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1
Wilson, Jennifer L. (Jennifer Lynn). "Network analyses for functional genomic screens in cancer." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104236.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 128-151).
Gene interference screens are a widely adopted and popular tool for uncovering gene function but imperfections in the technology limit the power of these investigations. There are many completed and on-going RNAi investigations across a multitude of biological systems because these experiments are scalable, cost-effective, and relatively easily adapted to multiple experimental environments. The most influential disadvantage is that many of the individual reagents are non-specific and interfere with genes other than the intended target. Efforts to improve limitations in RNAi have focused on statistical models and improving reagents, yet have not explored using biological context to select gene targets. This thesis uses network modeling and data integration to provide context for gene interference studies, and demonstrates the utility of this approach in two systems: Acute Lymphoblastic Leukemia (ALL) is a disease of undifferentiated B-cells that results from accumulation of genetic lesions, yet we have an incomplete understanding of all genes contributing to the disease and how they interact. To discover genetic mediators of this disease, we employ a genome-scale shRNA screen, and complement this data with differential mRNA expression and ChIP-seq data using network integration. The integrated model identifies processes not represented in any input set and predicts novel genes contributing to disease. We specifically validate the role of Wwpl as a tumor suppressor in ALL. Aberrant growth factor pathway activity drives cancer pathology and is the target of molecular cancer therapies. Specifically, the epidermal growth factor receptor (EFGR) pathway and its ligand, transforming growth factor alpha (TGF[alpha]) are clinically relevant to gastric cancer. We use an shRNA screen and Prize Collecting Steiner Forest (PCSF) algorithm to discover the pathway regulating TGF shedding. This pathway identifies common regulators of TGF[alpha] shedding and NF[chi]B regulation, yet targeting NF[chi]B and the EGFR pathway has thus far been unsuccessful in cancer therapies. Our network identifies IRAK1 as a viable path forward for modulating both TGF[alpha] and NF[chi]B in gastric cancer.
by Jennifer L. Wilson.
Ph. D.
2
Burrows, Anna. "Genome-Wide Loss-of-Function Genetic Screens Identify Novel Senescence Genes and Putative Tumor Suppressors." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10191.
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During every cell cycle and upon exogenous stress, tumor suppression programs are engaged to ensure genomic stability. In response to replicative aging and oncogenic stimuli, the p53 and Rb pathways are activated to prevent the proliferation of damaged cells. Several lines of evidence suggest that escape from senescence is a crucial early step in oncogenic progression. A major challenge in the cancer field is to combine genomic information regarding cancer-associated genetic changes with high-throughput functional studies, in order to confirm genetic requirements and pinpoint biological roles of these perturbed genes in oncogenesis. Furthermore, a complete genetic understanding of replicative senescence, and how it might be bypassed, is lacking. We describe here two genome scale loss-of-function genetic screens that interrogate these tumor suppressor programs. We utilized a unique sensitization approach to isolate senescence pathways and unmask compensatory mechanisms that may have been difficult to identify in previous studies. These genetic screens have generated comprehensive and validated datasets of putative senescence and p53 pathway genes. We present this dataset as a high-quality resource for further investigation into these biological pathways. We have uncovered several genes in distinct biological pathways which have not been demonstrated to have a functional role in senescence, and which may be putative tumor suppressors. We have identified BRD7 and BAF180, two SWI/SNF components, as critical regulators of p53. BRD7 and BAF180 are required for p53 activity and p21 expression during replicative and oncogene-induced senescence, and evidence suggests that they are inactivated in human cancer. In addition, we have uncovered a role for the deubiquitinating enzyme USP28 in the regulation of p53 accumulation during senescence, such that loss of USP28 results in bypass of the senescence program. We have also investigated several other novel senescence genes including SEMA6A, SEMA3b, and TMEM154. We have found that the expression of these genes is highly regulated during senescence by distinct means, including both p53-dependent and p53-independent mechanisms. These results demonstrate the efficacy of our sensitized screening approach, and also highlight the emerging view that the senescence program requires the combined action of multiple biological pathways for its execution.
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Pickering, H. "Identification of Chlamydia trachomatis immune targets through immunological and population-genomic screens and elucidation of potential roles in bacterial pathogenesis." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2017. http://researchonline.lshtm.ac.uk/3928322/.
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Ocular infection by Chlamydia trachomatis (Ct) results in trachoma, the leading infectious cause of blindness. Infection clears naturally, but repeated exposure in endemic areas and resulting inflammation promote tissue damage leading eventually to blinding sequelae. Antibiotic treatment as part of community-based intervention reduces prevalence of infection and disease but rarely eliminates the problem completely and progression to scarring and blindness does still occur. Sixty years of vaccine trials have produced variable results therefore new candidate antigens and better understanding of the underlying causes of infection and disease are required. Serum samples from trachoma-endemic communities in The Gambia were tested against the arrayed Ct proteome to identify antibody responses associated with protection from infection and from scarring disease. More focussed global antibody profiles were associated with partial immunity to infection. Several antibody targets were identified as individually associated with infection and disease outcome. Clinical Ct isolates collected from Guinea-Bissau were screened for evidence of natural selection to identify further immune targets and to validate those discovered through serological techniques. Evidence of positive selection was found for known Ct virulence factors, there was little evidence of balancing selection. Antibody targets associated with susceptibility to infection and scarring had evidence of purifying selection. One of the Ct antigens, CT442, identified as being an immune target and under natural selection was characterised further using cell-culture models. It was localised to the inclusion membrane through immunofluorescence microscopy, the primary point of contact with the host, and potentially interacted with pathways involved in intracellular vesicular trafficking based on interacting proteins identified through mass spectrometry. Ct infection is shown to stimulate a broad, polyclonal antibody response, individuals with more focussed responses are better protected from persistent infection and scarring progression. Purifying selection in antibody targets which associate with poor resolution of infection suggests two possible hypotheses for Ct evasion of immune responses. The decoy hypothesis, in which Ct actively promotes immune responses against irrelevant, decoy antigens to divert antibody responses away from protective antigens, and the blocking hypothesis, in which antibodies against certain Ct surface antigens block the binding of neutralising antibodies. Evidence of selection in CT442 show it is important but unlikely essential for Ct survival, the functions that are driving this evolution require further study.
4
Allan, Kristina Jean. "Enhancing Oncolytic Virotherapy Using Functional Genomic Screening." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37910.
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Li, Shuzhao. "A genomic screen for Zic1 target genes in neural development." Thesis, Montana State University, 2006. http://etd.lib.montana.edu/etd/2006/li/LiS0806.pdf.
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Pedro, Rodrigues Joana Cristina. "Yeast genome-wide telomere screens and insights into cancer." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3898.
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Telomeres are the very ends of linear eukaryotic chromosomes and when too short or dysfunctional they can trigger senescence (ageing). If the cell can bypass senescence, it can lead to genetic instability or cancer. Telomere capping proteins such as the CST (Cdc13, Stn1 and Ten1) complex and Yku70 are essential for the telomeres not to be recognised as double strand breaks. In this thesis I have used published yeast genome-wide screens to identify genes that are relevant to cancer and telomere biology. Overall 14 out of 19 genetic interactions identified by genome-wide screens could be confirmed by small scale experiments. This work mainly focuses on the telomeric roles of VPS74 and the PAF1 complex. Here, I show that the Golgi gene VPS74, whose human orthologue, GOLPH3, is an oncogene, genetically interacts with telomere capping genes and DNA damage response genes. I demonstrate that Vps74 is important for cell fitness of yku70Δ cells and that the low fitness of Vps74 depleted cells is dependent on the presence of DNA damage checkpoint proteins. I have also systematically investigated the roles of PAF1 complex (Cdc73, Paf1, Ctr9, Leo1 and Rtf1, in yeast) components on telomere biology. The conserved PAF1 complex affects RNA abundance in eukaryotes. I demonstrate that individual PAF1 complex components perform different functions at telomeres. I show that loss of Cdc73 improves fitness of telomere defective yeast cells, while loss of other PAF1 components has the opposite effect. Moreover, I show that Paf1 and Ctr9 strongly reduce telomeric repeat-containing non-coding RNA (TERRA), while Cdc73, Leo1 and Rtf1 have little effect. Paf1 and Ctr9 function independently of Sir4 to regulate TERRA and this is because they stimulate TERRA decay, as well as decay of other RNAs. Additionally, I found that Paf1 and Ctr9 decrease TEN1 and STN1 mRNA levels. I suggest that the PAF1 complex plays a specialized role at telomeres, with Paf1 and Ctr9 maintaining telomere integrity and Cdc73 decreasing the fitness of telomere defective cells.
