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/58589/.
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Wnt/β-catenin signalling plays a central role in the regulation of multicelluar organism development and in the maintenance of tissue homeostasis in adults. Dysregulation of Wnt signalling resulting in aberrant pathway activation is a key initiating step in the development of a diverse range of cancers, including colorectal cancer, and as such is an important target for therapeutic intervention. A novel Wnt pathway inhibitor, ‘MSC’, has been identified as blocking activated Wnt signalling, specifically through inhibiting the ability of CDK8 and CDK19 to activate nuclear β-catenin/TCF-dependent transcription. However, despite potently inhibiting Wnt-dependent transcription, the ability of MSC to reduce cellular viability was limited. This study aimed to determine genes that whose loss operated with MSC to reduce cell survival. A whole-genome RNAi chemical sensitisation screen identified 3 genes whose depletion in combination with MSC treatment conditionally reduced the viability of HCT116 cells in vitro. The outstanding hit of this screen was Histidyl Aminoacyl tRNA Synthetase (HARS). The identification of this enzyme as an MSC ‘interactor’ suggested links between Wnt signalling and the regulation of translation. BRAF and MED11 RNAi also conferred conditional sensitivity to MSC. Interestingly, MED11 is a component of the Mediator complex, a multiprotein transcription regulatory complex in which CDK8 functions to regulate β-catenin/TCF-dependent transcription, suggesting that mediator complex may be a key target of MSC action. A parallel overexpression screen was initiated to identify novel Wnt pathway activators, and subsequently used to map MSC resistance. Expression of the transcription factors GBX1 and HMGB2, determined to be novel regulators of TCF-dependent transcription, blocked MSC-mediated disruption of Wnt signalling. Overexpression of either gene in a clinical context might therefore be regarded as a contra-indication for MSC-class therapies. These studies have highlighted potential avenues for broadening the scope of MSC activity through the determination of survival and resistance mechanisms, thus the rational design of MSC-combination therapies could be of huge clinical benefit for the treatment of colorectal cancer.
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Stravalaci, Matteo. "Identification and characterization of mediators of toxicity of Aβ oligomers by genome wide screening in Caenorhabditis elegans." Thesis, Open University, 2017. http://oro.open.ac.uk/50285/.
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Soluble oligomers of the amyloid-β (Aβ) protein play a key role in the pathogenesis of Alzheimer’s disease (AD), although the underlying molecular mechanisms are poorly understood. In order to search for proteins involved in the formation and/or toxicity of Aβ oligomers, a transgenic C. elegans model of AD was used in which inducible expression of Aβ oligomers results in a complete paralysis; in these worms a genetic screen following chemical mutagenesis was applied to discover the genes involved in the Aβ-dependent paralysis (forward genetics). This analysis allowed identification of a mutated clone showing a complete lack of paralysis, despite this it not bear mutations in the Aβ coding region, and accumulates Aβ transcript and protein levels comparable to that of the non-mutated strain. This is the first in vivo model in which the expression of Aβ oligomers do not result in any toxic effect. The genome of the mutated worm was then sequenced and compared with that of the control strain to search for altered genes. Two genes, with no known function, were found to bear a stop codon mutation, likely resulting in the translation of an inactive protein. The rest of the mutations were missense mutations. Among them, point mutations were observed in some genes previously correlated with nematode lifespan and ageing. In C. elegans these biological processes are coordinated by the insulin/IGF-1-like signalling (IIS) pathway, which also regulates the response of the organism to toxic aggregated proteins. Thus, the activation of the IIS pathway was investigated in control and mutated worms. As expected, Aβ expression induced the up-regulation of two genes coding for small heat shock proteins (a class of chaperons known to be involved in AD) in the control strain, whereas these genes were actually down-regulated in the mutated strain. Since heat shock proteins are known to bind Aβ oligomers, these chaperons could directly mediate the formation of toxic amyloid species. Moreover, the results of the whole genome sequencing indicate that several proteins could act as potential novel mediators of Aβ toxicity and could open up new insights for research on age-related, neurodegenerative diseases.
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Sooda, Anuradha. "Discovery of novel HLA-B*57:01 restricted T-cell antigens through genome-wide screening of Epstein-Barr virus." Thesis, Sooda, Anuradha (2020) Discovery of novel HLA-B*57:01 restricted T-cell antigens through genome-wide screening of Epstein-Barr virus. PhD thesis, Murdoch University, 2020. https://researchrepository.murdoch.edu.au/id/eprint/54110/.
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A relationship between latent viral infections and drug hypersensitivity reactions (HSR) has been observed in several clinical situations. Abacavir hypersensitivity has been shown by several groups to be an HLA-B*57:01 restricted CD8+ T cell dependent process. Although compelling evidence suggests that carriage of HLA-B*57:01 is necessary, it is not sufficient for abacavir hypersensitivity. Based on previous structural and functional studies, it was hypothesised that heterologous immune responses of the pre-existing anti-viral T cell responses mediate abacavir hypersensitivity. Human herpesviruses (HHV) have been posited to be associated with the development of HSR because they have co-evolved with the human major histocompatibility complex and they persist in most people as lifelong latent, asymptomatic infections. In addition, they are reactivated in immunocompromised individuals and have been implicated in drug-viral interactions such as associated with Epstein-Barr virus (EBV). EBV is the most common HHV infecting more than 95% of the world’s population. The EBV genome is approximately 172 kb in length and encodes 89 proteins. However, the extent to which these proteins are surveyed by host-derived T cells has not been fully explored. The objective of this body of work was to generate ORFeome-wide map of memory CD8+ T cell responses for the identification of HLA-B*57:01 restricted antigens which could be involved in the cross-reactivity of abacavir drug hypersensitivity. Mapping revealed 14 novel HLA-B*57:01 restricted EBV antigens and confirmed the previously identified epitopes EBNA3B (VSFIEFVGW), EBNA2 (LASAMRML), and LMP1 (IALYLQQNW). A novel immunodominant epitope was detected in EBNA3C (QSRGDENRGW). To identify cross-reactive anti-viral T cell receptors, single cells specific for the immunodominant epitopes of EBNA3B (VSF) and EBNA3C (QSR) were sorted based on tetramer staining. Antigen-specific T cells were sequenced for the T cell receptors cloned into Jurkat cells. A modified heterologous T cell receptor reporter assay was developed to study the T cell responses against HLA-B*57:01 restricted EBV immunodominant epitopes. Dose-dependent T cell responses were detected against each EBV epitope, however, the TCRs were not cross-reactive to abacavir plus self-peptide presented in the context of HLA-B*57:01. The presence of abacavir was shown to modulate the EBV-specific TCR responses to the two EBV epitopes, which suggested the non-covalent binding of abacavir to HLA-B*57:01 may have down modulated the TCR responses. Although unable to demonstrate cross-reactivity of the EBV-specific TCRs with the abacavir plus self-peptide, this work has provided a road map for future experiments to elucidate the role of heterologous immunity in the immunopathogenesis of drug hypersensitivity and other aberrant acquired immune responses in humans.
