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Martin, Alarcon Daniel Alberto. "Tools for RNA and cell-free synthetic biology." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104124.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 58-63).
Amid the myriad recent developments in synthetic biology, progress has been fastest in the areas with the most versatile tools for understanding and engineering biological systems. RNA synthetic biology and synthetic minimal cells are areas where design is limited by the availability of tools to observe, program, and manipulate the systems in question. In this work I present expanded toolsets to achieve these goals. The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular, programmable protein architecture for targeting unmodified RNA sequences. I report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which I call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. I validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. I further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems. Genetic circuits are a fundamental tool in synthetic biology; an open question is how to maximize the modularity of their design, to facilitate their integrity, scalability, and flexibility. Liposome encapsulation enables chemical reactions to proceed in well-isolated environments. I here adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. I demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) so that they contain multiple-part genetic cascades, that these cascades can be controlled by external as well as inter-liposomal communication without cross-talk, and that these cascades can also be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability.
by Daniel Alberto Martin Alarcon.
Ph. D.
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Wesselhoeft, R. Alexander(Robert Alexander). "Synthetic circular RNA for protein expression." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/122710.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019Cataloged from PDF version of thesis.
Includes bibliographical references (pages 111-126).
Messenger RNA (mRNA) has broad potential for therapeutic and engineering applications. One fundamental limitation of mRNA is its relatively short half-life in biological systems, effected in part by rapid exonuclease-mediated degradation upon delivery. Circular RNA (circRNA), a type of single-stranded RNA with a contiguous structure that lacks the end motifs necessary for exonuclease recognition, may be resistant to this mechanism of degradation and therefore may exhibit superior stability. However, challenges in circularization, purification, and protein expression have impeded a thorough investigation of exogenous circRNA. By rationally designing ubiquitous accessory sequences to facilitate circularization, we engineered a permuted self-splicing intron that efficiently circularized RNAs up to 5kb in length in vitro.
With the addition of these accessory sequences, we were able to demonstrate nearly complete circularization of precursor RNAs containing an internal ribosome entry site (IRES) for translation initiation and a coding region such as erythropoietin or eGFP. We found that translation from optimized circRNA was robust, and circRNA protein expression stability far exceeded that of both unmodified and nucleoside modified linear mRNA in some cellular contexts. We monitored cytokine release and antiviral defense induction in sensitive cells transfected with circRNA purified by different methods and found that the immunogenicity and stability of circRNA preparations was dependent on the degree of purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses.
In contrast to purified unmodified linear mRNA, purified unmodified circRNA was invisible to several RNA sensors including RIG-i and endosomai toil-like receptors (TLRs) and did not provoke a significant cytokine response upon transfection. Using purified circRNA, we finally provided the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and showed that the duration of circRNA translation was extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs. In total, this work suggests that circRNA is a promising alternative to linear mRNA for therapeutic applications.
by R. Alexander Wesselhoeft.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
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DiAndreth, Breanna Elizabeth. "RNA sensing and programming platforms for mammalian synthetic Biology." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/123058.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019Cataloged from PDF version of thesis.
Includes bibliographical references (pages 153-173).
The field of synthetic biology aims to control cellular behavior using programmable gene circuits. Generally these gene circuits sense molecular biomarkers, process these inputs and execute a desired calculated response. This is especially relevant for gene and cell therapies where integrating multiple disease-related inputs and/or sophisticated control could lead to safer and more effective approaches. While mammalian synthetic biology has made great progress, few gene circuit-based therapies have entered the clinic. Regulatory issues aside, this lag may be due to several technical impediments. First, the computing part of circuits is often accomplished via transcriptional regulation, which presents challenges as we move toward the clinic. Second, the field relies on a limited set of sensors; the detection of other types of disease biomarkers will help robustly identify cell state.
Finally, the design cycle currently used to develop gene circuits is laborious and slow, which is not suitable for clinical development, especially applications in personalized medicine. In this thesis I describe how I address these three limitations. I develop a new posttranscriptional regulation platform based on RNA cleavage that I term "PERSIST" (Programmable Endonucleolytic RNA Scission-Induced Stability Tuning). CRISPR-specific endonucleases are adapted as RNA-level regulators for the platform and we demonstrate several genetic devices including cascades, feedback, logic functions and a bistable switch. I explore sensor designs for relevant biomolecules including mRNAs, miRNAs and proteins via the PERSIST and other platforms. Finally, I present a "poly-transfection" method, associated advanced data analysis pipelines, and computational models that make circuit engineering faster and more predictive.
Taken together, the expanded RNA toolkit that the PERSIST platform offers as well as advancements in sensing and circuit design will enable the more straightforward creation of robust gene circuits for gene and cell therapies.
by Breanna Elizabeth DiAndreth.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering
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Matsuura, Satoshi. "Synthetic RNA-based logic computation in mammalian cells." Kyoto University, 2019. http://hdl.handle.net/2433/242426.
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Garcia, Martin Juan Antonio. "RNA inverse folding and synthetic design." Thesis, Boston College, 2016. http://hdl.handle.net/2345/bc-ir:106989.
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Thesis advisor: Welkin E. JohnsonThesis advisor: Peter G. Clote
Synthetic biology currently is a rapidly emerging discipline, where innovative and interdisciplinary work has led to promising results. Synthetic design of RNA requires novel methods to study and analyze known functional molecules, as well as to generate design candidates that have a high likelihood of being functional. This thesis is primarily focused on the development of novel algorithms for the design of synthetic RNAs. Previous strategies, such as RNAinverse, NUPACK-DESIGN, etc. use heuristic methods, such as adaptive walk, ensemble defect optimization (a form of simulated annealing), genetic algorithms, etc. to generate sequences that minimize specific measures (probability of the target structure, ensemble defect). In contrast, our approach is to generate a large number of sequences whose minimum free energy structure is identical to the target design structure, and subsequently filter with respect to different criteria in order to select the most promising candidates for biochemical validation. In addition, our software must be made accessible and user-friendly, thus allowing researchers from different backgrounds to use our software in their work. Therefore, the work presented in this thesis concerns three areas: Create a potent, versatile and user friendly RNA inverse folding algorithm suitable for the specific requirements of each project, implement tools to analyze the properties that differentiate known functional RNA structures, and use these methods for synthetic design of de-novo functional RNA molecules
Thesis (PhD) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology
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Thomas, Gregory Stuart. "Targeting prostate cancer with synthetic RNA ligands." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/1508.
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Prostate cancer represents a serious health concern as the most diagnosed form of cancer in men and the second leading cause of cancer death in the Western world. Current treatments for prostate cancer are non-targeted and result in a number of undesirable, non-specific effects, highlighting the need for novel, targeted therapeutics in the treatment of prostate cancer.
Prostate Specific Membrane Antigen (PSMA) offers great promise in the targeting of prostate cancer for imaging and therapy. PSMA is a transmembrane carboxypeptidase with cell surface expression several orders of magnitude higher in cancerous prostatic epithelia than found in other tissue and PSMA is constitutively internalized into cells. The unique expression profile of PSMA and its constitutive internalization offer great value in the targeted delivery of therapeutics to prostate cancer cell.
In 2002, two synthetic RNA ligands, aptamers, were selected for their ability to inhibit the enzymatic activity of PSMA. In 2006, the utility of these aptamers in the delivery of cytotoxic siRNA across the cell membrane was demonstrated in vivo using aptamer-siRNA chimeras. However, those experiments were performed by intratumoral injection, and systemic administration will be necessary for use in the clinic.
In this thesis, we improve PSMA targeted chimeras to serve as more powerful therapeutics in the treatment of prostate cancer. We optimize existing aptamer-siRNA chimeras for increased potency and stability and improved pharmacokinetics to enable systemic administration. We truncate the PSMA binding aptamers for amenability to large-scale chemical synthesis. With emerging roles for PSMA enzymatic activity in the prostate cancer disease we identify aptamers that are suitable for chemical synthesis and retain inhibitory properties against PSMA. Finally, we assess the use of aptamers as synthetic ligands in the functional inhibition of PSMA mediated motility in prostate cancer.