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Giedraitis, Vilmantas. "Candidate gene analyses and genome-wide screens in multiple sclerosis /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-408-9/.
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Sawcer, Stephen James. "A linkage genome screen in multiple sclerosis." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627121.
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Jarvis, Morgan L. "Development of a novel screen protocol for the identification of genes causing replication associated genomic instability in Schizosaccharomyces pombe." Thesis, Kingston, Ont. : [s.n.], 2008. http://hdl.handle.net/1974/1227.
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Deligiannaki, Myrto. "Identification of novel septate junction components through genome-wide glial screens." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-183079.
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Epithelial barriers are central to the development of metazoans by compartmentalizing the body in distinct chemical milieus essential for the function of many organs. One such barrier is the blood-brain barrier, which isolates the nervous system from the body fluid to maintain its ionic homeostasis and ensure nerve pulse transmission. In Drosophila, the blood-brain barrier is formed late in embryogenesis by a thin epithelium of subperineurial glia that ensheath the nervous system. Similar to other epithelia, subperineurial glia seal the paracellular space by forming large multiprotein complexes at the lateral membrane, the septate junctions (SJs), which impede free diffusion and mediate barrier function.
To identify novel genes required for blood-brain barrier formation, we followed a genome-wide in vivo RNAi approach. We initially screened almost the whole genome for genes required in glia for adult viability and impressively identified 3679 potential candidates. Subsequently, we tested these candidates for requirement in subperineurial glia for adult survival and identified 383 genes. At a last step, we directly asked if blood-brain barrier formation is compromised in the knock-down of the genes by performing the embryonic dye penetration assay in a selection of candidates and identified five genes that play a role during barrier development. Three of these genes, macroglobulin complement-related (mcr) and the previously uncharacterized pasiflora1 and pasiflora2 are further characterized in the context of this thesis.
Here we show that all three proteins are novel components of the Drosophila SJ. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetra-spanning membrane proteins, while Mcr was reported to be a secreted protein in S2 cells required for phagocytosis and clearance of specific pathogens. Through detailed phenotypic analysis we demonstrate that the mutants show leaky blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins, phenotypes that are characteristic of SJ mutants. Consistent with the observed phenotypes, the genes are co-expressed in SJ-forming embryonic epithelia and glia and are required cell-autonomously to exert their function. In columnar epithelia, the proteins localize at the apicolateral membrane compartment, where they colocalize with other SJ proteins, and similar to known SJ components, their restricted localization depends on other complex members. Using fluorescence recovery after photobleaching experiments, we demonstrate for Pasiflora proteins that they are core SJ components, as they are required for complex formation and themselves show restricted mobility within the membrane of wild-type epithelial cells, but fast diffusion in cells with disrupted SJs. Taken together, our results show that Pasiflora1 and Pasiflora2 are novel integral SJ components and implicate a new family of tetraspan proteins in the development of cell junctions. In addition, we find a new unexpected role for Mcr as a transmembrane SJ protein, which raises questions about a potential intriguing link between epithelial barrier function, phagocytosis and innate immunity and has potential implications for the function of occluding junctions.
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Pedanou, Victoria E. "Identification of Novel Pathways that Promote Anoikis through Genome-wide Screens." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/889.
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Epithelial cells that lose attachment to the extracellular matrix (ECM) undergo a specialized form of apoptosis called anoikis. Anoikis has an important role in preventing oncogenesis, particularly metastasis, by eliminating cells that lack proper ECM cues. The basis of anoikis resistance remains to be determined and to date has not been linked to alterations in expression or activity of previously identified anoikis effector genes. Here, I utilized two different screening strategies to identify novel anoikis effector genes and miRNAs in order to gain a deeper understanding of anoikis and the potential mechanisms of anoikis resistance in cancer.
Using large-scale RNA interference (RNAi) screening, I found that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. I found that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis I show that knockdown of Kdm3a enhances metastatic potential. Finally, I find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, my results reveal a novel transcriptional regulatory program that mediates anoikis.
Next, I sought to discover miRNAs involved in anoikis by investigated changes in miRNA expression during anoikis using small RNA sequencing technology. Through this approach I discovered that miR-203 is an anoikis effector miRNA that is also highly down-regulated in invasive breast cancer cells. In breast epithelial cells, miR-203 is induced upon the loss of ECM attachment and inhibition of miR-203 activity leads to a resistance to anoikis. I utilized a dual functional- and expression- based RNA sequencing approach and found that miR-203 directly targets a network of pro-survival genes to induce cell death upon detachment. Finally, I found that the loss of miR-203 in invasive breast cancer leads to the elevation of several anoikis-related pro-survival target genes to contribute to anoikis resistance. Taken together, my studies reveal novel pathways through which cell death is induced upon detachment from the ECM and provide insight into potential mechanisms of anoikis resistance in cancer.
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Pedanou, Victoria E. "Identification of Novel Pathways that Promote Anoikis through Genome-wide Screens." eScholarship@UMMS, 2010. http://escholarship.umassmed.edu/gsbs_diss/889.
Full textAbstract:
Epithelial cells that lose attachment to the extracellular matrix (ECM) undergo a specialized form of apoptosis called anoikis. Anoikis has an important role in preventing oncogenesis, particularly metastasis, by eliminating cells that lack proper ECM cues. The basis of anoikis resistance remains to be determined and to date has not been linked to alterations in expression or activity of previously identified anoikis effector genes. Here, I utilized two different screening strategies to identify novel anoikis effector genes and miRNAs in order to gain a deeper understanding of anoikis and the potential mechanisms of anoikis resistance in cancer.
Using large-scale RNA interference (RNAi) screening, I found that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. I found that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis I show that knockdown of Kdm3a enhances metastatic potential. Finally, I find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, my results reveal a novel transcriptional regulatory program that mediates anoikis.
Next, I sought to discover miRNAs involved in anoikis by investigated changes in miRNA expression during anoikis using small RNA sequencing technology. Through this approach I discovered that miR-203 is an anoikis effector miRNA that is also highly down-regulated in invasive breast cancer cells. In breast epithelial cells, miR-203 is induced upon the loss of ECM attachment and inhibition of miR-203 activity leads to a resistance to anoikis. I utilized a dual functional- and expression- based RNA sequencing approach and found that miR-203 directly targets a network of pro-survival genes to induce cell death upon detachment. Finally, I found that the loss of miR-203 in invasive breast cancer leads to the elevation of several anoikis-related pro-survival target genes to contribute to anoikis resistance. Taken together, my studies reveal novel pathways through which cell death is induced upon detachment from the ECM and provide insight into potential mechanisms of anoikis resistance in cancer.
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Li, Hubo. "Genome-Wide RNAi Screens for Novel Regulators of Acute Myeloid Leukemia." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226105.