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Lawson, Jonathan Luke Done. "Genome-wide microscopy screening identifies links across processes including a conserved connection between DNA damage control and the microtubule cytoskeleton." Thesis, University of Cambridge, 2015. https://www.repository.cam.ac.uk/handle/1810/253007.
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Previous PhD students in the lab created a method for large-scale, high-content microscopy screening of a cell library consisting of over 3000 single mutant strains of the fission yeast, Schizosaccharomyces pombe. Each strain has one nonessential gene knocked-out, allowing investigation of the resulting phenotypes. I report the implementation and completion of this screen; developing methods to ensure reliable and accurate results through inclusion of many controls across multiple screening repeats. In total, over 4.5 million images from approximately 19 000 biologically independent cell populations were imaged and analysed. All strains screened contained GFP-labelled tubulin (GFP-Atb2) allowing visualisation of the microtubule polymer network and its organisation in cells, a feature that is conserved across eukaryotes and simplified in S. pombe, making it easy to study. Examination of cell outlines and microtubule patterns was used to study three cell processes: the shape of cells, the organisational pattern of interphase microtubules and the cell cycle stage of cells, as judged by microtubule pattern. Comparison with extensive data from wild-type cells led to the identification of 262 factors that influence one or more of these cell processes. I go on to biologically validate some of the outcomes from the screen, leading to a publication in Developmental Cell reporting the screen, its findings and the online genomic resource SYSGRO. I then focus on a group of mutants that suggest a connection between the DNA damage response (DDR) and microtubule organisation. From here I show that the DDR induces elongation of microtubule bundles in response to the DDR kinases, ATM and ATR. I begin to reveal factors that may mediate this response and finally, I provide evidence to suggest that the same mechanism is conserved in cultured human cells (Hc3716-hTERT), which may go some way to explaining clinical results showing a beneficial effect of microtubule destabilisation in conjunction with cancer therapies.
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Gerzenstein, Sabrina Melisa. "Pharmacogenomics of the Intraocular Pressure Response to Glucocorticoids." Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_theses/285.
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Glucocorticoids (GCs) have been widely used as a therapeutic agent for diverse inflammatory ocular diseases. However, a high percentage of patients undergoing this treatment develop high intraocular pressure (IOP), which if left unsupervised may lead to glaucoma. It is believed that the IOP elevation in response to GC treatment has a genetic determinant. In order to test this hypothesis, we analyzed in 52 patients the presence of single nucleotide polymorphisms (SNPs) in the glucocorticoid receptor gene (GR), the principal mediator of GCs uptake by the cells. We studied six GR SNPs previously reported to be associated with sensitivity and resistance to GCs: GluArg22/23GluLys (codon 22-23), Asn363Ser (codon 363), IVS2+646C>G (intron 2/BclI), IVS3-46G>C (intron 3), IVS4-16G>T (intron 4), Asn766Asn (Codon 766). Nevertheless, the results of this preliminary study did not show any specific correlation between SNPs in the GR gene and IOP elevation. Therefore, we proceeded to perform a whole genome SNP screen with the DNA samples of these patients to search for possible target genes responsible for the elevated IOP after GC treatment. As a result, we identified forty-eight SNPs in thirty-three genes that correlate with the high IOP response. The gene showing the strongest association is a poorly known G-protein coupled receptor. In addition, four SNPs hit a single transporter gene. Other candidate genes identified are a translation elongation factor, an F-box protein, an oxysterol binding protein, and a solute carrier family gene. These results support our hypothesis that IOP elevation following GC treatment is a genetically determined response. GCs are a common treatment for innumerable medical conditions; we believe that a genetic association between GC treatment and its physiological response may be important for improving treatment management and drug development for retinal diseases as well as for other medical ailments. However, further studies need to be performed to analyze in depth the association between the candidate genes identified in this study and the steroid response.
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Van, Anh Dao [Verfasser], and Siegfried [Akademischer Betreuer] Roth. "Genome-wide RNAi screening and the analysis of candidate genes for dorsoventral patterning in Tribolium castaneum / Dao Van Anh. Gutachter: Siegfried Roth." Köln : Universitäts- und Stadtbibliothek Köln, 2014. http://d-nb.info/1054420459/34.
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Bicchieri, M. E. "A WHOLE-GENOME APPROACH TO IDENTIFY MICRORNA 'MODIFIERS' OF BREAST CANCER STEM CELL SELF-RENEWAL." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/265272.
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An emerging notion in the breast cancer field is that a rare subpopulation of cells within the tumor bearing stem cell (SC)-like properties, cancer stem cells (CSCs), are responsible for the degree of aggressiveness of the tumor, as well as the emergence of therapeutic resistance and disease relapse. Therefore, the development of novel therapeutic strategies that specifically target the CSCs population within a tumor could be the key to achieve an effective cure for breast cancer.
One strategy for targeting CSCs could be to inhibit their altered self-renewal mechanism and induce a quasi-normal differentiation process in tumor cells. However, the mechanisms that control the replicative mode of SC division and the degree of “stemness” of tumours are poorly characterized.