Our results demonstrate the ability of aptamer-siRNA chimeras to specifically kill PSMA-expressing cells with cytotoxic siRNA upon systemic injection. We confirm a newly reported role for PSMA in the promotion of cell motility and demonstrate the ability of aptamers to effectively neutralize PSMA-mediated motility. The results presented within argue strongly for the functional utility of aptamers in the treatment of prostate cancer.
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Harris, Andreas William Kisling. "The design of gene regulatory networks with feedback and small non-coding RNA." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:e3a323b1-9067-415d-8728-6c70c1b6cf23.
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The objective of the field of Synthetic Biology is to implement novel functionalities in a biological context or redesign existing biological systems. To achieve this, it employs tried and tested engineering principles, such as standardisation and the design-build-test cycle. A crucial part of this process is the convergence of modelling and experiment. The aim of this thesis is to improve the design principles employed by Synthetic Biology in the context of Gene Regulatory Networks (GRNs). Small Ribonucleic Acids (sRNAs), in particular, are focussed on as a mechanism for post-transcriptional expression regulation, as they present great potential as a tool to be harnessed in GRNs. Modelling sRNA regulation and its interaction with its associated chaperone Host-Factor of Bacteriophage Qβ (Hfq) is investigated. Inclusion of Hfq is found to be necessary in stochastic models, but not in deterministic models. Secondly, feedback is core to the thesis, as it presents a means to scale-up designed systems. A linear design framework for GRNs is then presented, focussing on Transcription Factor (TF) interactions. Such frameworks are powerful as they facilitate the design of feedback. The framework supplies a block diagram methodology for visualisation and analysis of the designed circuit. In this context, phase lead and lag controllers, well-known in the context of Control Engineering, are presented as genetic motifs. A design example, employing the genetic phase lag controller, is then presented, demonstrating how the developed framework can be used to design a genetic circuit. The framework is then extended to include sRNA regulation. Four GRNs, demonstrating the simplest forms of genetic feedback, are then modelled and implemented. The feedback occurs at three different levels: autoregulation, through an sRNA and through another TF. The models of these GRNs are inspired by the implemented biological topologies, focussing on steady state behaviour and various setups. Both deterministic and stochastic models are studied. Dynamic responses of the circuits are also briefly compared. Data is presented, showing good qualitative agreement between models and experiment. Both culture level data and cell population data is presented. The latter of these is particularly useful as the moments of the distributions can be calculated and compared to results from stochastic simulation. The fit of a deterministic model to data is attempted, which results in a suggested extension of the model. The conclusion summarises the thesis, stating that modelling and experiment are in good qualitative agreement. The required next step is to be able to predict behaviour quantitatively.
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Klauser, Benedikt [Verfasser]. "RNA Synthetic Biology using the Hammerhead Ribozyme : Engineering of Artificial Genetic Switches / Benedikt Klauser." Konstanz : Bibliothek der Universität Konstanz, 2015. http://d-nb.info/1112745483/34.
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Cacan, Ercan. "Evolutionary synthetic biology: structure/function relationships within the protein translation system." Thesis, Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/45838.
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Production of mutant biological molecules for understanding biological principles or as therapeutic agents has gained considerable interest recently. Synthetic genes are today being widely used for production of such molecules due to the substantial decrease in the costs associated with gene synthesis technology. Along one such line, we have engineered tRNA genes in order to dissect the effects of G:U base-pairs on the accuracy of the protein translation machinery. Our results provide greater detail into the thermodynamic interactions between tRNA molecules and an Elongation Factor protein (termed EF-Tu in bacteria and eEF1A in eukaryotes) and how these interactions influence the delivery of aminoacylated tRNAs to the ribosome. We anticipate that our studies not only shed light on the basic mechanisms of molecular machines but may also help us to develop therapeutic or novel proteins that contain unnatural amino acids. Further, the manipulation of the translation machinery holds promise for the development of new methods to understand the origins of life.
Along another line, we have used the power of synthetic biology to experimentally validate an evolutionary model. We exploited the functional diversity contained within the EF-Tu/eEF1A gene family to experimentally validate the model of evolution termed ‘heterotachy’. Heterotachy refers to a switch in a site’s mutational rate class. For instance, a site in a protein sequence may be invariant across all bacterial homologs while that same site may be highly variable across eukaryotic homologs. Such patterns imply that the selective constraints acting on this site differs between bacteria and eukaryotes. Despite intense efforts and large interest in understanding these patterns, no studies have experimentally validated these concepts until now. In the present study, we analyzed EF-Tu/eEF1A gene family members between bacteria and eukaryotes to identify heterotachous patterns (also called Type-I functional divergence). We applied statistical tests to identify sites possibly responsible for biomolecular functional divergence between EF-Tu and eEF1A. We then synthesized protein variants in the laboratory to validate our computational predictions. The results demonstrate for the first time that the identification of heterotachous sites can be specifically implicated in functional divergence among homologous proteins.
In total, this work supports an evolutionary synthetic biology paradigm that in one direction uses synthetic molecules to better understand the mechanisms and constraints governing biomolecular behavior while in another direction uses principles of molecular sequence evolution to generate novel biomolecules that have utility for industry and/or biomedicine.
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Wang, Qingqing. "Alternative Splicing Regulation in Programmed Cell Death and Neurological Disorders: A Systems Biology Approach." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10849.
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Alternative splicing (AS) is a major source of biological diversity and a crucial determinant of cell fate and identity. Characterizing the role of AS regulatory networks in physiological and pathological processes remains challenging. The work presented here addresses this challenge using systems biology analyses of AS regulatory networks in programmed cell death and neurological disorders. The first study describes a genome-wide screen based on splicing-sensitive reporters to identify factors that affect the AS of apoptosis regulators Bclx and Mcl1. The screen identified over 150 factors that affect apoptosis through modulating the pro- and anti-apoptotic splicing variants of these apoptosis regulators. This screen revealed a new functional connection between apoptosis regulation and cell-cycle control through an AS network. It also unearthed many disease-associated factors as AS effectors. The second study describes the functions of the Polyglutamine-binding protein 1 (PQBP1)-mediated AS regulatory network in neurological disorders. PQBP1 is a factor linked to intellectual disability and was unexpectedly identified as an AS effector from the screen described above. We found that PQBP1 influences the splicing of many mRNAs and is associated with a wide range of splicing factors. Depletion of PQBP1 in mouse primary cortical neurons caused defects in neurite outgrowth and altered AS of mRNAs enriched for functions in neuron projection regulation. Disease-mutants of PQBP1 lose associations with splicing factors and cannot complement the aberrant AS patterns and neuron morphology defects in PQBP1 depleted-neurons. This study revealed a novel function of PQBP1 in AS regulation associated with neurite outgrowth and indicated that aberrant AS underlies the pathology of PQBP1-related neurological disorders. A final study examines the dynamics of the Drosophila Sex-lethal AS regulation network using a combination of experimental tools and mathematical modeling. This study demonstrates that the features of Sxl AS regulation have great potentials in building synthetic memory circuits in mammalian cells to track cell fate. Collectively, this work describes the landscape of three diverse AS regulatory networks in various biological processes. The results and methods presented here contribute to our rapidly advancing knowledge of AS regulation in biology and human disease.
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Ferry, Quentin R. V. "RNA-based engineering of inducible CRISPR-Cas9 transcription factors for de novo assembly of eukaryotic gene circuits." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:b89c1b17-ea75-4049-a5d0-7cd1b5d0bd8e.