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Acute myeloid leukemia (AML) is a heterogeneous disease with complex molecular mechanisms. Recent advent of genomic technologies, such as copy number profiling, whole genome sequencing, and gene expression profiling has accumulated a plethora of large-scale data in AML cell lines and patient samples. However, the functional relevance of most genes identified by these methods has yet to be determined. To systematically characterize the genetic requirement in AML, we conducted genome-wide shRNA screens in 17 AML cell lines in parallel with 199 cell lines of other cancer types. We identified over 150 genes that were required for proliferation specifically by AML, but not other cancer cell lines. We further interrogated the requirements of primary screen hits in vivo with a secondary screen in a xenotransplantation model driven by the MLL-AF9 oncogenic fusion. Integrating both of the RNAi screens and additional gene expression data, we identified transcription factor ZEB2 as a top candidate for regulating AML proliferation. In human AML cells, ZEB2 inhibition impairs proliferation and promotes granulocytic differentiation. Mechanistically, we showed that ZEB2 interacts with the CtBP co-repressor complex, and transcriptionally represses genes involved in cell adhesion and migration. ZEB2’s relevance in AML is further demonstrated by its overexpression in MLL-rearranged AML, and by the epigenetic silencing of its negative regulators, miR-200 family microRNAs, in AML. Our results extend the role of ZEB2 beyond regulating epithelial-mesenchymal transition, and establish ZEB2 as a novel regulator of AML proliferation and differentiation.
MicroRNA-like off-target effect is a major caveat of RNAi screens, which often leads to false positive discoveries. However, systematic analysis of off-target effects in large-scale RNAi screen data can also lead to the discovery of microRNAs with functional relevance. By analyzing the off-target effects in our AML screen, we identified several microRNAs as candidate suppressors for AML proliferation. We show that miR-105, miR-140, miR-501, and miR-532 are novel regulators of the myeloid oncogene MYB. In particular, miR-105 inhibits AML cell growth and miR-532 is associated with myeloid differentiation. The combination of the ZEB2 and microRNA work emphasizes the power of RNAi screens in the exploration of novel cancer regulators.
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Bowen, Emma. "A genomic screen for the identification of novel components of the S-phase checkpoint." Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/8190/.
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Serra, Ryan W. "Investigating Cancer Molecular Genetics using Genome-wide RNA Interference Screens: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/676.
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The development of RNAi based technologies has given researchers the tools to interrogate processes as diverse as cancer biology, metabolism and organ development. Here I employ genome-wide shRNA screens to discover the genes involved in two different processes in carcinogenesis, oncogene-induced senescence [OIS] and epigenetic silencing of tumor suppressor genes [TSGs].
OIS is a poorly studied yet significant tumor suppressing mechanism in normal cells where they enter cell cycle arrest [senescence] or programmed cell death [apoptosis] in the presence of an activated oncogene. Here I employ a genomewide shRNA screen and identify a secreted protein, IGFBP7, that induces senescence and apoptosis in melanocytes upon introduction of the oncogene BRAFV600E. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 has potent pro-apoptotic and anti-tumor activity in mouse xenograft models using BRAFV600E-postive melanoma cell lines. Finally, IGFBP7 is epigenetically silenced in human melanoma samples suggesting IGFBP7 expression is a key barrier to melanoma formation.
Next I investigated the factors involved in epigenetic silencing in cancer. The TSG p14ARFis inactivated in a wide range of cancers by promoter hypermethylation through unknown mechanisms. To discover p14ARF epigenetic silencing factors, I performed a genome-wide shRNA screen and identified ZNF304, a zinc finger transcription factor that contains a Krüppel-associated box [KRAB] repressor domain. I show that ZNF304 binds to the p14ARF promoter and recruits a KRAB co-repressor complex containing KAP1, SETDB1 and DNMT1 for silencing. We find oncogenic RAS signaling to promote the silencing of p14ARF by USP28-mediated stabilization of ZNF304. In addition I find ZNF304 to be overexpressed in human colorectal cancers and responsible for hypermethylation of over 50 TSGs known as Group 2 CIMP marker genes. My findings establish ZNF304 as a novel oncogene that directs epigenetic silencing and facilitates tumorigenicity in colorectal cancer.
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Serra, Ryan W. "Investigating Cancer Molecular Genetics using Genome-wide RNA Interference Screens: A Dissertation." eScholarship@UMMS, 2006. http://escholarship.umassmed.edu/gsbs_diss/676.
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The development of RNAi based technologies has given researchers the tools to interrogate processes as diverse as cancer biology, metabolism and organ development. Here I employ genome-wide shRNA screens to discover the genes involved in two different processes in carcinogenesis, oncogene-induced senescence [OIS] and epigenetic silencing of tumor suppressor genes [TSGs].
OIS is a poorly studied yet significant tumor suppressing mechanism in normal cells where they enter cell cycle arrest [senescence] or programmed cell death [apoptosis] in the presence of an activated oncogene. Here I employ a genomewide shRNA screen and identify a secreted protein, IGFBP7, that induces senescence and apoptosis in melanocytes upon introduction of the oncogene BRAFV600E. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 has potent pro-apoptotic and anti-tumor activity in mouse xenograft models using BRAFV600E-postive melanoma cell lines. Finally, IGFBP7 is epigenetically silenced in human melanoma samples suggesting IGFBP7 expression is a key barrier to melanoma formation.
Next I investigated the factors involved in epigenetic silencing in cancer. The TSG p14ARFis inactivated in a wide range of cancers by promoter hypermethylation through unknown mechanisms. To discover p14ARF epigenetic silencing factors, I performed a genome-wide shRNA screen and identified ZNF304, a zinc finger transcription factor that contains a Krüppel-associated box [KRAB] repressor domain. I show that ZNF304 binds to the p14ARF promoter and recruits a KRAB co-repressor complex containing KAP1, SETDB1 and DNMT1 for silencing. We find oncogenic RAS signaling to promote the silencing of p14ARF by USP28-mediated stabilization of ZNF304. In addition I find ZNF304 to be overexpressed in human colorectal cancers and responsible for hypermethylation of over 50 TSGs known as Group 2 CIMP marker genes. My findings establish ZNF304 as a novel oncogene that directs epigenetic silencing and facilitates tumorigenicity in colorectal cancer.
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Amberkar, Sandeep [Verfasser], and Roland [Akademischer Betreuer] Eils. "Integrative bioinformatics analyses of genome-wide RNAi screens / Sandeep Amberkar ; Betreuer: Roland Eils." Heidelberg : Universitätsbibliothek Heidelberg, 2014. http://d-nb.info/1180032608/34.
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Xiong, Zikai. "Genome-wide recessive screens for DNA mismatch repair genes in mouse ES cells." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612055.
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Ng, Cheuk-Him (Andy). "Genome-Wide Screen Identifies Novel Genes Involved in Mitochondrial Quality Control." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/33204.
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In addition to ATP generation, mitochondria are essential in various cellular processes ranging from biosynthetic pathways, apoptosis, cell cycle progression, and calcium buffering. Studies in living cells have now firmly established that mitochondria exist as a dynamic network sculpted by fission and fusion reactions, rather than separated, individual organelles. Not surprisingly, mutations in genes involved in mitochondrial dynamics and quality control lead to human diseases such as Charcot-Marie-Tooth disease type 2A, Optic atrophy, and autosomal recessive Parkinson disease. I have designed a high-throughput protocol to permit genome-wide screening for novel genes that are required for normal mitochondrial morphology. I have executed a genome-wide RNA interference screen and identified several novel genes required for mitochondrial dynamics in addition to known regulators of mitochondrial dynamics. A detailed high-throughput genome-wide screening protocol is presented. I have shown that TID1, a gene identified from the screen, has a dual-role in maintaining the integrity of mitochondrial DNA and preventing the aggregation of complex I subunits. My analysis of the mitochondrial role of TID1 supports the existence of a TID1- mediated stress response to ATP synthase inhibition. The genome screen also identified the novel gene ROMO1 as essential for normal mitochondrial morphology. I have shown that ROMO1 may have an additional role in maintaining mitochondrial spare respiratory capacity, possibly by affecting cellular substrate availability. Finally, in a collaborative effort, we have shown that homozygous mutations in the mitochondrial fusion gene MFN2 lead to multiple symmetric lipomatosis (MSL) associated with neuropathy. Mechanistically, this mutation reduces MFN2 homocomplex formation. Taken together, these results show the utility of genome-wide screening in identifying genes involved in mitochondrial quality control.