Recent research has highlighted the role of microRNAs (miRNAs), a class of small non-coding RNAs, as key regulators of gene expression in a variety of cellular processes, including SC self-renewal and differentiation. miRNAs negatively regulate gene expression at a post-transcriptional level and their expression is often deregulated in disease, making them ideal candidates as tumour biomarkers.
Despite recent studies uncovered new microRNA molecules linked to stem cell biology, we definitively miss a defined picture of which microRNAs are involved in the regulation of breast cancer stem cell self-renewal and their contribution to tumorigenesis.
The overall goal of this project was to identify key miRNA “modifiers” of breast cancer SC self-renewal that could either inhibit or enhance the self-renewal potential of cancer stem cells, with the purpose of identifying key molecules involved in the acquisition/regulation of stem-cell traits and bona fide novel therapeutic targets.
We used a lentiviral microRNA library, composed of approx. 650 precursor microRNAs, to perform a functional whole-genome screening based on phenotypic competition assays on a very aggressive breast cancer cell line with stem-like properties (SUM159). Infected cells were challenged in an in-vitro 3D competition assay based on self-renewal ability of CSCs (mammosphere propagation). In the competition assays, miRNAs that supported stem cells expansion were positively selected during passages, while microRNAs that inhibited self-renewal were depleted overtime. In parallel we also performed a 2D assay based on cell proliferation to gain insights into the ability of miRNAs to alter the proliferation of cancer cells in adherent conditions. For each screening the positive and the negative selected miRNAs were identified by means of Next Generation Sequencing analysis.
The screening yielded to 20 candidate microRNAs selected as potential modifiers of self-renewal: 18 presumably acting by decreasing self-renewal (and hypothetically with tumor suppressing functions) and 2 by increasing self-renewal (and potentially with oncogenic functions). A proof-of-principle validation revealed that 6 out of 10 tested cloned, confirmed their effects even when analyzed as single clones, underlining the potentiality of the whole-genome phenotype screening.
We focused our attention on the two most promising candidates and, in order to search for the mechanism through which these microRNAs exert their function, we performed an RNA-seq analysis of transcriptional changes induced upon their overexpression. We revealed that one microRNA in particular was able to regulate hundreds of genes and control independently different pathway related to self-renewal, migration and proliferation, suggesting that this miRNA could effectively act at multiple levels to silence the self-renewal potential of cancer stem cells and, likely, inhibit the proliferation and migratory ability of the tumor, too.
As further experiments, we will definitively need to completely understand the role of this microRNA in cancers with the potential of being effective even on the most aggressive breast cancer disease.
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Ghosh, Aniket [Verfasser], Mikael [Akademischer Betreuer] Simons, Herbert [Akademischer Betreuer] Jäckle, and Stefan [Akademischer Betreuer] Eimer. "Genome-wide RNAi screening reveals glial phosphoethanolamine ceramide is critical for axonal ensheathment / Aniket Ghosh. Gutachter: Mikael Simons ; Herbert Jäckle ; Stefan Eimer. Betreuer: Mikael Simons." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2012. http://d-nb.info/1044305770/34.
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Ghosh, Aniket Verfasser], Mikael [Akademischer Betreuer] [Simons, Herbert [Akademischer Betreuer] Jäckle, and Stefan [Akademischer Betreuer] Eimer. "Genome-wide RNAi screening reveals glial phosphoethanolamine ceramide is critical for axonal ensheathment / Aniket Ghosh. Gutachter: Mikael Simons ; Herbert Jäckle ; Stefan Eimer. Betreuer: Mikael Simons." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2012. http://nbn-resolving.de/urn:nbn:de:gbv:7-webdoc-3784-4.
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Löllgen, Ruth Mari Caroline [Verfasser], x. [Gutachter] Arnold, x. [Gutachter] Wiedenmann, and x. [Gutachter] Zeitz. "Genome-wide screening of loss of heterozygosity in human midgut carcinoid tumors with fluorescent technique / Ruth Mari Caroline Löllgen ; Gutachter: x. Arnold, x. Wiedenmann, x. Zeitz." Berlin : Humboldt-Universität zu Berlin, 2004. http://d-nb.info/1206197919/34.
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Cerrato, Giulia. "Oleate : An Atypical Cellular Stress Inducer That Stalls Protein Secretion Oleate-Induced Aggregation of LC3 at the Trans-Golgi Network Is Linked to a Protein Trafficking Blockade A Genome-Wide RNA Interference Screen Disentangles the Golgi Tropism of LC3 Live Cell Imaging of LC3 Dynamics." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL023.