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Synthetic biology in mammalian cells holds great promise for reverse engineering biological processes and rewiring cellular behaviors for therapeutic purpose. An essential aspect in our ability to reprogram the cellular code is the availability of highly orthogonal, inducible transcriptional regulators. CRISPR-based strategies employing effector-domain tethering to the single guide RNA (sgRNA)-dCas9 complex have greatly advanced this field by allowing for precise activation or repression of any gene via simple sgRNA reprograming. However, the implementation of inducible CRISPR-based transcriptional regulators (CRISPR-TRs) has so far been restricted to dCas9 protein engineering and conditional effector tethering. Although elegant, these approaches are limited by dCas9 promiscuous loading of sgRNAs, which hinders their use for the creation of independent multi-gene transcriptional programs. To address this limitation, I have developed a modular framework for the rational design of inducible CRISPR-TR, based on simple and reversible modifications of the sgRNA sequence. At the core of this conceptual framework lies the ability to inactivate native sgRNAs by appending on their 5'-end a short RNA segment, which folds to form a spacer-blocking hairpin (SBH). Base-pairing between the extension and the sgRNA spacer prevents docking of the CRISPR-TR on-target, fully abrogating its activity. Subsequently, I have created inducible SBH variants (iSBH) by replacing the hairpin loop with conditional RNA cleaving units. Using a variety of sensing-loops, I was able to engineer a panel of switchable iSBH-sgRNAs, designed to activate specifically in the presence of protein, oligonucleotide, and small molecule inducers. Leveraging the versatility of this method, I demonstrate that iSBH-sgRNAs expression can be multiplexed to assemble synthetic gene circuits implementing parallel and orthogonal regulation of multiple endogenous gene targets. Finally, I have distilled the design principles derived throughout this project to develop a web tool that automates the creation of iSBH- sgRNAs. Already a valuable addition to the synthetic biology toolkit, iSBH-based inducibility should in theory also be applicate to all CRISPR-Cas9 derivatives (genome editing, epigenetic alteration, DNA labelling, etc.) as well as other newly characterized RNA-guide nucleases from the CRISPR family.
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Junetha, Syed Jabarulla. "Chemical Biology Approaches for Regulating Eukaryotic Gene Expression." 京都大学 (Kyoto University), 2015. http://hdl.handle.net/2433/202664.
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Duncan, John Nichlaus. "Co-evolution of small molecule responsive riboswitches by chemical and genetic selection." Thesis, University of Manchester, 2011. https://www.research.manchester.ac.uk/portal/en/theses/coevolution-of-small-molecule-responsive-riboswitches-by-chemical-and-genetic-selection(9f0be71a-519d-4766-b862-3b38cfcc57cd).html.
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Riboswitches are regulatory structures present in the 5′-UTR of a wide range of bacterial mRNAs. They consist of a small-molecule binding aptamer domain, which affects the conformation of a nearby expression platform to control gene expression through a transcriptional or translational mechanism. Because of their ability to bind selectively to very small concentrations of ligand, in a protein-independent manner, they have great potential for use as novel small-molecule controllable gene expression systems. This thesis describes how a combination of chemical genetics and genetic selection were used to develop and test a novel riboswitch-based gene-expression system. Several constructs were generated which could respond in vivo to a variety of non-natural small heterocyclic compounds and output via a simple fluorescence based assay in a dose-dependent manner. Methods for controlling the overall protein expression landscape of the riboswitch-based gene-expression system are outlined. In addition, the rational design of mutant riboswitch aptamers with improved ligand-binding capabilities is described alongside attempts to modulate the structural stability of the expression platform. Riboswitches need to be highly discriminatory to function effectively in vivo, binding to one ligand from a cellular pool of thousands. Mutant riboswitches were created that responded specifically to the ligands ammeline or azacytosine, and were found to have no induction in the presence of adenine, the wild-type riboswitch ligand. This in vivo ligand orthogonality was confirmed by subsequent in vitro studies. The ligand-induced structural changes undertaken by the mutant riboswitch aptamer domains were subsequently characterised using a variety of in vitro methods including SHAPE, ITC and x-ray crystallography. Finally, the feasibility of using riboswitch gene-expression systems in fully synthetic applications was demonstrated through the construction and analysis of small synthetic gene clusters and operons. The in vivo expression of two fluorescent proteins under independent riboswitch control was studied under single and dual induction for a range of ligand concentrations. The ability to control the expression of multiple genes is highly desirable in the emerging field of synthetic biology, the results described here indicate that riboswitches are ideally suited to complement current gene expression tools.
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Izard, Jerome. "Contrôle de la croissance et régulation génique chez Escherichia coli." Thesis, Grenoble, 2012. http://www.theses.fr/2012GRENV061/document.
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La faculté d’adaptation aux conditions environnementales des bactéries provient de lacomplexité de leur réseau de régulation génique, impliquant de nombreux régulateursspécifiques et la machinerie d’expression génique. Nous avons montré que le gène crl, codantun régulateur global d’Escherichia coli, est exprimé de façon transitoire lors de laphase exponentielle. Notre étude a permis d’identifier deux régulateurs responsables dece profil parmi une centaine testés. Ainsi, le complexe CRP-AMPc, réprime de façon indirectel’expression de crl, tandis que la nucléoprotéine Fis se fixe sur le promoteur de crlet active sa transcription directement. Le profil d’expression de crl étant similaire à celuide nombreux régulateurs globaux, nous nous sommes intéressés au rôle de la machinerieglobale d’expression des gènes et à son impact sur la croissance. Dans ce but, nous avonsconstruit un système nous permettant de contrôler la croissance d’E. coli en modulantl’expression des sous-unités _ et _’ de l’ARN polymérase et donc le niveau de transcriptiondans la cellule. Lorsque l’ARN polymérase est en faible concentration, le taux decroissance devient quasiment nul et les cellules filamentent. Ce contrôle de la croissanceest dose dépendant et a été mis en évidence autant à l’échelle de la population qu’à cellede la cellule unique. Nous avons enfin étudié par RNA-seq l’impact du niveau d’ARNpolymérase sur la transcription de l’ensemble du génome de cette souche. Cette étudemontre que toutes les classes fonctionnelles de gènes sont affectées par notre système, àl’exception des gènes qui codent les protéines ribosomalesBacteria can adapt to many different environmental conditions. This capacity of adaptationis conferred to the organism by a complex regulatory network, composed of specificregulators and the global gene expression machinery. We have studied the expression dynamicsof Crl, a global regulator of Escherichia coli, and observed a peak of transcriptionduring the exponential phase of growth. In order to identify potential regulators of crlexpression, we have measured the expression profile of crl in about one hundred differentmutant strains. This screen has revealed that CRP-cAMP represses indirectly the transcriptionof crl and the nucleoprotein Fis activates transcription of the crl promoter bybinding to the crl promoter region. We noted that the expression of most global regulatorsof E. coli have an expression profile similar to the one of Crl. We have thereforestudied the relationship between global gene expression machinery and cellular growth.We constructed a bacterium where the transcription of the two large subunits of RNApolymerase, _ et _’, is under external control. A small concentration of RNA polymeraseleads to a small growth rate of this engineered bacterium and the cells start to filament,whereas a high concentration of RNA polymerase produces phenotypically wild-type cells.We have characterized the control of growth rate by our system at the population level andin single cells. An analysis of the global transcription pattern of this strain by RNA-seqshows that the transcription of genes in all functional classes, with the possible exceptionof genes coding for ribosomal proteins, are almost equally affected by the modificationsof the intracellular concentration of RNA polymerase
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Al-Mamari, Ahmed. "Biocontainment system for bacterial antigen delivery carriers." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28793.
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Genetically modified organisms (GMOs) are confined physically in order to contain their spread in nature and to minimise chances of horizontal gene transfer. However, with the potential that GMOs hold as cheap, reliable and efficient micro-machines, their eventual uncontrolled release into the wider space is becoming more likely. Indeed, their application as environmental sensors is largely increasing. Nevertheless, the field of synthetic biology may also afford solutions to the problem. A major potential application of GMOs is the delivery of antigens to human and animal hosts, through the utilization of live, engineered microbes. Recombinant technology is promising for several reasons including their capacity to be less reactogenic, more potent, safer and genetically definable. Also, they have the potential to provide protection against multiple targets simultaneously, are relatively inexpensive and can be eradicated with antibiotics, as the need arises. Besides, delivery of vaccines to mucosal surfaces is more efficient. Mutant Salmonella expressing heterologous antigens have been shown to induce protection against a variety of pathogens. Nevertheless, limited containment systems are available that can be applicable for bacterial antigen carriers. This project aims to design safeguards for the bacterial antigen delivery systems that limit ORF translatability and self-inactivates/destructs upon exit from the host. In this work, double quadruplet codons were suppressed by orthogonal tRNAs, providing a barrier for gene translation in the recipient cells when antigen is horizontally transferred. Furthermore, three kill switches were designed that are activated by a decrease in temperature from 37 °C. First, Sau3AI endonuclease was activated by protein self-splicing at low temperature mediated by Mtu recA intein. The activation of the endonuclease led to three-fold logarithmic decrease in the number of viable cells within two hours of gene expression. Second, RNA-dependent activation of RNase 7 showed a reduction in the number of viable cells at low temperature of three logarithmic folds. RNase 7 was controlled by the cspA 5’UTR, which sequesters ribosome binding site at 37 °C and allows translation at low temperature. Third, CspA 5’UTR was shown to regulate expression of TEV protease at 37 °C and low temperature. This led to bacterial cellular inhibition within two hours of TEV induction and five-fold logarithmic reduction in the number of viable cells at low temperature. In addition, for the first time and contrary to previous studies, the TEV protease was shown to inhibit cellular growth. It was also shown that biofilm formation was drastically impaired by the TEV activity. The three killing switches and the quadruplet translation system are poised to function as robust safeguards for bacterial antigen delivery systems.