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Newbury, Dianne F. "A genome wide screen for loci involved in specific language impairment." Thesis, University of Oxford, 2002. http://ora.ox.ac.uk/objects/uuid:2b30517a-cecd-49f3-8a5d-556cef6d6723.
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Approximately 4% of English-speaking children are affected by Specific Language Impairment (SLI); a disorder in the development of language skills despite adequate opportunity and normal intelligence. Several studies have indicated the importance of genetic factors in SLI; a positive family history confers an increased risk of development, and monozygotic concordance consistently exceeds that of dizygotic twins. However, like many behavioural traits, SLI is assumed to be genetically complex with several loci contributing to the overall risk. This thesis aims to clarify the genetic mechanisms underlying Specific Language Impairment by the exploitation of recent advances in technological, genetic and statistical techniques. This goal is achieved, for the main part, through the completion of the first-ever, systematic genome-wide screen for loci involved in the disorder. A collection of 98 families was drawn from both epidemiological and clinical populations, all with probands who display severe deficits in language skills. Genome-wide linkage analyses were completed for three language-related measures and identified two regions which may harbour susceptibility gene variants for SLI, one on chromosome 16 and a second on chromosome 19. Both of these loci yielded maximum LOD scores of 3.55 and exceeded the threshold for suggestive linkage under all types of analysis performed. Fine mapping of the chromosome 19 locus with a high-density map of microsatellite markers provided further support for the role of this region in SLI but failed to narrow the area of linkage. The second section of the thesis therefore explores alternative genetic strategies that may facilitate the localisation of susceptibility variants from the genomic regions identified. Mutation screening and association analyses were performed for two candidate genes within a subset of 48 families affected by SLI. The first ⎼ numblike (NBL), or numb-related (NUMB-R) (MIM 604018) ⎼ was selected from the region of linkage on chromosome 19q and the second ⎼ Forkhead-bOX domain P2 (FOXP2) (MIM 605317) ⎼ has recently been shown to be mutated in a family with a severe speech and language disorder. Finally, I describe the mapping of a translocation breakpoint within a child affected by a severe language impairment and orofacial dyspraxia. This breakpoint lies on chromosome 2q and coincides with a putative region of linkage in both language impairment and autism. In the long-term it is hoped that techniques similar to those described here will allow the identification of the gene variants which underlie SLI allowing to the development of better diagnosis and treatment for those children with language impairments.
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Kiritsy, Michael C. "Functional Genomics of Mammalian Innate Immunity." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1102.
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The breadth of genetic diversity in the mammalian immune response stands out amongst the ubiquity of variation seen in the genome, evidence that microbial infections have been a major driver of evolution. As technology has facilitated an understanding of the etiology of immunological diversity, so too has it enabled the assessment of its varied functions. Functional genomics, with its ability to assess both cause and effect, has revolutionized our understanding of fundamental biological phenomena and recalibrated our hypotheses. We build upon the model of host immunity established by rare genetic variants that are causative of immunodeficiencies, but that incompletely consider the complexities of the genome. To expand our understanding, we performed a series of forward genetic screens to identify regulators of distinct functions of the innate immune system. Our studies discovered genes with novel functions in antigen presentation and immunoregulation, including several involved in central metabolism. Studies in macrophages and dendritic cells identified mitochondrial respiration as a positive regulator of the interferon-gamma response, and cells incapable of respiration failed to activate T cells. Notably, human mutations in several of these genes are responsible for immune dysfunction. In summary, this work uses new methods in genetic engineering to systematically assess the regulation of innate immunity. Our results suggest that variation in these regulatory pathways is likely to alter immunity in states of health and disease. Thus, our work validates a new approach to identify candidate genes relevant to immune dysfunction.
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Meisner, Sarah. "The genetics of susceptibility to leprosy." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298995.
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DuPree, Michael G. "A candidate gene study and a full genome screen for male homosexuality." Connect to this title online, 2002. http://etda.libraries.psu.edu/theses/approved/WorldWideIndex/ETD-209/index.html.
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Tran, Grant. "Modeling drug efficacy in the tumour microenvironment with Saccharomyces cerevisiae genome-wide screens in hypoxic conditions." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/60210.
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Hypoxia, the state of reduced oxygen, is a microenvironment found in many solid tumours and is correlated with an increased risk in patient mortality. This is due to an increase in resistance to radiotherapy and chemotherapy as well as a decrease in drug efficacy. The mechanisms and cellular factors (gene products) associated with this reduced chemotherapeutic efficacy in hypoxia remains unclear. This research looks to identify cellular processes and pathways that cancerous cells are able to exploit in order to survive and thrive in this microenvironment. The eukaryotic model baker’s yeast Saccharomyces cerevisiae combined with a genome-wide approach was used to screen the yeast knockout collection for specific genotypes that are sensitive to the hypoxic environment alone, and in combination with commonly used chemotherapeutics. Pathways and processes identified in these screens include transcriptional regulation, cytoskeleton maintenance, ribosomal biogenesis, macromolecular complex assembly and the heat shock response. The combination of heat and hypoxia was found to result in a synergistic effect that drastically affected cell fitness. DNA-damaging chemotherapeutics screened in hypoxic conditions showed reduced efficacy. Genotypes most sensitive to drugs in the hypoxic environment fall into Gene Ontology (GO) terms categorized in the response to the specific mechanism of the drug. This includes DNA repair processes such as homologous repair, post-replicative repair and mismatch repair. The mechanistic specificity uncovered in these screens suggests that the hypoxic environment exacerbates drug-specific stresses, and the identified genotypes highlight gene products and pathways critical for these responses. Cell survival and success in this microenvironment therefore requires adaptations to these exacerbated stresses, a phenomenon successfully accomplished by resistant tumour cells. This research contributes to our understanding of cellular biology under this cancer microenvironment, and provides data to highlight the challenges in using chemotherapeutics to treat tumours.Pharmaceutical Sciences, Faculty of
Graduate
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Deligiannaki, Myrto Verfasser], and Ulrike [Akademischer Betreuer] [Gaul. "Identification of novel septate junction components through genome-wide glial screens / Myrto Deligiannaki. Betreuer: Ulrike Gaul." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2015. http://d-nb.info/1072376601/34.
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Prüßing, Katja [Verfasser]. "Genome-wide screen for modifiers of Abeta42-induced neurodegeneration in Drosophila / Katja Prüßing." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2012. http://d-nb.info/1026309840/34.
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Fernandes, Caroline. "Genome-wide screen for novel regulators of Parkinson's disease genes in «Drosophila melanogaster»." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103725.