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Les diverses classes d’acides gras (chaines carbonées saturées ou cis-/trans- insaturées) influencent la physiologie au niveau de la cellule et de l’organisme de façon différente. Curieusement, ces catégories distinctes ont un effet important (mais différent) sur l’autophagie, le mécanisme intracellulaire de dégradation qui maintient l’homéostasie énergétique et protège les cellules contre le stress. L’oléate, l’acide gras cis-insaturé endogène et alimentaire le plus abondant, possède la propriété atypique d’induire une redistribution de la protéine LC3 (signe particulier d’autophagie) de manière non-canonique et préférentiellement dans l’appareil de Golgi. Puisqu’il a été montré que, d’une part, les acides gras cis-insaturés présentent des effets bénéfiques pour la santé et que, d’autre part, les acides gras trans-insaturés et saturés induisent des effets toxiques, nous avons décidé d’explorer les mécanismes à la base de la redistribution de LC3 au niveau de l’appareil de Golgi induite par l’oléate. Cette analyse pourrait nous éclairer sur l’origine des différents effets des acides gras sur la santé. Pour cela, un criblage robotisé du génome entier par ARNs interférents a permis d’identifier plusieurs gènes impliqués dans le transport des protéines lié à l’appareil de Golgi, et également dans la réponse intégrée au stress.Des expériences supplémentaires ont montré que l’oléate impacte la morphologie subcellulaire de l’appareil de Golgi, en corrélation avec le blocage de la sécrétion protéique conventionnelle (dépendante du Golgi) lorsque que la cargaison est bloquée au niveau du réseau trans-golgien. L’inhibition de la sécrétion protéique a été observée dans plusieurs systèmes expérimentaux, tant in vitro qu’in vivo. De plus, un criblage visant à rechercher des agents chimiques capables d’induire les mêmes effets cellulaires que l’oléate, a permis d’identifier plusieurs composés appartenant à diverses classes pharmacologiques. De la même manière que l’oléate ces composés induisent un blocage de la sécrétion protéique conventionnelle, renforçant l’idée que cette voie de perturbation du Golgi joue un rôle pharmacologique important. En conclusion, ces résultats montrent que l’oléate représente une classe de molécules agissant sur l’appareil de Golgi pour y induire l’agrégation de LC3, tout en bloquant en même temps la sécrétion protéiqueDistinct classes of fatty acids (FAs) (saturated or cis-/trans-unsaturated carbon chains) impact on cellular and organismal physiology in a different manner. Interestingly, these diverse categories have a profound (but different) effect on autophagy, the conserved intracellular degradation mechanism that maintains energy homeostasis and protects cells against stress. Oleate, the most abundant endogenous and dietary cis-unsaturated FA, has the atypical property to induce the redistribution of the LC3 protein (peculiar sign of autophagy) in a non-canonical fashion and preferentially to the Golgi apparatus. Intrigued by these observations, which might be related to the health-improving effects of cis-unsaturated FAs (and the notorious toxicity of trans-unsaturated and saturated FAs), we decided to explore the mechanisms causing the oleate-induced relocation of LC3 to the Golgi apparatus. To achieve this goal, a robotized RNA interference genome-wide screen led to the identification of multiple genes involved in the Golgi-related protein transport, as well as in the integrated stress response. Follow-up experiments revealed that oleate affected the subcellular morphology of the Golgi apparatus, correlating with a blockade of conventional (Golgi-dependent) protein secretion that caused secretory cargo to be stalled at the level of the trans-Golgi network. The inhibition of protein secretion was observed using several experimental systems, both in vitro and in vivo. Moreover, a systematic screen searching for other chemical entities that mimic the oleate-induced cellular effects led to the identification of several compounds belonging to rather different pharmacological classes. These “oleate mimetics” also shared with oleate the capacity to block conventional protein secretion, supporting the notion that this pathway of Golgi perturbation is indeed of pharmacological relevance. In conclusion, this research work shows that oleate represents a class of molecules that act on the Golgi apparatus to cause the recruitment of LC3 and to stall protein secretion
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Ndiaye, Fatou Kiné. "Étude post-GWAS des gènes de susceptibilité au diabète de type 2 : rôle phare dans la fonction de la cellule β pancréatique." Thesis, Lille 2, 2017. http://www.theses.fr/2017LIL2S039/document.
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Les études d’association pangénomique (GWAS) ont permis la mise en évidence de nouvelles voies putativement importantes dans la physiopathologie du diabète de type 2, par l’identification de variants génétiques fréquents (SNP) de susceptibilité au diabète de type 2, mais souvent avec peu ou pas d'informations sur le mécanisme sous-jacent expliquant le lien entre ces variants génétiques et le phénotype diabétique. En effet ces SNP sont souvent non codants et ont un effet modeste sur le risque de diabète de type 2, ce qui rend difficile leur étude d’un point de vue fonctionnel. Dès le début des GWAS, il a été suggéré que ces gènes associés au diabète de type 2, étaient des « gènes de la cellule β pancréatique » sans que des études fonctionnelles n’aient été faites de manière systématique. Dans ce contexte, nous avons mené une étude de fishing pour déblayer cette quantité importante de données provenant des GWAS et d’identifier des gènes potentiellement importants, pouvant être de nouvelles cibles thérapeutiques. Le premier objectif de ma thèse a été l’étude de l’expression des gènes de susceptibilité au diabète de type 2 dans un panel de tissus humains comprenant des tissus pancréatiques et des tissus sensibles à l’insuline. Pour cela nous avons utilisé une technique de quantification non biaisée de l’expression génique dans le but de montrer si ces gènes associés au diabète de type 2 avaient une expression enrichie (proportion de gènes de susceptibilité au diabète de type 2 surexprimés dans les cellules β versus les autres tissus) dans les cellules β pancréatiques. Nous avons ensuite réalisé des études fonctionnelles sur la trentaine de gènes de susceptibilité au diabète de type 2 les plus exprimés dans notre modèle cellulaire par des tests de sécrétion d’insuline, des études de la viabilité cellulaire, du séquençage d’ARN (RNA-seq) et du western blotting dans la lignée de cellules β pancréatiques humaines EndoC-βH1. Les EndoC-βH1 sont des cellules en mesure de sécréter de l’insuline en réponse au glucose et à d’autres sécrétagogues. Nous les avons utilisé afin d’étudier le rôle de ces gènes de susceptibilité au diabète de type 2 dans la fonction de la cellule β pancréatique, en particulier dans la sécrétion insulinique. Notre étude d’expression a montré que l’expression des gènes de susceptibilité au diabète de type 2 est enrichie de manière significative dans les cellules β pancréatiques et la lignée EndoC-βH1. Pour cinq gènes du diabète de type 2 (TBC1D4, TCF19, KCNK16, CDKN2A et SLC30A8) ayant une présence et un effet déjà connus dans la fonction des cellules β, nous avons démontré une variation significative de la sécrétion