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Laos, Roberto, and Steven Benner. "The first self-replicating molecule and the origin of life." Revista de Química, 2014. http://repositorio.pucp.edu.pe/index/handle/123456789/99479.
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El origen de la vida en la Tierra es una de las preguntas más difíciles presentadas a la ciencia. En los últimos 60 años, ha habido un progreso considerable en entender cómo moléculas relativamente sencillas, que son relevantes para la vida, pueden ser generadas espontáneamente o pueden llegar a la Tierra desde el espacio. Además, los análisis de la evolución de la historia de ácidos nucleicos, los cuales almacenan la información genética, apuntan a un ancestro común universal ya extinto. Los estudios del origen de la vida ofrecen muchas pistas que apuntan hacia un origen común, quizás no solo en el Tierra sino también en algún otro punto del sistema solar. Debido al largo tiempo transcurrido desde que la Tierra empezó a albergar vida, las pistas más antiguas de los primeros organismos se han perdido. Es muy poco probable encontrar exactamente cómo fue este primer organismo. Sin embargo, en los últimos años la biología sintética ha logrado progresar mucho en la modificación de biomoléculas, en particular, los ácidos nucleicos. Es posible que pronto podamos construir y comprender un sistema minimalista en el cual las moléculas puedan copiarse a sí mismas dentro de una célula rudimentaria. El estudio de un sistema así podría permitirnos develar el origen de los primeros organismos.The origin of life on Earth is one of the most challenging questions in science. In the last 60 years, considerable progress has been made in understanding how simple molecules relevant to life can be generated spontaneously or are known to arrive to Earth from space. Additionally, analysis of the evolution history of nucleic acids, which are the repository of genetic information, points to a now extinct, universal common ancestor for all life on Earth. The studies of the origin of life offer many clues towards a common origin, perhaps not just on Earth but somewhere else in the solar system. However due to the length of time that the Earth has harbored life, the oldest clues of the first organisms are mostly gone. It is unlikely to find exactly what this first organism was like. Nevertheless, in the last few years, synthetic biology has made remarkable progress at modifying biomolecules, particularly nucleic acids. It is possible that soon we will be able to construct and understand a minimalistic system in which molecules can copy themselves in a protocell. The study of such systems could shed light into the origin of the first organisms.
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Sadegh, Cameron. "Directed differentiation of mouse embryonic stem cells into neocortical output neurons." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11064.
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During development of the neocortex, many diverse projection neuron subtypes are generated under regulation of cell-extrinsic and cell-intrinsic controls. One broad projection neuron class, corticofugal projection neurons (CFuPN), is the primary output neuron population of the neocortex. CFuPN axons innervate sub-cortical targets including thalamus, striatum, brainstem, and spinal cord.
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Moreno, de Palma Isabel. "Metabolic channeling for biofuel production : Co-localization of Pdc and Adh." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-319519.
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Enhancing productivity in bioprocesses, especially for biofuel production, is crucial for achieving an environmentally and economically sustainable biotechnology industry.Metabolic channelling occurs in nature when the intermediate between two consecutive enzymes in a pathway is directed from the first enzyme to the second avoiding diffusion in the cytosol. This would be very advantageous in bioprocesses as it would increase efficiency of a particular pathway, reducing side products and protecting the cells from potential toxic intermediates. In recent years different strategies for emulating channelling effect wereproposed and used with very promising results. Clustering of enzymes seems to be the simplest way to create metabolic channelling. In this master thesis, four different strategies to co-localize enzymes in clusters are compared. The metabolic pathway chosen as a model was ethanol production by pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh). Chimeric proteins were genetically engineered and transformed in E. coli creating different strains. Ethanol production by the different strains was measured to compare production efficiency. Cell growth and protein expression were used for further understanding of the results. Strengths and weaknesses of each strategy and proposals for further improvement were discussed.
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Fritz, Sarah E. "Molecular basis of the DExH-box RNA helicase RNA helicase A (RHA/DHX9) in eukaryotic protein synthesis." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437413252.
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D'Abramo, Claudia M. "Biochemical characterization of the BVDV RNA-dependent RNA polymerase during initiation and elongation of RNA synthesis." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111870.
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The RNA-dependent RNA polymerase (RdRp) of viruses belonging to the Flaviviridae family, including the hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) is critical for viral replication. The major goal of this PhD study was to biochemically characterize the role of the polymerase during initiation and elongation of RNA synthesis, utilizing the BVDV RdRp as a model system. We showed that the BVDV polymerase efficiently incorporates chain-terminating nucleoside analogues, which ultimately arrest RNA synthesis. The incorporated chain-terminators, however, can be removed from the primer terminus in the presence of pyrophosphate (PPi). These results suggest that the phosphorolytic excision of incorporated chain-terminators is a possible mechanism that can diminish the efficiency of this class of compounds against viral RdRps. The chain-terminators then served as valuable tools in subsequent experiments to analyze the functional role(s) of the RdRp-associated GTP-specific binding site (G-site) and the consequences of GTP binding during the initiation of RNA synthesis. The results provide biochemical evidence for the existence of a G-site in the BVDV enzyme, and suggest that GTP binding controls template positioning during de novo initiation. Finally, through the development of a novel ribonuclease-based footprinting assay, it was determined that catalytically active complexes contact the newly synthesized RNA during elongation of RNA synthesis with approximately 6-7 base pairs. The polymerase moves along the template according to the position where RNA synthesis is arrested. Taken together, this study provides novel insight into mechanisms involved during initiation and elongation of RNA replication of viruses belonging to the Flaviviridae family. The ability of RdRps to excise incorporated chain-terminators points to possible shortcomings of nucleoside analogue inhibitors that are under development as antiviral agents for the treatment of infection with HCV.
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Baboo, Sabyasachi. "Nuclear translation." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:5266f049-d576-44fd-ab26-11cf7a27f678.
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In bacteria, protein synthesis can occur tightly coupled to transcription. In eukaryotes, it is believed that translation occurs solely in the cytoplasm; I test whether some occurs in nuclei and find: (1) L-azidohomoalanine (Aha) – a methionine analogue (detected by microscopy after attaching a fluorescent tag using ‘click’ chemistry) – is incorporated within 5 s into nuclei in a process sensitive to the translation inhibitor, anisomycin. (2) Puromycin – another inhibitor that end-labels nascent peptides (detected by immuno-fluorescence) – is similarly incorporated in a manner sensitive to a transcriptional inhibitor. (3) CD2 – a non-nuclear protein – is found in nuclei close to the nascent RNA that encodes it (detected by combining indirect immuno-labelling with RNA fluorescence in situ hybridization using intronic probes); faulty (nascent) RNA is destroyed by a quality-control mechanism sensitive to translational inhibitors. I conclude that substantial translation occurs in the nucleus, with some being closely coupled to transcription and the associated proof-reading. Moreover, most peptides made in both the nucleus and cytoplasm are degraded soon after they are made with half-lives of about one minute. I also collaborated on two additional projects: the purification of mega-complexes (transcription ‘factories’) containing RNA polymerases I, II, or III (I used immuno-fluorescence to confirm that each contained the expected constituents), and the demonstration that some ‘factories’ specialize in transcribing genes responding to tumour necrosis factor α – a cytokine that signals through NFκB (I used RNA fluorescence in situ hybridization coupled with immuno-labelling to show active NFκB is found in factories transcribing responsive genes).