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Introduction: Compromised mitochondrial integrity leads to neuronal cell death in Parkinson's disease and other neurodegenerative diseases. Cells have several quality control mechanisms in place to counteract dysfunction and preserve a healthy mitochondrial network. Recent studies have implicated the autosomal recessive Parkinson's disease genes, Parkin and Pink1, in a common pathway regulating mitochondrial quality control. Together, Parkin and Pink1 promote isolation and selective degradation of damaged mitochondria, but the mechanisms governing this process are unclear. Methods: The goal of our study was to use a genetically sensitized Drosophila model of Parkinson's disease to identify novel regulators of the Pink1/Parkin pathway. We screened deficiencies spanning the second and third chromosome for dominant enhancers and suppressors of the Pink1/parkin wing posture phenotype, which derives from mitochondrial defects in these mutants. Results: From this screen we identified several cytological regions that strongly interact with Parkin and/or Pink1. Of these, four were further dissected revealing five interacting genes. Among them, opa1 and drp1 have been previously implicated in the Pink1/Parkin pathway. The other three genes, p60, β4galNacTA, and debra, represent novel regulators of Parkin and Pink1 function. Conclusion/implications: The unbiased identification of previously known Pink1/Parkin interactors demonstrates the validity of this approach. Moreover, the discovery of novel genes involved in the Pink1/Parkin pathway will allow better understanding of the mechanisms governing mitochondrial quality control and will aid in the development of therapeutic treatments.Introduction : L'altération de la fonction mitochondriale entraîne la dégénérescence de certains neurones chez les personnes atteintes de maladies neurodégénératives dont la maladie de Parkinson. Les cellules saines sont dotées d'un système de contrôle leur permettant de faire face et de réparer les dysfonctionnements des mitochondries et ainsi de préserver leur intégrité. Des études récentes ont révélé l'implication de deux gènes à l'origine de syndromes parkinsoniens autosomiques récessifs, Pink1 et Parkin, dans une voie de signalisation commune contrôlant le maintien de la fonction mitochondriale. Pink1 et Parkin interviennent ensemble dans l'isolation et la dégradation des mitochondries défectueuses. Cependant, à l'heure actuelle, les mécanismes moléculaires contrôlant ce processus restent à élucider. Méthodes : Le but de notre étude a été d'identifier de nouveaux régulateurs de la voie de signalisation Pink1/Parkin par crible génétique dans un modèle drosophile de la maladie de Parkinson. Pour cela, nous avons criblé une collection de lignées de drosophiles déficientes sur les chromosomes deux et trois pour leur capacité à modifier (augmenter ou diminuer) le phénotype de posture de l'aile caractéristique de la mutation de Pink1/Parkin. Résultats: Nous avons identifié plusieurs régions cytologiques qui interagissent fortement avec Parkin et/ou Pink1. Quatre de ces régions ont été disséquées de façon à révéler cinq gènes. Parmi eux, opa1 et drp1 avaient déjà été impliques dans la voie de signalisation Pink1/Parkin. Les trois autres gènes p60, β4galNacTA et debra représentent de nouveaux régulateurs de la fonction de Pink1 et Parkin. Conclusion/implications : D'une part, l'identification non biaisée de gènes déjà connus comme interagissant avec Pink1/Parkin démontre la validité de cette approche. D'autre part, la découverte de nouveaux gènes candidats de la voie Pink1/Parkin pour le maintien de l'intégrité mitochondriale permettra de mieux comprendre les mécanismes moléculaires contrôlant ce processus et aidera à l'élaboration de traitements.
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Mendez, Jamie Elizabeth. "Investigation of Hsf1 Interacting Partners via a Genome-wide Yeast Two-hybrid Screen." Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4543.
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Heat shock factor 1 (HSF1) is the master transcriptional regulator of the heat shock response (HSR), an evolutionarily conserved cellular stress response. HSF1 promotes the expression of a variety of molecular chaperones that aid in restoring protein homeostasis upon exposure to proteoxic stress. However, all of the proteins responsible for regulating the HSR together with HSF1 are unknown. A genome-wide yeast two hybrid screen was performed to identify new S. cerevisiae Hsf1 protein interacting partners. Two GAL4 DNA binding domain-Hsf1 fusion proteins (baits) were constructed with mutations in the Hsf1 C-terminal activation domain to dampen Hsf1 mediated auto-activation of the reporter gene. Each haploid bait strain was mated with a haploid prey strain containing one of ~6,000 S. cerevisiae open reading frames fused to the GAL4 activation domain (prey). Interaction between the bait and prey reconstituted the GAL4 protein enabling it to bind to a GAL4 DNA binding site and activate the HIS3 reporter gene. The identified proteins from 4 screens were pooled generating 240 putative Hsf1 interacting partners. This list was narrowed to 38 candidates by selecting the 15 strongest interactions identified based on colony size and 33 candidates conserved in C. elegans. Hsf1 interactions with the 14 candidates in which protein expression was confirmed were then re-tested by a manual yeast two-hybrid assay. Hsf1 interactions with Sti1, Rim2 and Prp46 were repeatable in this manual assay. A study of the impact of knockdown of each of their C. elegans homolog on the HSR was performed using RNAi in an hsp70-promoter::GFP reporter strain of C. elegans. Preliminary results suggest that knockdown of Sti1 may impact the HSR in the worm. Further study of Sti1 and other potential Hsf1 interacting partners identified in this screen is warranted.
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McKnight, N. C. "A genome-wide screen for starvation-induced autophagy : identifies new modulators of autophagy." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302281/.
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Autophagy is a catabolic mechanism by which cytoplasmic components are sequestered and transported by a double-membrane vesicle called an autophagosome to the lysosome for degradation. This recycling of organelles and macromolecules provides the cell with amino acids in times of nutrient deprivation though we do not fully know how the process is triggered or controlled. It is a highly regulated process in mammalian cells and its deregulation has been shown to contribute to multiple diseases. In order to find new regulators of mammalian autophagy, I performed a genome-wide screen using the Dharmacon human siRNA library in a stable human cell line expressing GFP-LC3, a specific marker for autophagosomal membranes. First I incubated the cells with the siRNA pools then I starved the cells of amino acids. This initiated the formation of GFP-LC3-labelled autophagosomes that I quantified using the Cellomics VTiScan microscope and accompanying software. I measured the effect of specific siRNA-mediated knock-down on multiple parameters including spot count. Accounting for cell death and normalising the data, I generated a Z-score for each siRNA pool and retested the best 500 autophagy-increasing and 500 autophagydecreasing siRNAs as above. The 190 strongest siRNA pools were deconvoluted leaving 20 hits that reproduced the phenotype with three or four out of four duplexes. These 20 hits were then assayed for endogenous LC3 lipidation in a different cell line and the ability of their siRNA to reduce mRNA levels was determined. Four increasers of GFP-LC3 spots increased endogenous LC3 lipidation, suggesting that these proteins are either negative regulators of autophagy or inhibit the maturation or degradation of autophagosomes. Five decreasers of GFP-LC3 spots also inhibited endogenous LC3 lipidation and I have characterised two of these proteins required for autophagy. SCOC colocalises with early autophagy markers and may be providing a scaffold for autophagy machinery. WAC, through its reported binding partners, may be playing a role in both the autophagic and ubiquitin/proteasome pathways.
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Drake, Peter. "The first genome-wide screen for animal peroxins using RNAi in Drosophila melanogaster." Thesis, University of Sheffield, 2013. http://etheses.whiterose.ac.uk/3777/.
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Peroxisomes are spherical, membrane-bound organelles found ubiquitously in Eukaryotes. They have very important metabolic functions, the loss of which has severely morbid consequences for human patients. Genes involved in peroxisome biosynthesis are evolutionarily conserved. There are now more than thirty known proteins (peroxins) involved in peroxisome biosynthesis (including peroxisomal matrix protein import, peroxisome multiplication and inheritance). Higher organisms are expected to have many more peroxins than yeasts and plants that have well established peroxisome families. Yet fewer peroxins are known of and consequently their interactions are poorly understood. Recent technological advances in RNA interference (RNAi) and high-throughput screening have meant entire organisms can now be screened in negative gene function assays. The inauguration in 2010 of the Sheffield RNAi Screening Facility (SRSF) has meant that an on-site facility at Sheffield University can facilitate genome-wide screening of Drosophila melanogaster, an organism for which technologies into genetic manipulation are well established. The use of cell-culture and high-content, automated epifluorescence microscopy means that D. melanogaster cells can be screened for peroxins using well-established fluorescence assays. Two genome-wide screens were performed on S2R+ cells in an attempt to uncover novel peroxins in fruitflies which may elucidate further peroxisomes in mammals. Both screens used fluorescent peroxisomal matrix markers. The first was transfected transiently and the second stably, improving the expression profile in the cellular population. A small number of aberrant phenotypes were quickly identified and many knockdown phenotypes had a milder presentation. The most statistically significant aberrant candidates were chosen to be compiled into a custom designed microtitre plate. We made many copies of this and we repeated the assay in triplicate. We were able to identify 3 newly aberrant phenotypes of knockdown of which 2 appear to effect peroxisomes directly. One of these appears to behave in a PEX16 like manner and we decided to investigate this further.