d’insuline après extinction génique, en concordance avec la littérature. Par ailleurs, nous avons pu mettre en évidence quatre gènes de susceptibilité au diabète de type 2 (PRC1, SRR, ZFAND3 et ZFAND6) montrant une baisse significative de la sécrétion d’insuline après extinction génique et dont la présence ou la fonction dans la cellule β était pour l’heure inconnue. Les analyses RNA-seq ont montré une association significative de l’extinction de ces gènes avec des réseaux moléculaires liés à la physiopathologie du diabète de type 2 (par exemple : l’apoptose des cellules pancréatiques, l’insulinémie, la glycolyse, le stress du réticulum endoplasmique…). Et l’évaluation de l’expression de nos quatre gènes dans des îlots de souris obèses (ob/ob) ou traitées à la streptozotocine a montré une corrélation positive de leur expression avec celle de l’insuline. Notre étude a démontré que les études fonctionnelles post-GWAS sont importantes et permettent de définir le lien de causalité des gènes de susceptibilité avec la maladie, et ainsi de mener à des progrès sur la compréhension de la physiopathologie de la maladie [...]Genome-wide association studies (GWAS) have identified a plethora of single nucleotide polymorphisms (SNPs) associated with the risk of type 2 diabetes, but most often with little information about the mechanism underlying the relationship between these genetic variants associated with type 2 diabetes and the diabetic phenotype. Indeed, these SNPs are often noncoding and have a modest effect on the risk of type 2 diabetes, making difficult their functional study. At the beginning of the GWAS era, it has been suggested that susceptibility genes for type 2 diabetes are strongly involved in pancreatic β cell gene function, while no functional studies had been systematically performed. In this context, we conducted a “fishing” study to decipher this large amount of data generated by GWAS and to pinpoint potentially important genes that may be new therapeutic targets. The first objective of my thesis was to study the expression of type 2 diabetes susceptibility genes in a panel of human tissues comprising pancreatic and insulin-sensitive tissues using an unbiased technique of quantification of genes expression in order to show that these genes associated with type 2 diabetes were enriched in pancreatic β-cells. We then performed functional studies on the thirty mostly expressed genes in our cell model by insulin secretion tests, cell viability test, RNA sequencing (RNA-seq) and Western blotting in the human pancreatic β cell line (EndoC-βH1). These cells are able to secrete insulin in response to glucose and other secretagogues. Our goal was to study the role of these type 2 diabetes susceptibility genes in pancreatic β cell function, particularly in insulin secretion. Our expression study of type 2 diabetes susceptibility genes showed that their expression is significantly enriched in pancreatic β cells and the EndoC-βH1 cell line. For five genes associated with type 2 diabetes (TBC1D4, TCF19, KCNK16, CDKN2A and SLC30A8) with an already known presence and function in pancreatic β cell, we showed a significant variation in glucose-stimulated insulin secretion after gene silencing, in agreement with the literature. In addition, we identified four type 2 diabetes associated genes (PRC1, SRR, ZFAND3 and ZFAND6), with a significant decrease in insulin secretion after gene silencing without already know function in pancreatic β cell. RNA-seq has shown a significant association between the extinction of these genes and molecular networks related to the pathophysiology of type 2 diabetes (e.g. apoptosis of pancreatic cells, insulinemia, glycolysis, endoplasmic reticulum stress response...). The assessment of the expression of our four genes in the islets of obese mice (ob/ob) or treated with streptozotocin shows a positive correlation between their expression and the expression of insulin. Our study has shown that post-GWAS functional studies are important and can help to define the causal link between these genes and the disease, and therefore to make progress in the understanding of the pathophysiology of type 2 diabetes. This study allowed us to identify genes whose function in β cell was not anterior known and which are involved in pancreatic β cell function and the pathophysiology of type 2 diabetes
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Merker, Sören [Verfasser], Klaus-Peter [Gutachter] Lesch, and Erhard [Gutachter] Wischmeyer. "Genome-wide screenings in attention-deficit/hyperactivity disorder (ADHD): investigation of novel candidate genes SLC2A3 and LPHN3 / Sören Merker. Gutachter: Klaus-Peter Lesch ; Erhard Wischmeyer." Würzburg : Universität Würzburg, 2014. http://d-nb.info/110974997X/34.
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"Genome wide screening of genetic aberrations in nasopharyngeal carcinoma." 2002. http://library.cuhk.edu.hk/record=b6073440.
Full textAbstract:
Bik-Yu Hui."July 2002."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (p. 187-203).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.
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Chung, Pei-Chen, and 鍾北辰. "Genome-wide screening for yeast essential genes involved in chronological aging." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/z924r9.
Full textAbstract:
碩士國立陽明大學
生命科學系暨基因體科學研究所
105
Aging is a leading factor for many diseases, which plagues people around the world. Budding yeast Saccharomyces cerevisiae (S. cerevisiae) has been used to study the mechanisms of aging for more than two decades because of its well-established genome information, and various genome-wide mutation library. Chronological lifespan (CLS), the length of time that non-dividing cell survives, is one of the important paradigms of aging studies in S. cerevisiae. There are 1,235 genes annotated to regulate CLS, while only 5 genes are essential genes. I think the result are biased since most studies use yeast deletion library to perform large-scale screening. However, the essential genes are vital for cell survive and not involved in deletion library. In this study, we took advantage of Yeast Tet-promoter Hughes Collection, in which tetracycline-regulatable promoter was used to replace each native promoter of individual essential gene, to study the role of essential genes in aging process. We first developed a microscope-based high throughput CLS assay. Results demonstrated that CLS of yTHC strains were really affected by gene mis-regulation caused by promoter replacement. Further analysis showed that the low viability group have more total and physical interaction partners and suppose that proteins involved in protein complex are more sensitive to gene mis-regulation. Functional enrichment analysis also showed that some essential genes, which involved in protein complexes, might affect CLS once they got mis-regulated. I proposed that dosage imbalance of proteins involved in protein complex might easily cause proteotoxicity stress and result in shorten CLS. The results provided insights on the roles of essential genes for CLS, gave us a hint that some essential genes were more sensitive to gene mis-reguation and worth for further studies.
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Li, Wei-Hsiao, and 李偉孝. "Identification of Radioprotectors in Human Hepatocellular Carcinoma by Genome-Wide RNAi Screening." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/v458jn.