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Boehm, Christian Reiner. "Gene expression control for synthetic patterning of bacterial populations and plants." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267842.
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The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.
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Jia, Yiping. "Mechanistic studies of DNA-dependent transcription initiation and RNA synthesis by bacteriophage T7 RNA polymerase /." The Ohio State University, 1998. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487953204281995.
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Kim, Young-Chan. "Protein-ligand and protein-protein interactions involved in de novo initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp)." [Bloomington, Ind.] : Indiana University, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3204540.
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Thesis (Ph. D.)--Indiana University, Dept. of Chemistry, 2006.Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0249. Adviser: C. Cheng Kao. "Title from dissertation home page (viewed Feb. 9, 2007)."
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Zhang, Ze. "The control of ribosomal RNA synthesis in mammalian cells." Thesis, University of Southampton, 2013. https://eprints.soton.ac.uk/350477/.
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The biogenesis of ribosomes is a fundamental process that occurs in all living cells. In mammalian cells, it is a highly complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNAs) with about 80 ribosomal proteins (RPs). More than 150 non-ribosomal proteins are involved in the processing of rRNAs. The main focus of this project is to use adult rat ventricular cardiomyocytes (ARVCs) as a model to address how mTOR complex 1 (mTORC1) and other signalling pathways regulate the synthesis of rRNAs. A new technique has been developed to monitor the synthesis of new rRNAs using 4-thiouridine (4-SU) and I have applied it in both HeLa cells and heart muscle cells to study the control of ribosome synthesis. HeLa cells were treated with different mTOR inhibitors to identify effects on the transcription and/or decay of rRNA. We analysed both the synthesis rate and the decay rate of new RNAs made by Pol I and Pol III using real-time RT-PCR. Interestingly, rapamycin not only blocked the synthesis of 18S, 28S and 5S rRNA, but also induced the decay of newly synthesized rRNAs. This demonstrates that mTORC1 regulates Pol I and Pol III transcription, as well as the decay of rRNA. In cardiomyocytes, hypertrophic agents such as phenylephrine (PE) strongly activate protein synthesis and lead to heart cell growth. The boost of protein synthesis drives the increase of cell size and leads to hypertrophy. Cardiac hypertrophy (CH) is a major risk factor for heart failure. Therefore, it is important to understand the mechanisms that how hypertrophic agents which cause the overgrowth of heart muscle increase ribosome production. Although it is known that inhibiting mTORC1 largely blocks the rapid activation of protein synthesis by PE, here it did not affect the synthesis of new 18S rRNAs. However, inhibitors of the MEK/Erk pathway and p90RSK each block the new rRNA synthesis. These data reveal that, in contrast to many other types of cell, ribosome biogenesis is controlled by MEK/ERK/p90RSK signalling, not mTORC1, in cardiomyocytes. Taken together, the data presented here may provide cues for potential valuable therapy of cardiac left ventricular hypertrophy.
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Hannoush, Rami Nabil. "RNA folding : synthesis, structure and biological properties of hairpins based on 2',5'-linked RNA loops." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82890.
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Ribonucleic acids (RNA) are polymers of ribonucleotides linked together by 3',5'-phosphodiester linkages and play a prominent role in protein biosynthesis. Much less common are ribonucleic acids based on 2',5'-phosphodiester linkages (2',5'-RNA). In this study, the synthesis, physical and biological properties of several 2' ,5'-linked oligoribonucleotides are described.One aspect of this thesis deals with the synthesis and structural studies of 2',5'-dodecaribonucleotides. Their base sequence was designed to promote intramolecular folding by base pairing leading to the formation of "hairpin loops". The hairpins consist of a tetranucleotide loop and a 4 base-pair duplex (stem). The thermodynamic stability and conformation of these hairpins were assessed by UV, CD and NMR spectroscopy. Melting experiments (UV) revealed that with a few exceptions, hairpins comprised of 2',5'-(UUCG) loops exhibit greater thermodynamic stability (e.g. Tm) than the corresponding hairpins with 3' ,5'-linked loops. The 'extra' stability imparted by the 2',5'-UUCG) loop was found to be virtually independent of the sugar composition of the stem. For example, the 2',5'-tetraloop stabilizes hairpins whether their duplex stem is based on double-stranded DNA or RNA. In contrast, the 3',5-tetraloop stabilizes hairpins only when their stem duplex is double-stranded RNA. NMR studies revealed that the 2',5'-tetraloop (UUCG) is self-stabilized by a wobble U·G base pair, extensive base stacking and sugar-base contacts. A more striking feature is the protrusion of the cytosine residue into the solvent without participating in any of the loop stabilizing interactions. This identifies the 2',5 '-linked (UUCG) loop as a novel structural motif and provides the first demonstration that 2',5' can fold back on itself to form a hairpin structure of unusual thermodynamic stability.
The ability of hairpin structures to inhibit human immunodeficiency virus (HIV-1) reverse transcriptase (RT) was also evaluated. Four potent hairpins based on 3',5'- and 2' ,5'-RNA were able to inhibit the RNase H activity of HIV-1 reverse transcriptase, a key enzyme for the proliferation of the human immunodeficiency virus (HIV-1). The polymerase activity of HIV-1 RT was not affected by this class of oligonucleotides. The hairpins were identified from a nucleic acid library synthesized via diversity-oriented solid-phase synthesis. These compounds represent the first examples of hairpins, 12 nucleotides in length, that inhibit specifically the RNase H activity of HIV-1 RT without affecting its polymerase activity.
Another study in this work dealt with yeast RNase III (Rnt1p), an enzyme implicated in the mechanism of action of RNA interference (RNAi). Through these investigations, it was discovered that Rntlp cleaves the DNA strand of DNA:RNA hybrids. This was totally unexpected since Rntlp, like other RNase III enzymes, was thought to be specific only towards the cleavage of double-stranded RNA. These studies also increased our knowledge about the mechanism by which the enzyme cleaves the target RNA (or DNA) strand and suggest that the vicinal 2'-hydroxyl group on the ribose sugar does not participate directly in the cleavage reaction.
Finally, the formation of C-tetrad structures (i-motif) was induced through the design and synthesis of extra-stable hairpin loops containing deoxycytidine rich stems. The corresponding hairpins containing ribocytidine folded into duplexes rather than C-tetrad structures. These studies lead to the first detection and identification of antiparallel 2',5 '-RNA duplexes based on hemiprotonated C·C+ base pairs.
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York, Ashley D. "A study of viral and cellular factors in the regulation of the influenza virus RNA-dependent RNA polymerase." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:5958fafd-4c91-4434-910e-29e2dd0539b9.
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The segmented negative-sense vRNA genome of influenza A virus is replicated via a complementary RNA (cRNA) intermediate by the viral RNA-dependent RNA polymerase (RdRP). The replicative intermediate has been thought to exist as a complementary ribonucleoprotein (cRNP) complex. Development of an RNA-based affinity-purification strategy for the isolation of viral ribonucleoprotein complexes enabled the structural and functional characterisation of the previously uncharacterised cRNP complex. The cRNP exhibits a filamentous double-helical organisation with defined termini, containing the viral RdRP at one end and a loop structure at the other end. In vitro characterisation of cRNP activity led to a proposal of a model of vRNA synthesis that relies on a trans-activating RdRP. The viral RdRP is an important host range determinant indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Affinity-purification followed by mass spectrometry was performed to identify cellular proteins that interact with the viral RdRP. 171 cellular proteins were found to co-purify with the viral RdRP, the most abundant of which were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, mitochondrial and ribosomal proteins. Among the phosphatases, three subunits of the cellular serine/threonine-protein phosphatase 6 (PP6) were identified. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and siRNA-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. Taken together, these results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.
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Edery, Isaac. "Role of cap function during eukaryotic protein synthesis and precursor messenger RNA splicing." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75970.