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Powers, Ralph Wilson. "Genome-wide screens reveal that reduced TOR signaling extends chronological and replicative life span in S. cerevisiae /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/5044.
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Gore, Tanvi. "Analyse génétique du trafic intracellulaire du morphogène Hedgehog chez la Drosophile." Thesis, Nice, 2015. http://www.theses.fr/2015NICE4110.
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Hedgehog (Hh) est un morphogène conservé au cours de l’évolution, et qui est impliqué dans un grand nombre de processus développementaux. Ma thèse vise à comprendre comment la sécrétion, et la mise en place du gradient d’Hh sont régulées à partir de son site de production, en utilisant la drosophile comme modèle animal. Pour identifier les régulateurs positifs impliqués dans la maturation du signal Hh, nous avons conçu et réalisé un crible génétique couvrant l’ensemble du génome, par ARNs interférents (ARNi). Grâce à ce crible, nous avons identifié la petite protéine GTPase Rab8 qui serait impliquée spécifiquement dans le routage intracellulaire de Hh. Selon notre modèle proposé, la protéine Hh serait secrétée de 2 façons. Sa sécrétion du coté apical est nécessaire à l'activation de gènes cibles à longue distance, alors que sa sécrétion du coté baso-latéral permettrait l'activation de gènes cibles à courte distance. La façon par laquelle Hh est transportée de la membrane apicale à la membrane basale à l’intérieur des cellules productrices n’est pas connue. La perte de fonction de Rab8 dans les cellules productrices de Hh induit une augmentation de l’activation des gènes cibles à courtes distances, alors l’expression des gènes cibles activés à longues distances est réduite. De plus, en utilisant des expériences sur tissus vivant pour suivre la dynamique de l'internalisation de la protéine endogène d’Hh, nous avons constaté que la perte de Rab8 n'a pas d’effet sur sa sécrétion primaire, mais entraine des défauts dans l’endocytose de Hh, affectant, par la suite, la mise en place du gradient morphogénétiqueHedgehog (Hh) is a conserved secreted morphogen involved in an array of developmental processes. Using Drosophila as a model, during my thesis we aimed to ask how the secretion, extraction and transport of Hh protein are regulated at the site of its production. To understand the positive regulators of Hh secretion and transport we designed and performed a genome-wide RNAi screen in Drosophila to identify new regulators of Hh transport and identified the small GTPase Rab8 as a novel component required for Hh trafficking. According to our proposed model, there are two pools of secreted Hh. The apical pool is needed for long range target gene activation, and basolateral pool for short range target gene activation. It is not clear how Hh is sorted apico-basally in the producing cells. Interfering with Rab8 function in the Hh producing cells extends Hh short range targets. Conversely, it reduces the long range Hh targets, suggesting that interfering with Rab8 function in the Hh producing cells impairs Hh trafficking, thus hampering the fine tuning between the two secreted pools of Hh. Moreover, using live assays to track the dynamics of endogenous Hh internalization, we observed that loss of Rab8 in Hh producing cells does not affect its primary secretion, but causes defects in Hh endocytosis, subsequently affecting its gradient activity. We hypothesize a model where Hh is targeted for primary secretion to the apical side of the wing disc, which then is internalized, and this internalized Hh is then directed for recycling which is essential for its long range activity
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Scheich, Christoph. "High-throughput evaluation of protein folding conditions and expression constructs for structural genomics." Phd thesis, [S.l. : s.n.], 2004. http://pub.ub.uni-potsdam.de/2004/0055/scheich.pdf.
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Ivatt, R. M. "A whole genome RNAi screen to identify novel promoters of PINK1/Parkin-mediated mitophagy." Thesis, University of Sheffield, 2013. http://etheses.whiterose.ac.uk/4899/.
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The PINK1/Parkin pathway is genetically linked to recessive forms of Parkinson's disease (PD) and plays a central role in the maintenance of mitochondrial homeostasis. Following toxification with the membrane uncoupler CCCP, PINK1, a serine/threonine kinase, becomes stabilised on the outer membrane of damaged mitochondria. This stabilisation is necessary for the translocation of Parkin, an E3- ubiquitin ligase, to mitochondria prior to their removal via mitophagy. In order to identify novel pathway components upstream of Parkin translocation, we performed a genomewide RNAi screen using Drosophila S2R+ cells. Upon completion, we validated primary screen hits with multiple rounds of secondary screening, assessing a range of mitochondrial homeostatic processes. In follow-up screens, corresponding human orthologs were assessed for their ability to influence Parkin translocation and mitophagy in HeLa cells, demonstrating conserved pathway function. Human screen hits selected for in depth analysis included several genes acting in a common lipogenesis pathway. These are the transcription factor and master pathway regulator SREBF1, the Skp1-Cul1-F-box E3-ligase protein FBXW7 and the serine/threonine kinase GSK3A. Follow-up analyses of these genes suggest a role for SREBPdependent lipid synthesis in PINK1/Parkin-mediated mitophagy, likely acting upstream of PINK1. Importantly, SREBF1 has recently been identified as a risk locus for sporadic forms of PD (Do et al, 2011). Hence, this study reveals a novel mechanistic link between familial- and sporadic causes of PD, with mitochondrial dysfunction at its centre.
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Yamanoi, Kouji. "Suppression of ABHD2, identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer." Kyoto University, 2017. http://hdl.handle.net/2433/227585.
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Yang, Dingyi. "Identifying novel immune modulating factors in a genome-wide Staphylococcus aureus screen in human neutrophils." Thesis, University of Sheffield, 2017. http://etheses.whiterose.ac.uk/19447/.
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Sharma, Sumana. "Genome-scale identification of cellular pathways required for cell surface recognition." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/271825.
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A range of biochemically diverse molecules located in the plasma membrane— such as proteins, glycans, and lipids—mediate cellular recognition events, initiation of signalling pathways, and the regulation of processes important for the normal development and function of multicellular organisms. Interactions mediated by cell surface receptors can be challenging to detect in biochemical assays, because they are often highly transient, and membrane-embedded receptors are difficult to solubilise in their native conformation. The biochemical features of low-affinity extracellular protein interactions have therefore necessitated the development of bespoke methods to detect them. Here, I develop a genome-scale cell-based genetic screening approach using CRISPR-Cas9 knockout technology that reveals cellular pathways required for specific cell surface recognition events. Using a panel of high-affinity monoclonal antibodies, I first establish a method from which I identify not only the direct receptor but also other required gene products, such as co-receptors, post-translational modi cations, and transcription factors contributing to antigen expression and subsequent antibody-antigen recognition on the surface of cells. I next adapt this method to identify cellular factors required for receptor interactions for a panel of recombinant proteins corresponding to the ectodomains of cell surface proteins to the endogenous surface receptors present on a range of cell lines. In addition to finding general cellular features recognised by many ectodomains, I also identify direct interaction partners of recombinant protein probes on cell surfaces together with intracellular genes required for such associations. Using this method, I identify IGF2R as a binding partner for the R2 subunit of GABAB receptors, providing a mechanism for the internalisation and regulation of GABAB receptor signalling. The results here demonstrate that this single approach can identify the molecular nature and cell biology of surface receptors without the need to make any prior assumptions regarding their biochemical properties.
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Tran, Anh Thuy. "Genome-wide RNA-interference screen for human host factors vital to influenza A virus-induced cell death and viral replication." Journal of Virology, 2013. http://hdl.handle.net/1993/18323.
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Influenza virus is a globally significant infectious agent with the potential to cause catastrophic pandemic outbreaks. Present treatment of influenza infections is restricted to only four anti-viral drugs, but there are increasing global reports of anti-viral resistance in several seasonal strains and also the 2009 pandemic swine-origin influenza virus H1N1. Possible future pandemic outbreaks, emerging new strains and drug resistance underscore the need to understand this complex virus and its pathogenicity with the goal that novel targets can be uncovered for future therapeutic development.