Full textAbstract:
碩士國立臺灣大學
生物化學暨分子生物學研究所
101
Hepatocellular carcinoma (HCC), the third most prevalent malignant tumor in Taiwan, has a poor prognosis due to high rates of recurrence and metastasis. Radiotherapy is one of the modalities in cancer treatments. With the recent development of stereotactic radiosurgery technology, physicians can deliver precise doses of energy to an exact location (i.e. the tumor) and thus limit collateral damage to surrounding normal tissues. Despite such an advanced technology, radiotherapy has not yet been incorporated into standard management guidelines of HCC because of the unsatisfactory clinical outcomes. In order to improve the efficacy of radiotherapy in treating HCC, we carried out a genome-wide RNA-interference (RNAi) screen in Huh7 cells (a human HCC cell line) and identified many genes as radiation protector candidates (i.e. knockdown of such genes increases the sensitivity of Huh7 cells to a predetermined dose of radiation) which might be used as potential targets for drug design to enhance radiation sensitivity of HCC. We identified twenty-one candidates from the screening results, including sixteen radioprotectors and five radiosensitizers. All of the candidates were validated by colony formation assay. Among these candidates, clusterin is the most significant radio-protector. Here we demonstrated that suppression of clusterin expression enhances the radiation-mediated cell death. Furthermore, the phenomenon caused by clusterin suppression is specific for ionizing radiation, but not for ultraviolet (UV) radiation. However, the detailed mechanisms and functions of clusterin need to be further studied and elucidated in the future.
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Ghosh, Aniket. "Genome-wide RNAi screening reveals glial phosphoethanolamine ceramide is critical for axonal ensheathment." Doctoral thesis, 2012. http://hdl.handle.net/11858/00-1735-0000-000D-EF82-A.
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Ramos, Pedro André Dias. "Genome-wide shRNA screening identifies genes involved in fulvestrant resistance in breast cancer." Master's thesis, 2016. http://hdl.handle.net/10362/19135.
Full textAbstract:
Breast cancer accounts as the most prevalent cancer and the leading cause of cancer death worldwide among women. Estrogen is one main factor responsible for tumour growth in breast cancer patients through stimulation of estrogen receptor (ER) signalling. ER positive (ER+) breast cancer patients are eligible for anti-estrogenic drugs. Fulvestrant (Faslodex®) represents a second-line therapy for the treatment of postmenopausal women with ER+ advanced breast cancer. Unfortunately, a significant
number of ER+ patients will develop resistance to second-line fulvestrant treatment. It is therefore important to understand the molecular mechanisms of resistance and to identify biomarkers capable of predicting response to this treatment.
The aim of this project is to establish a genome-wide shRNA functional screening to identify key proteins central in resistance mechanisms and potentially predictive biomarkers capable of identifying ER+ patients that are responsive or resistant to fulvestrant treatment. To do so, a MCF-7-based fulvestrant resistant breast cancer cell line was used. MCF-7/LCC1 and MCF-7/LCC9 were transduced with shRNA libraries covering over 15,000 mRNAs and treated with fulvestrant. This led to depletion and/or
enrichment of shRNAs targeting genes evaluated by next generation sequencing (NGS). Deconvolution of NGS data from genomic DNA of LCC1 and LCC9 cells transduced by shRNA libraries led to identification of 206 genes that may have functional significance in fulvestrant resistance. Ingenuity Pathway Analysis of the candidate genes identified HSD17B10 and HSPE1 as key-molecules in networks related to cell proliferation and death. We have found that these genes are upregulated in different fulvestrant-resistant cell lines when compared to their fulvestrant-sensitive parental cell line at gene and protein expression levels using RT-qPCR and Western blotting. This expression is enhanced in fulvestrant presence, suggesting that these proteins may have critical importance in the resistance phenotype. Further studies on these proteins may elucidate on how to overcome fulvestrant resistance.
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Wu, Yi-Hsin, and 吳以新. "Establishing CRISPR interference-based genome-wide screening platform for identification of novel genes in macrophage alternative polarization." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/m99x3g.
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碩士國立臺灣大學
分子醫學研究所
107
Macrophages are crucial players in immune regulation. They have a wide spectrum of activation states depend on the diverse surrounding stimuli they receive. Classical activation (M1) and alternative activation (M2) are described as two extremes of their polarized states, which elicit pro-inflammatory responses and anti-inflammatory responses respectively to maintain tissue homeostasis. Regnase-1 is a ribonuclease essential in controlling immune responses by regulating mRNA decay of proinflammatory cytokines, and it is reported to be important in promoting macrophage M2 polarization in which ER stress, ROS and autophagy are involved. However, detailed regulatory mechanism of this pathway is remained unclear. The goal of our study is to perform a genome-scale CRISPRi-dCas9 screening to explore new regulators in Regnase-1 mediated M2 polarization. By flow cytometry detection of M2 markers expression, we can identify genes that after CRISPRi disruption and Regnase-1 overexpression lead to decreased M2 expression, as potential regulators in this pathway. We have tested and compared the M2 phenotypes of four mice macrophage cell lines and examined the M1/M2 discrimination of several M2 markers by flow cytometry analysis. Our results demonstrated the M2 discriminating ability of Egr2 and CD206, which by flow cytometry detection can together be used to distinguish M2 phenotypes in both BMDMs and immortalized BMDMs. We have also established CRISPRi-Regnase-1 and inducible Regnase-1 overexpression system for further proof-of-principle screening and the preparations of the large-scale screening. Our data also infer a potential relation between ER stress related protein and M2 polarization, which is to be further investigated in the future works.
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Sanchez, Torres Viviana. "Escherichia coli Enhanced Hydrogen Production, Genome-wide Screening for Extracellular DNA, and Influence of GGDEF Proteins on Early Biofilm Formation." Thesis, 2010. http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-8889.
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Escherichia coli is the best characterized bacterium; it grows rapidly, and it is easy to
manipulate genetically. An increased knowledge about the physiology of this model organism
will facilitate the development of engineered E.coli strains for applications such as production of
biofuels and biofilm control. The aims of this work were the application of protein engineering
to increase E. coli hydrogen production, the identification of the proteins regulating extracellular
DNA production (eDNA), and the evaluation of the effect of the proteins synthesizing the signal
3'-5'-cyclic diguanylic acid (c-di-GMP) on biofilm formation.