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The cap structure, m$ sp7$GpppX (where X = any nucleotide), is present at the 5$ sp prime$ end of all eukaryotic cellular messenger RNAs (except organellar). Previous studies have demonstrated that during protein synthesis the cap structure is recognized by a $ sim$24 kDa cap binding protein (CBP), termed eukaryotic initiation factor 4E (eIF-4E). With the use of a newly developed cap-analogy affinity matrix a high molecular weight complex that contains eIF-4E was purified, termed eIF-4F. In addition to eIF-4E this CBP complex consists of eIF-4A and a 220 kDa polypeptide (p220). Using an RNA unwinding assay direct evidence was obtained indicating that eIF-4F, eIF-4A and eIF-4B functionally interrelate resulting in helicase activity. Secondary structure in the 5$ sp prime$ untranslated region of eukaryotic mRNAs inhibits translation, therefore this unwinding activity is most likely required for efficient 40S ribosomal subunit attachment to mRNA. To fascilitate the purification and biophysical characterization of eIF-4E, the yeast homolog was overexpressed in E. coli. mRNA secondary structure can also inhibit translation in trans by another mechanism. This was shown for the unique structure (TAR) at the 5$ sp prime$ end of all mRNAs from the human immunodeficiency virus-1 (HIV-1). The mechanism of translation inhibition involves the activation of the double-stranded RNA dependent kinase (dsI), which catalyzes the phosphorylation of eIF-2. This is the first demonstration of a specific naturally occurring mRNA sequence that can activate dsI. A novel translational regulatory mechanism is proposed. Finally, the cap structure is also required for efficient precursor mRNA splicing in HeLa nuclear extracts. These and other studies indicate that the cap structure plays a multifunctional role during regulation of gene expression.
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Apura, Patrícia de Faria Pais. "Controlling gene expression in enterobacteriaceae: studies on sRNAs and strategies for synthetic biology." Master's thesis, Faculdade de Ciências e Tecnologia, 2014. http://hdl.handle.net/10362/12400.
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Dissertation presented to obtain the Master Degree in Molecular, Genetics and BiomedicineTranscriptional and post-transcriptional control of gene expression dictate the levels of proteins in the cell. Therefore the modulation of gene expression can have important consequences for biotechnological and/or pharmaceutical purposes. Among the types of cellular RNAs, small RNAs (sRNAs) have been an emerging class of bacterial gene expression regulators, which mostly act by base-pairing with one or more mRNA target(s) affecting their translation and/or their stability. Here, we focus on the study of SraL sRNA, more specifically in the validation of putative targets for this sRNA obtained in a previous transcriptomic analysis. Until now SraL was only shown to regulate the mRNA levels of Trigger Factor, an important protein chaperone. The information here reported give strong evidence for SraL involvement in the cysteine biosynthetic pathway, which requires further investigation. Nevertheless, our results could not provide a validation of those putative targets previously obtained by transcriptomic analyses. Optimization of protein expression requires not only an increase of the stability of mRNA transcripts but also an optimal behavior of function-encoding DNA segments, which are often context-dependent. Building on the work of others, we have designed a set of combinatorial promoters and 5’UTRs and evaluated their effects/outcomes using Superfolder GFP as reporter. Our data shows a clear variability of protein levels within our set of constructs. The highest levels of protein were associated with the implementation of an insulation sequence flanking the promoter region and the introduction of 5’ stabilizing structures at the mRNA level. Further investigation concerning the alteration of the rate of the mRNA decay by depletion of the function of participating nucleases, might constitute an advantageous approach. The knowledge collected will be extremely important to design robust modules which substantially increase protein production. This field is rapidly growing and much remains to be discovered about these important regulatory processes.
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Schonauer, Melissa. "Intersection of RNA Processing and Fatty Acid Synthesis and Attachment in Yeast Mitochondria." Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/194674.
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Intersections of distinct biological pathways in cells allow for nodes of metabolic regulation. This work describes the discovery of the intersection of two pathways in yeast mitochondria: RNA processing and fatty acid synthesis and attachment. Analysis of the components of the pathways is presented here along with a model illustrating the connection as a potential mode of regulation of mitochondrial gene expression.A genome-wide screen of respiratory-deficient
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Schulz, Daniel [Verfasser], and Patrick [Akademischer Betreuer] Cramer. "Transcriptome surveillance in S. cerevisiae by RNA synthesis and degradation coupling and selective termination of non-coding RNAs / Daniel Schulz. Betreuer: Patrick Cramer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2013. http://d-nb.info/1045561460/34.
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Chen, Yuanyuan. "Characterizing RNA Structure and synthesis by Raman Microscopy." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1277761094.
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Lotz, Thea Sabrina [Verfasser], Beatrix [Akademischer Betreuer] Süß, and Felicitas [Akademischer Betreuer] Pfeifer. "Development of photo-responsive synthetic RNA devices / Thea Sabrina Lotz ; Beatrix Süß, Felicitas Pfeifer." Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2018. http://d-nb.info/1177992124/34.
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Lin, Lina. "Synthesis, Structure, Function and Biomedical Studies of Nucleic Acid Derivatized with Selenium." Digital Archive @ GSU, 2010. http://digitalarchive.gsu.edu/biology_diss/77.
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Nucleic acids are macromolecules in cells for storing and transferring genetic information. Moreover, nucleic acids, especially RNAs, can fold into well-defined 3D structures and catalyze biochemical reactions. As ubiquitous biological molecules in all living systems, nucleic acids are important drug targets, and they can also be used in diagnostics and therapeutics. Structural information of nucleic acids provides the foundation for DNA and RNA function studies. X-ray crystallography has been a useful tool for structural studies of bio-macromolecules at atomic level. There are two major problems in macromolecular crystal structure determination: phasing and crystallization. Although selenium derivatization is routinely used for solving novel protein structures through the MAD phasing technique, the phase problem is still a critical issue in nucleic acid crystallography. The covalent selenium-derivatization of nucleic acids has been proven to be a useful strategy for solving the phase problem in nucleic acid X-ray crystallography. Besides the facilitation of nucleic acid crystallography, there is also a wide range of other applications for selenium-derivatized nucleic acids (SeNA). The investigation presented in this dissertation mainly focuses on the following research subjects (1) Synthesis and characterization of selenium-derivatized nucleic acids for X-ray crystallography, especially phosphoroselenoate RNAs. They are generated and used for crystallization. (2) Application of selenium-derivatized RNA for RNA interference. Phosphoroselenoate RNAs are tested for RNAi activities. (3) Synthesis and characterization of the uridine 5’-triphosphate modified with selenium at position 4. (4) Facile synthesis and antitumor activities of selenium modified deoxyribonucleosides. MeSe-thymidine nucleosides have shown antitumor activity in cell assays.
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Boussebayle, Adrien [Verfasser], Beatrix [Akademischer Betreuer] Suess, and Loewer [Akademischer Betreuer] Alexander. "Development of RNA aptamers and synthetic riboswitch using Capture-SELEX / Adrien Boussebayle ; Beatrix Suess, Loewer Alexander." Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2018. http://d-nb.info/1182537537/34.
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Bouakaz, Elli. "Choice of tRNA on Translating Ribosomes." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6324.
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Knight, William A. "Synthesis of unnatural amino acids for genetic encoding by the pyrrolysyl-tRNA/RNA synthetase system." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3794.
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The complexity of all biomolecules in existence today can be attributed to the variation of the amino acid repertoire. In nature, 20 canonical amino acids are translated to form these biomolecules, however, many of these amino acids have revealed posttranslational modifications (i.e. acetylation, methylation) after incorporation. Amino acids that exhibit PTM are known for their involvement in cellular processes such as DNA repair and DNA replication; these PTMs are commonly found on histones within the chromatin complex. Utilization of in vivo site-specific incorporation has recently reported functionality of post-translationally modified amino acids.1 xii Here we report the synthesis and in vivo site-specific incorporation of the histone PTM, 2-hydroxyisobutyrl lysine (Khib), with the pyrrolysyl tRNA/ RNA synthetase system. This translational machine can better serve to probe Khib for functional benefits. Additionally, this thesis focuses much of its attention on the development of unnatural amino acids (UAA) with optogenetic characteristics. These UAAs, if site-specifically incorporated, can be used to control enzymes and proteins through rapid light perturbation (365nm UV light). Furthermore, discussed is the synthesis of photo-caged threonine and photo-caged serine as potential substrates for the pyrrolysyl translational machinery.