Extensive lung tissue damage during influenza virus infection is proposed to contribute to the development of aberrant host immune responses. Strong evidence now demonstrates the significance of the cellular death pathway in promoting efficient influenza virus replication and disease progression. Viruses rely heavily on the machinery of their host for productive replication, which is also an Achilles’ heel that could be targeted for treatment. In pursuit of unraveling the complex nature of influenza virus replication, I carried out a global shRNA screen to identify specific host factors and signaling pathways that are involved in influenza-induced cell death and replication. In this study I identified 138 genes required for influenza viruses to induce infected host cell death. These genes were found to be involved in Protein Kinase A, NF-kB and PI3K signaling cascades. These signaling pathways are well known regulators of cell death and survival, which suggests influenza viruses may carefully regulate these pathways to reach a balance that suit their requirements for efficient proliferation, eventually at the cost of the host cell. I chose five candidate genes—BAD, MxB, TNFSF12-13,TNFSF13, and USP47—that were associated with apoptosis and the major signaling pathways determined in my network analysis to further verify the genome-wide screen as well as elucidate the role of these potentially novel host factors in influenza virus replication.
I show in my study that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and both virus replication and viral protein production also are dramatically reduced. I also report here that MxB depletion protected cells from virus-mediated cytopathology and resulted in significant inhibition of influenza virus replication for H1N1 and H3N2 subtypes. Additionally, I report that TNFSF12-13, TFNSF13, and USP47, similarly, are required for efficient influenza virus replication and induction of cell death. Depletion of these proteins resulted in significant inhibition of viral propagation and conferred protection of host cells to virus killing.
Overall, my study has provided a list of novel host factors that play significant roles during influenza virus infection. Further studies on these potential genes and their encoded protein products may uncover possible new targets for drug development for future therapeutic treatment. In addition to providing greater understanding of influenza virus infection, these studies will also highlight important fundamentals of cellular processes that may be broadly applicable to other fields of research.
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Lee, Liam Changwoo. "Functional identification of molecular oncotargets associated with the resistance to ALK inhibition in neuroblastoma via genome-wide CRISPR-Cas9 screens." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267921.
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Recent whole-exome sequencing studies of hundreds of high-risk neuroblastoma (hNB) patients have identified Anaplastic Lymphoma Kinase (ALK) as the only directly ‘druggable’ target with a significant mutation rate (9%). ALK is a receptor tyrosine kinase whose dysregulation has been implicated as the driver lesion in a variety of cancer types, including Non-Small Cell Lung Cancer (NSCLC) and various paediatric malignancies. As a kinase normally only expressed during early development in the foetal brain, ALK is an ideal therapeutic target and it has proven relatively simple to target therapeutically. However, resistance to ALK-targeted therapy, particularly in ALK+ NSCLC patients has frequently been observed. The majority of the acquired resistance mechanisms noted in NSCLC patients rely on bypass signalling pathways, which are tissue-context dependent. To proactively identify and develop strategies to counter these varied yet expected resistance mechanisms in other ALK-driven tumours, we must gain a better insight of the bypass-track mechanism(s) in a tumour-specific manner. The present study aimed to functionally identify putative resistance mechanisms against ALK inhibitors via extensive CRISPR/Cas9-based genome-wide knockout (GeCKO) or overexpression screens (SAM) in the human neuroblastoma cell line, SHSY-5Y, to develop novel therapeutic strategies for ALK mutant NBs. The GeCKO screen identified a total of 39 genes and miRNAs, and the SAM overexpression screen identified 25 genes that induce resistance to ALK inhibitors. These putative resistance-inducing candidates were then aligned with a publicly available expression dataset of hNB patients (n = 476) to identify those with prognostic significance (Kaplan-Meier event-free survival analysis), specifically those that are indicative of relapse risk. Furthermore, all candidates identified from the screen were individually validated in vitro. Two of the candidates, one from each of the knockout and overexpression screens, were further investigated. Inhibition of hsa-miR-1304-5p, identified from GeCKO screen, induced resistance to ALK inhibitors. Interestingly, interference of has-miR-1304-5p, in the absence of ALK inhibitors, also enabled enhanced cell viability whilst the transfection of its mimic led to a significant reduction of viability across 17 distinct NB cell lines. Through genome-wide cDNA microarrays, in silico predictions, and UTR-luciferase assays, this study identified hsa-miR-1304-5p to be a major regulator of the Ras/MAP Kinase pathway. Overexpression of PIM1, identified from the SAM screen, in NB cell lines induced resistance to ALK inhibitors and this phenotype could be reversed on transducing cells with RNAi against PIM1. Interestingly, inhibition of PIM1 in wild-type cell lines via RNAi or pharmacological compounds led to substantially enhanced potency of ALK inhibitors suggesting PIM1 inhibitors as combinatorial agents with ALK inhibitors for the therapy of treatment-naive hNB. Through protein analysis of all identified downstream targets of PIM1, this study revealed NB-specific actions of the PIM1 oncoprotein that include the inactivation of the pro-apoptotic protein BAD. In summary, this study has identified mechanisms of resistance to ALK inhibitors as well as novel front-line therapeutic strategies for hNB patients that should be implemented clinically.
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Norton, Matthew. "Genome-wide RNAi Screen Identifies Romo1 as a Novel Regulator of Mitochondrial Fusion and Cristae Integrity." Thesis, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23701.
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Mitochondria exist in a dynamic network regulated by the opposing processes of mitochondrial fusion and fission. Regulation of mitochondrial morphology is critical for metabolism, quality control and cell survival, among other cellular processes. Large GTPases are responsible for shaping the mitochondrial network. Mitofusins 1 and 2 and Opa1 regulate outer and inner mitochondrial membrane fusion, respectively. Conversely, Drp1 is recruited to mitochondria to carry out fission. Although many proteins have been implicated in these processes, there are still many unknowns. We sought to identify novel regulators of mitochondrial morphology and conducted a genome-wide RNAi screen to identify candidate genes. We identified Reactive Oxygen species Modulator 1 (ROMO1) as a novel regulator of mitochondrial fusion and cristae integrity. In the absence of ROMO1, the mitochondrial network fragments and cristae are lost. These defects lead to impaired mitochondrial respiration and sensitization to cytochrome c release and downstream apoptosis. ROMO1 is regulated by mitochondrial REDOX at 4 cysteine residues that couple REDOX signaling to mitochondrial morphology. We have characterized ROMO1 as an interactor with the MINOS complex, required for cristae junction maintenance, and the inner mitochondrial membrane fusion GTPase OPA1. Through these interactions ROMO1 couples cristae junction security to mitochondrial fusion.
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Rose, Elaine. "Identifying Mechanisms of Resistance to Oncolytic Virotherapy in Acute Leukemia Through a Genome-wide CRISPR Screen." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/38112.
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Approximately half of all adults diagnosed with acute leukemia (AL) relapse after standard chemotherapy, highlighting the need for alternative treatment options. We have previously shown that vaccination with irradiated autologous tumour cells infected with an oncolytic virus (OV) can elicit a durable, tumour specific, T cell- mediated response in a mouse model of AL. In the context of this AL infected cell vaccine (ICV) model, infection of autologous cells ex vivo with an OV is essential for stimulating a lasting immune response. While the murine AL line L1210 can be robustly infected with Maraba MG1, creating a potent infected cell culture, this ICV still has room for improvement as ICV-vaccinated mice with high tumour burden still die from leukemia. Therefore, we sought to utilize a genome wide CRISPR-Cas9 screen to identify genetic factors that mediate OV resistance in this model of AL. L1210 cells stably expressing Cas9 were transduced with the mouse GeCKOv2 library, which contains 130,209 gRNAs against 20,611 genes within the mouse genome. Following selection, cells were treated with Maraba MG1 and genomic DNA from resistant populations was sequenced to identify genes enriched in resistant cells relative to mock treated cells. Our screen identified several genes that mediate susceptibility to OV infection including those involved in viral entry (Ldlr), receptor-mediated endocytosis (Atp6v1g2), intracellular signaling (Cav1), the cytoskeleton (Filip1 and Tmod4), as well as autophagy and exosome production (Atg5). We aim to use the findings from this work to improve therapeutic efficacy in otherwise OV resistant tumour models as well as identify biomarkers, to determine the feasibility of administering an ICV using patient derived tumour cells.