The Escherichia coli hydrogen production rate was increased 9 fold through random
mutagenesis of fhlA. Variant FhlA133 (Q11H, L14V, Y177F, K245R, M288K, and I342F)
enhances hydrogen production by increasing transcription of the four transcriptional units
regulated by FhlA. The amino acid replacements E363G and L14G in FhlA increased hydrogen
production 6 fold and 4 fold, respectively.
The complete E. coli genome was screened to identify proteins that affect eDNA
production. The nlpI, yfeC, and rna mutants increased eDNA production and the hns and rfaD
mutants decreased eDNA production. Deletion of nlpI increases eDNA 3 fold while
overexpression of nlpI decreases eDNA 16 fold. Global regulator H-NS is required for eDNA with E. coli since deletion of hns abolished eDNA production while overexpression of hns
restored eDNA to 70 percent of the wild-type levels. Our results suggest that eDNA production in E.
coli is related to direct secretion.
Deletions of the genes encoding the diguanylate cyclases YeaI, YedQ, and YfiN
increased swimming motility and eDNA as expected for low c-di-GMP levels. However,
contrary to the current paradigm, early biofilm formation increased dramatically for the yeaI (30
fold), yedQ (12 fold), and yfiN (18 fold) mutants. Hence, our results suggest that c-di-GMP
levels should be reduced for initial biofilm formation because motility is important for initial
attachment to a surface.
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Löllgen, Ruth Mari Caroline [Verfasser]. "Genome wide screening of loss of heterozygosity in human midgut carcinoid tumors with fluorescent technique / von Ruth Mari Caroline Löllgen." 2004. http://d-nb.info/972556702/34.
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Peter, Josephine Jasmine. "Identification of yeast genes enabling efficient oenological fermentation under nitrogen-limited conditions." Thesis, 2018. http://hdl.handle.net/2440/113360.
Full textAbstract:
Nitrogen deficiency can often lead to slow or sluggish fermentation, resulting in wine out of specification and at risk of oxidation and microbial contamination. Problems due to nitrogen deficiency can be rectified by optimising grape chemistry (through vineyard fertilization), or more commonly supplementing the fermentation with ammonium salts. An alternative is to use wine yeast that can utilize nitrogen efficiently and complete fermentation more reliably. However, to develop ‘nitrogen efficient’ yeast, it is important to understand how such yeast can utilize nitrogen effectively by identifying genes that influence fermentation performance over a range of nitrogen concentrations. Past research related to the identification of genes influencing nitrogen efficiency under fermentative conditions is largely confined to laboratory yeast. Investigation of the ~5,000 non-essential genes in yeast is possible through research tools such as deletion libraries (collections of strains, each with a single gene deletion). Several genomewide studies have successfully used deletion libraries in the auxotrophic background of laboratory yeast to investigate phenotypes in response to exposure to single stress factors associated with fermentation. However, the need to supplement with amino acids to overcome auxotrophies makes quantitative physiological studies in nitrogen limiting conditions impractical. Therefore, in this study, we have used a prototrophic deletion collection in both laboratory and wine yeast backgrounds to identify genes influencing fermentation performance. Screening (micro-fermentation; 600 μL) of the prototrophic laboratory yeast deletion library (BY4741; 5,372 deletants) and the partial wine yeast library (AWRI1631; 1,844 deletants) for growth and consumption of sugar and nitrogen under limiting (75 mg FAN L⁻¹) and non-limiting nitrogen (450 mg FAN L⁻¹) conditions identified deletants with improved fermentation. To better understand the role of individual genes in fermentation, candidate gene sets from each screen were compared to each other and to other published data sets from genome wide transcriptomic analyses related to fermentation. Wine yeast deletants that enabled shortened micro-fermentation duration in low nitrogen conditions were further investigated, since the experiment best represented nitrogen deficient grape must associated with problematic fermentation. Fifteen deletants completed fermentation quicker than the wildtype (c.a. a 15-59% time reduction) when tested in larger (100 mL) fermentations. This group of genes were annotated to biological processes including protein modification, transport, metabolism and ubiquitination (UBC13, MMS2, UBP7, UBI4, BRO1, TPK2, EAR1, MRP17, MFA2 and MVB12), signalling (MFA2) and amino acid metabolism (AAT2). Among the genes identified, MFA2 (mating a-factor), which conferred a 34% decrease in fermentation duration, was further investigated. We were interested to understand how deletion of this mating type gene affected fermentation since a link between these metabolic pathways would be novel. The 15 strains identified in this study, which were fermentation proficient in a ‘wine-like', limited nitrogen condition, provide a basis to better understand how yeast adapt to nitrogen limitation during fermentation. Furthermore, the corresponding genes can be targeted in second generation strain improvement programs, using tools such as CRISPR (yet to be approved by relevant regulatory bodies) to generate nitrogen efficient yeast to reduce the need to supplement low nitrogen fermentations.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2018.
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Merker, Sören. "Genome-wide screenings in attention-deficit/hyperactivity disorder (ADHD): investigation of novel candidate genes SLC2A3 and LPHN3." Doctoral thesis, 2014. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-100129.