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Kamelgarn, Marisa Elizabeth. "MUTATIONS OF FUS CAUSE AGGREGATION OF RNA BINDING PROTEINS, DISRUPTIONS IN PROTEIN SYNTHESIS, AND DYSREGULATION OF NONSENSE MEDIATED DECAY." UKnowledge, 2019. https://uknowledge.uky.edu/toxicology_etds/27.
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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron death and subsequent muscle atrophy. Approximately 15% of ALS cases are inheritable, and mutations in the Fused in Sarcoma (FUS) gene contribute to approximately 5% of these cases, as well as about 2% of sporadic cases. FUS performs a diverse set of cellular functions, including being a major regulator of RNA metabolism. FUS undergoes liquid- liquid phase transition in vitro, allowing for its participation in stress granules and RNA transport granules. Phase transition also contributes to the formation of cytoplasmic inclusions found in the cell bodies of FUS ALS patients motor neurons. The nature of these inclusions has remained elusive, as the proteins localized to them have not been identified. Additionally, the functional consequence of the accumulation of cytoplasmic FUS inclusions has not been established, nor is it understood how they contribute to selective motor neuron death.
We carried out two related, but independent studies to characterize the proteins that may be included in FUS-positive inclusions. In this first study, we utilized immunoprecipitation of wild-type and mutant FUS in the presence and absence of RNase, followed by LC MS/MS. The identified proteins represent those that directly or indirectly interact with FUS, with relatively high affinity that can be pulled down with immunoprecipitation. A wide variety of interacting proteins were identified and they are involved in a multitude of pathways including: chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. Their interaction with FUS varied greatly in their requirements for RNA. Most notably, FUS interacted with hnRNPA1 and Matrin-3, proteins also known to cause familial ALS. Immunofluorescent staining of proteins interacting with mutant FUS were localized to cytoplasmic inclusions. We concluded that mis-localization of these proteins potentially lead to their dysregulation or loss of function, thus contributing to FUS pathogenesis.
In the second study, we developed a protocol to isolate dynamic FUS inclusions and employed LC MS/MS to identify all proteins associated with FUS inclusions. We identified a cohort of proteins involved in translation, splicing, and RNA export to be associated with the FUS inclusions. Further pathway and disease association analysis suggested that proteins associated with translation and RNA quality control pathways may be the most significant. Protein translation assays using both N2A and ALS patient fibroblasts demonstrated suppression of protein biosynthesis in mutant FUS expressing cells. However, translation initiation was not impaired. To understand how protein synthesis is suppressed by mutant FUS mediated defects in RNA metabolism, we examined changes in a well conserved RNA turnover pathway namely: nonsense mediated decay (NMD). We found that NMD is hyperactivated in cells expressing mutant FUS, likely due to chronic suppression of protein translation shifting the pathways autoregulatory circuit to allow for hyperactivation. We concluded that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation. These defects together likely contribute to motor neuron death.
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Siegmund, Vanessa [Verfasser]. "DNA and RNA Polymerases with Expanded Substrate Scope : Synthesis of Modified Nucleic Acids Using Engineered Polymerases Generated by Directed Evolution / Vanessa Siegmund." Konstanz : Bibliothek der Universität Konstanz, 2013. http://d-nb.info/1043443320/34.
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Seekircher, Stephanie [Verfasser], and Petra [Akademischer Betreuer] Dersch. "Identification of regulatory factors that control the synthesis of the small regulatory RNA CsrC in Yersinia pseudotuberculosis / Stephanie Seekircher ; Betreuer: Petra Dersch." Braunschweig : Technische Universität Braunschweig, 2014. http://d-nb.info/1175821209/34.
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Vasanthavada, Keshav. "Generation of cDNA chips from the black widow spider, latrodectus hesperus, for gene discovery and expression profiling using microarray technology, and molecular characterization of a novel silk glue protein." Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/624.
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eDNA microarray technology has generated a tremendous amount of interest among biologists because of its promise to monitor the entire genome on a single chip, thus enabling researchers to have a better picture of the interaction among thousands of genes simultaneously. In the current study, this technology was used to print over 3,000 unknown genes from various silk glands of the black widow spider to profile their expression patterns and to identify novel candidates. Spiders are remarkable creatures because of their ability to make different silks, each with a specific function. Some of these silks have amazing mechanical properties, comparable to those of the finest synthetic materials. Several silk genes have been cloned from various spiders over the last few years, and the contribution of each of those genes in silk production has been identified. However, the majority of cellular and biochemical processes involved in silk
manufacture remain a mystery. In our research, we attempt to identify genes that might be involved in silk assembly, on a global scale and investigate more about those genes and their interplay with other key biological molecules involved in silk manufacture. Our study showed that silking spiders for a certain period of time resulted in down-regulation of two important silk genes, ECP-1 and ECP-2. Both these genes are key molecules implicated for their role in maintaining the egg case architecture in the black widow spider.,-and we believe that these genes are also directly or indirectly involved in the manufacture of dragline silk. Microarray analyses also enable the discovery of several other interesting molecules, two of which could be accessory proteins involved in silk formation. Furthermore, in a separate study we also characterized a novel silk glue protein with unique ensemble repeats. In conclusion, we believe that the findings of this study will indeed be significant to silk researchers and material scientists alike and it will enhance our knowledge in understanding the mystery behind silk production.
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Fernando, Chaminda. "Identification of the Pla2 Responsible For Prostanoid Synthesis in Response to Inflammatory Cytokines." VCU Scholars Compass, 2005. http://scholarscompass.vcu.edu/etd/803.
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Preliminary studies from our laboratory showed that cPLA2α may be responsible for approximately 50-60% of the PGE2 production in response to inflammatory cytokines. Thus, we hypothesized that a closely-related PLA2 is responsible for 40-50% of the PGE2 produced in response to inflammatory cytokines. To this end, we utilized RNAi technology, extensively optimized, to down regulate the expression of closely-related isoforms of phospholipase A2 in A549 cells and used an enzyme linked immuno sorbent assay (ELISA) to quantitate the PGE2 produced. These studies found that cytosolic phospholipase A2α (cPLA2α) regulated 97.7% of the prostaglandin E2 (PGE2) produced in response to inflammatory cytokines (e.g. IL-1β or TNFα), as well as regulating the basal levels of this prostanoid. Furthermore, cPLA2γ, cPLA2δ, and iPLA2 were found to also to regulate the basal levels of PGE2 production. On the other hand, cPLA2β was not involved in prostanoid synthesis in A549 cells either in the presence or absence of inflammatory cytokines. Thus, our studies show that cPLA2α plays the pivotal role in the production of PGE2 in response to inflammatory cytokines, and suggests that cPLA2α may be a possible drug target in diseases such as asthma, inflammation, and cancer.
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García, Mayo Susana. "Synthesis of gold nanoparticles for rapid genotyping of M. tuberculosis using rolling circle amplification and nanoflare technology." Thesis, Stockholms universitet, Avdelningen för materialkemi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-191049.
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Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis, with an incidence in a quarter of the world population. Despite the scientific and technological advances, an effective diagnostic method has not yet been found that allows an early diagnosis and, also, to detect the strain present in the patient. The combination of nanotechnology with molecular diagnostics has shown promising advances offering new possibilities, such as the development of nanoflares. Nanoflares represent a new class of molecular probes, composed of gold nanoparticles functionalized with a recognition sequence that can be amplified by rolling circle amplification (RCA) technique, producing a fluorescence signal. This thesis focuses in the synthesis of gold nanoparticles, with different coatings and sizes, as well as their subsequent application in the preparation and optimization of nanoflares for the genotyping of synthetic M. tuberculosis targets using RCA technique. The different preparations of nanoflares have an impact in the assay sensitivity, showing two times increase in sensitivity for citrate-coated nanoparticles with respect to those coated with PEG. Furthermore, it was observed that the sensitivity is directly related to the synthesized particle size. Sensitivity is also affected by the application of a purification post-treatment of the synthesis product. This post-treatment reduces the sensitivity of nanoflares by up to 37% but, by contrast, extends its useful life. The results obtained are shown as a proof of concept for a future cost-effective, rapid and robust in situ diagnostic method that identifies the strain of tuberculosis present in the patient.