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Pfannenstiel, Brandon T., Xixi Zhao, Jennifer Wortman, Philipp Wiemann, Kurt Throckmorton, Joseph E. Spraker, Alexandra A. Soukup, et al. "Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus." AMER SOC MICROBIOLOGY, 2017. http://hdl.handle.net/10150/626452.
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The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique. IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin.
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Zahoor, Muhammad kashif. "Genome wide analysis for novel regulators of growth and lipid metabolism in drosophila melanogaster." Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00664844.
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The evolutionary conserved insulin and nutrient signaling network regulates growth andmetabolism. Nutrients are directly utilized for growth or stored, mostly as triglycerides. InDrosophila, activation of insulin/nutrient signaling in the fat body (the fly equivalent of liverand adipose tissue), causes an increase in fat stores composed of several small-size lipiddroplets (LDs). Conversely, fasting produces an increase in LD size and a decrease in fatcontents. The TOR kinase and its substrate S6 kinase (S6K) play a central role in this response,and particularly in Drosophila, they have been shown to orchestrate cell-autonomous andhormone-controlled growth. However, despite extensive research studies on different modelorganisms (mouse, fly, worm) to decipher the molecular and physiological functions of S6K,nothing is known about how its degradation is regulated.Taking advantage of the inducible RNA interfering (RNAi) library from NIG (Japan), we haveperformed three genetic screens to identify novel regulators of steroidogenesis, lipidmetabolism and dS6K-dependent growth. First, RNAi lines were screened in the ring gland; anorgan that controls the progression of the developmental steps by producing the steroidhormone ecdysone. Out of 7,000 genes screened, 620 positive candidates were identified toproduce developmental arrest and/or overgrowth phenotypes. Then, we challenged 4,000 genesby RNAi screening able to recapitulate the larger sized LD phenotype as obtained uponstarvation, leading to the identification of 24 potential candidates. Finally, the RNAi lines werescreened for their ability to enhance a growth phenotype dependent of the Drosophila S6K(dS6K). Out of 7,000 genes screened, 45 genes were identified as potential negative regulatorsof dS6K. These genes were further used to design a novel protein-protein interaction networkcentered on dS6K through the available data from yeast-2-hybrid (Y2H) assay. The most potentinteractors were then analyzed by treatment of cultured S2 cells with the corresponding doublestrand RNA (dRNA). Western blotting thus, allowed us to discriminate between the geneproducts that regulate dS6K levels versus those that regulate its phosphorylation, as a hallmarkfor its kinase activity. Interestingly, archipelago (ago), which encodes a component of an SCFubiquitinligase known to regulate the degradation of dMyc, Cyclin E and Notch, was identifiedas a negative regulator of dS6K-dependent growth. Based on the Y2H available data showingthat Ago and dS6K interact each other and the presence of a putative Ago-interaction motif indS6K, we hypothesized that Ago causes an ubiquitin-mediated degradation of dS6K. Ourmolecular data showed that loss of ago caused an elevated level of dS6K, which confirms arole of Ago in controlling dS6K degradation. Altogether our findings emphasize the importanceof the saturating screening strategies in Drosophila to identify novel regulators of metabolicand signaling pathways.
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Adamson, Brittany Susan. "A Genome-Wide Study of Homologous Recombination in Mammalian Cells Identifies RBMX, a Novel Component of the DNA Damage Response." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10723.
Full textAbstract:
Repair of DNA double-strand breaks is critical to the maintenance of genomic stability, and failure to repair these DNA lesions can cause loss of chromosome telomeric regions, complex translocations, or cell death. In humans this can lead to severe developmental abnormalities and cancer. A central pathway for double-strand break repair is homologous recombination (HR), a mechanism that operates during the S and G2 phases of the cell cycle and primarily utilizes the replicated sister chromatid as a template for repair. Most knowledge of HR is derived from work carried out in prokaryotic and eukaryotic model organisms. To probe the HR pathway in human cells, we performed a genome-wide siRNA-based screen; and through this screen, we uncovered cellular functions required for HR and identified proteins that localize to sites of DNA damage. Among positive regulators of HR, we identified networks of pre-mRNA-processing factors and canonical DNA damage response effectors. Within the former, we found RBMX, a heterogeneous nuclear ribonucleoprotein (hnRNP) that associates with the spliceosome, binds RNA, and influences alternative splicing. We found that RBMX is required for cellular resistance to genotoxic stress, accumulates at sites of DNA damage in a poly(ADP-ribose) polymerase 1-dependent manner and through multiple domains, and promotes HR by facilitating proper BRCA2 expression. Screen data also revealed that the mammalian recombinase RAD51 is commonly off-targeted by siRNAs, presenting a cautionary note to those studying HR with RNAi and highlighting the vulnerability of RNAi screens to off-target effects in general. Candidate validation through secondary screening with independent reagents successfully circumvented the effects of off-targeting and set a new standard for reagent redundancy in RNAi screens.
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Amai, Takamitsu. "Development of genome editing technology of mitochondrial DNA in Saccharomyces cerevisiae." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263707.
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Voß, Felizia Katharina [Verfasser]. "Molecular identification of the volume-regulated anion channel VRAC by a genome-wide siRNA screen / Felizia Katharina Voß." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1068809906/34.
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Buchholz, Frank, Mikołaj Słabicki, Mirko Theis, Dragomir B. Krastev, Sergey Samsonov, Emeline Mundwiller, Magno Junqueira, et al. "A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-180795.
Full textAbstract:
DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.
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Rambani, Komal. "A genome wide screen in C. elegans identifies cell non-autonomous regulators of oncogenic Ras mediated over-proliferation." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1461288131.
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Buchholz, Frank, Mikołaj Słabicki, Mirko Theis, Dragomir B. Krastev, Sergey Samsonov, Emeline Mundwiller, Magno Junqueira, et al. "A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia." Public Library of Science, 2010. https://tud.qucosa.de/id/qucosa%3A28927.
Full textAbstract:
DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.
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Boquete, Vilarino Lorena. "Development of shRNA screens to identify effectors of three complex traits : neighbour suppression of tumour growth and proliferation and protection from lipotoxicity in β-cells." Thesis, University of Exeter, 2016. http://hdl.handle.net/10871/27635.
Full textAbstract:
RNA interference (RNAi) is a natural mechanism of cellular defence against exogenous double stranded RNA (dsRNA). The discovery of small dsRNA molecules which can be processed by the RNAi pathway in mammalian cells was one of the key advances in the study of functional genomics. These molecules can be designed to downregulate the expression of specific genes. Collections or libraries of dsRNA molecules targeting an extensive number of genes are now available. Using these libraries, numerous studies have implemented high-throughput screens for the study of molecular effectors of numerous phenotypes. The process of designing an RNAi screen requires the consideration of several critical factors during both the experimental and analysis phases. The experimental screen should aim to reproduce the biological phenomenon studied as closely as possible by choosing an adequate model and screening conditions. Phenotype evaluation and assessment of knockdown effects need careful consideration. The results obtained from large-scale RNAi screens are often complex. An analysis pipeline should be implemented which integrates the biological basis of the phenomenon and facilitates the interpretation of the data. This project designed and implemented an unbiased shRNA screen in two in vitro models relevant to carcinogenesis and diabetes. The first screen implemented used a model of neighbour suppression to study the molecular effectors of the response in tumorigenic cells to growth suppression cues from the surrounding tissue, a cellular interaction relevant in early tumorigenesis. The second screen studied two phenotypes relevant to diabetes: proliferation and resistance to lipotoxicity of β-cells in a reversibly immortalised cell line. An integrative analysis pipeline was also developed to apply network biology and functional enrichment analysis methods for the interpretation of the data obtained from both screens.