Full textAbstract:
Attention-deficit/hyperactivity disorder (ADHD) is a highly prevalent childhood-onset neurodevelopmental disorder that involves a substantial risk of persisting into adolescence and adulthood. A number of genome-wide screening studies in ADHD have been conducted in recent years, giving rise to the discovery of several variants at distinct chromosomal loci, thus emphasising the genetically complex and polygenic nature of this disorder. Accordingly, promising novel candidate genes have emerged, such as the gene encoding the glucose transporter isoform 3 (SLC2A3) and the gene encoding the latrophilin isoform 3 (LPHN3). In this thesis, both genes were investigated in form of two separated projects. The first focused on SLC2A3 polymorphisms associated with ADHD and their potential physiological impact. For this purpose, gene expression analyses in peripheral cell models were performed as well as functional EEG measurements in humans. The second project concerned the murine gene Lphn3 including the goal of developing a mouse line containing a genetically modified Lphn3 with conditional knockout potential. In this respect, a specific DNA vector was applied to target the Lphn3 gene locus in murine embryonic stem (ES) cells as a prerequisite for the generation of appropriate chimeric mice. The results of the first project showed that SLC2A3 duplication carriers displayed increased SLC2A3 mRNA expression in peripheral blood cells and significantly altered event-related potentials (ERPs) during tests of cognitive response control and working memory, possibly involving changes in prefrontal brain activity and memory processing. Interestingly, ADHD patients with the rs12842 T-allele, located within and tagging the SLC2A3 gene, also exhibited remarkable effects during these EEG measurements. However, such effects reflected a reversed pattern to the aforementioned SLC2A3 duplication carriers with ADHD, thus indicative of an opposed molecular mechanism. Besides, it emerged that the impact of the aforementioned SLC2A3 variants on different EEG parameters was generally much more pronounced in the group of ADHD patients than the healthy control group, implying a considerable interaction effect. Concerning the second project, preliminary results were gathered including the successful targeting of Lphn3 in murine ES cells as well as the production of highly chimeric, phenotypically unremarkable and mostly fertile mouse chimeras. While germline transmission of the modified Lphn3 allele has not yet occurred, there are still several newborn chimeric mice that will be tested in the near future. In conclusion, the findings suggest that SLC2A3 variants associated with ADHD are accompanied by transcriptional and functional changes in humans. Future research will help to elucidate the molecular network and neurobiological basis involved in these effects and apparently contributing to the complex clinical picture of ADHD. Moreover, given the increasing number of publications concerning latrophilins in recent years and the multitude of research opportunities provided by a conditional knockout of Lphn3 in mice, the establishment of a respective mouse line, which currently is in progress, constitutes a promising approach for the investigation of this gene and its role in ADHDDas Aufmerksamkeitsdefizit/Hyperaktivitätssyndrom (ADHS) ist eine hoch prävalente und bereits in der Kindheit beginnende Neuroentwicklungsstörung, die eine erhebliche Persistenz ins Jugend- und Erwachsenenalter aufweist. In den vergangenen Jahren wurde eine Vielzahl von genomweiten Studien zu ADHS durchgeführt, welche zur Identifizierung zahlreicher genetischer Varianten an unterschiedlichen chromosomalen Loci geführt und somit die genetisch komplexe polygene Natur dieser Störung zur Geltung gebracht haben. Auf diese Weise traten auch neue Kandidatengene zutage, wie zum Beispiel das Gen für die Glukosetransporter-Isoform-3 (SLC2A3) und das Gen, welches Latrophilin-3 kodiert (LPHN3). Innerhalb dieser Thesis wurden beide Gene in Form von zwei voneinander getrennten Projekten untersucht. Das erste Projekt beschäftigte sich mit humanen ADHS-assoziierten SLC2A3-Polymorphismen und ihrer potentiellen physiologischen Bedeutung. Für diesen Zweck wurden Genexpressionsanalysen in peripheren Zellmodellen sowie funktionelle EEG-Messungen im Menschen durchgeführt. Im zweiten Projekt ging es um das murine Gen Lphn3 mit dem Ziel, eine Mauslinie zu entwickeln, die ein genetisch verändertes Lphn3 mit konditionalem Knockout-Potenzial aufweist. In diesem Zusammenhang wurde ein spezifischer DNA-Vektor verwendet, der auf den Lphn3-Genlocus in murinen embryonalen Stammzellen (ES-Zellen) abzielte, was eine Voraussetzung für die Erzeugung von geeigneten chimären Mäusen darstellt. Die Ergebnisse des ersten Projektes legten nahe, dass SLC2A3-Duplikationsträger erhöhte SLC2A3-mRNA-Expression in peripheren Blutzellen aufweisen sowie signifikant veränderte ereigniskorrelierte Potentiale während eines Tests kognitiver Reaktionskontrolle sowie eines Arbeitsgedächtnis-Tests, was möglicherweise von veränderter präfrontaler Hirnaktiviät bzw. Gedächtnis-Prozessierung begleitet wird. Interessanterweise zeigten ADHS-Patienten mit T-Allel des im SLC2A3-Gen liegenden SNPs rs12842 ebenfalls deutliche Effekte während dieser EEG-Messungen, allerdings in entgegengesetzter Form zu den zuvor genannten SLC2A3-Duplikationsträgern mit ADHS, was auf einen gegensätzlichen molekularen Mechanismus hindeutet. Zudem stellte sich heraus, dass der Einfluss der zuvor genannten SLC2A3-Varianten auf verschiedene EEG-Parameter innerhalb der ADHS-Gruppe generell deutlich stärker ausgeprägt war als in der gesunden Kontrollgruppe, also einen beachtlichen Interaktionseffekt impliziert. Bezüglich des zweiten Projektes konnten bisher Zwischenergebnisse erzielt werden: das erfolgreiche Targeting des Lphn3-Gens in murinen ES-Zellen sowie die Produktion hochchimärer, phänotypisch unauffälliger und größtenteils fertiler Maus-Chimären. Obgleich die Keimbahntransmission des modifizierten Lphn3-Allels bislang noch nicht eingetreten ist, gibt es noch eine Reihe an neugeborenen chimären Mäusen, die in nächster Zeit erst noch getestet werden müssen. Zusammenfassend deuten die Ergebnisse darauf hin, dass Variationen des SLC2A3-Gens, die mit ADHS assoziiert sind, mit transkriptionellen und funktionellen Veränderungen im Menschen einhergehen. Zukünftige Forschungsarbeiten werden dabei helfen, die molekularen Netzwerke und neurobiologischen Grundlagen zu verdeutlichen, die an diesen Effekten beteiligt sind und offenbar zu dem komplexen klinischen Bild von ADHS beitragen. Angesichts der steigenden Zahl an Publikationen über Latrophiline in den letzten Jahren und der unzähligen Forschungsmöglichkeiten, die ein konditionaler Knockout von Lphn3 in Mäusen bietet, stellt die derzeit laufende Etablierung einer entsprechenden Mauslinie einen vielversprechenden Ansatz dar, dieses Gen und seine Rolle für ADHS zu untersuchen