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Schillewaert, Stéphanie. "Etude de la maturation et de l'assemblage du ribosome eucaryote: caractérisation fonctionnelle de nouveaux facteurs trans-." Doctoral thesis, Universite Libre de Bruxelles, 2011. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209826.
Full textAbstract:
La synthèse du ribosome est un processus compliqué, très hiérarchisé et essentiel à toutes les cellules vivantes. La complexité de ce processus tient notamment au fait que les différentes étapes de la biogenèse du ribosome eucaryote sont temporellement et spatialement organisées dans des compartiments cellulaires différents (le nucléole, le nucléoplasme et le cytoplasme). Il est toutefois connu que le pré-ARNr 35S (le précurseur de trois des quatre ARNr, les ARNr 18S, 5.8S et 25S) est pris en charge dès sa synthèse par des facteurs impliqués dans sa maturation. Ainsi, la formation d’un ribosome requiert l’association, sur le transcrit naissant, des facteurs de synthèse, au nombre de 400. Ces facteurs essentiels interagissent transitoirement avec l’ARNr et ne font pas partie des particules ribosomiques matures impliquées dans la traduction. Leur rôle est d’assister le remodelage constant du pré-ribosome et le processus d’assemblage des sous-unités.Parmi ces facteurs de synthèse, nous avons caractérisé en détail, chez la levure et chez l’homme, la protéine Las1 impliquée dans la maturation des deux extrémités de l’ITS2, séquence qui sépare les ARNr 5.8S et 25S/28S. Chez la levure, en absence de la protéine Las1, les analyses de profils de polysomes révèlent un déficit de sous-unité 60S et l’apparition d’« halfmères ». Les techniques de purification d’affinité et de gradient de sédimentation nous indiquent que Las1 est associée aux pré-ribosomes 60S et qu’elle interagit avec de nombreux facteurs de synthèse de la petite, de la grande sous-unité ou des deux. De plus, Las1 copurifie avec des pré-ribosomes qui contiennent aussi les exoribonucléases 5’-3’ Rat1/Rai1 et Xrn1. Rai1 coordonne la maturation aux deux extrémités de l’ARNr 5.8S. Nous suggérons que Las1 appartient à un macrocomplexe connectant spatialement des sites de clivages éloignés sur la séquence primaire du pré-ARNr qui seraient rapprochés suite au reploiement de l’ITS2.
Un autre aspect de ce travail de thèse consiste en l’étude de l’assemblage des particules ribonucléoprotéiques et plus spécifiquement du pré-ribosome et des sous-unités ribosomiques eucaryotes. Nous avons utilisé la technique d’immunoprécipitation de chromatine (Ch-IP) pour caractériser l’assemblage d’une structure appelée le « SSU processome ». Celui-ci correspond à un pré-ribosome en formation ainsi que l’assemblage des protéines ribosomiques sur l’ARNr naissant.
Enfin, nous avons étudié le rôle d’une plateforme d’activation de méthyltransférases d’ARN et de protéines, la protéine Trm112 dans la ribogenèse. Nous avons montré que chez la levure, Trm112 est impliquée dans la synthèse du ribosome et dans la progression de la mitose. En absence de cette protéine, les pré-ARNr sont dégradés par un mécanisme de surveillance. Trm112 copurifie avec plusieurs facteurs de synthèse du ribosome dont la méthyltransférase Bud23, impliquée dans la modification post-transcriptionnelle de l’ARNr18S. Trm112 est requise pour cette méthylation et nous postulons que la protéine Bud23 est incapable de se lier aux pré-ribosomes en l’absence de Trm112.
Doctorat en Sciences
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Quynh, Tran Hoang Thi. "Identification and functional characterization of trans-acting factors required for eukaryotic ribosome synthesis." Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210540.
Full textAbstract:
Eukaryotic ribosome synthesis is a complex process that consumes a lot of energy and involves several hundreds of trans-acting factors that transiently associate with nascent ribosomes. Biogenesis of ribosomal subunits (the small 40S and the large 60S) starts with transcription of a long precursor ribosomal RNA (pre-rRNA) by RNA polymerase I (Pol I) in the nucleolus. This is a key step that globally controls yeast ribosome synthesis. The pre-rRNA, ‘the 35S transcript’, encodes the mature sequence (18S, 5.8S, and 25S) rRNA constituents of both the 40S and 60S subunits. The 35S transcript is subsequently modified, cleaved (processed) and assembled with numerous structural ribosomal proteins and ribosome synthesis factors (trans-acting factors) to form various ribosomal particles (pre-ribosomes, precursors to the 40S and 60S subunits) along ribosome assembly pathway. In the budding yeast Saccharomyces cerevisiae, it has been reported recently that the processing of the 35S nascent transcript and the assembly of pre-ribosomes occur concomitantly with Pol I transcription in the nucleolus. In this process, the growing Pol I transcript gradually assembles with pre-40S structural ribosomal proteins and ribosomal synthesis factors to form the so-called ‘SSU-processome’ or ‘90S pre-ribosome’, the earliest precursor of the 40S subunit. The SSU-processome/90S pre-ribosome localizes to the nucleolus and consists of the 35S pre-rRNA, the U3 small nucleolar (sno) RNA, about a dozen of 40S ribosomal proteins and more than forty ribosome synthesis factors. The U3 snoRNA and pre-40S ribosome synthesis factors are all implicated in the processing of the 35S precursor (at sites A0, A1 and A2) and therefore in the synthesis of the 18S rRNA component of the 40S subunit. Significantly, the association of the U3 snoRNA with the growing 35S transcript is important for pre-40S assembly, whereas its dissociation from the processed transcript (following cleavage at sites A0-A2) is crucial for the overall structural remodeling of the 18S rRNA and for the formation of pre-40S ribosomes from the earliest precursor 90S particles.
This thesis mostly addresses the identification and functional characterization of Esf2 and Bfr2, two novel 40S synthesis factors, components of the SSU-processome/90S pre-ribosome in yeast. Both proteins localize to the nucleolus and their genetic depletions lead to failure in the production of 40S subunits. In the absence of either factor, the 35S pre-rRNA is not processed at sites A0-A2 and the 18S rRNA is not synthesized. Also, pre-ribosome assembly is affected and pre-40S ribosomes fail to mature properly. Strikingly, in the absence of either factor, the U3 snoRNA remains associated with unprocessed 35S transcript within pre-ribosomes indicating that Esf2 and Bfr2 are required to dissociate U3 from pre-ribosomes. This process also involves RNP (ribonucleoprotein particle) unwinding activities of the putative RNA helicase Dbp8.
La biogenèse du ribosome eucaryote est un processus complexe qui consomme beaucoup d’énergie et implique plusieurs centaines de facteurs trans qui s’associent de manière transitoire avec les pré-ribosomes en cours de formation. La biogenèse des sous-unités ribosomiques (la petite sous-unité 40S et la grande sous-unité 60S) débute dans le nucléole par la synthèse d’un long précurseur d’ARN ribosomique (le pré-ARNr, dit 35S chez la levure Saccharomyces cerevisiae) par l’ARN Polymérase I (Pol I). Ceci constitue une étape clé dans le contrôle global de la synthèse du ribosome chez la levure. Le pré-ARNr 35S renferme les séquences des ARNr matures 18S (ARNr de la sous-unité 40S) et 5.8S et 25S (deux des trois ARNr de la sous-unité 60S). Le pré-ARNr 35S subit un long processus de maturation et d’assemblage au cours duquel il est modifié, clivé (on parle du « processing » du pré-ARNr) et s’assemble avec des protéines ribosomiques (« RP », composants structuraux des sous-unités ribosomiques matures) et de nombreux facteurs de synthèse (facteurs trans) pour former différentes particules pré-ribosomiques (précurseurs des sous-unités 40S et 60S).
Chez la levure S. cerevisiae, il a récemment été montré que le processing du pré-ARNr 35S et l’assemblage des pré-ribosomes se produisent de manière concomminante avec la transcription Pol I dans le nucléole. Ainsi, le transcrit Pol I en cours de synthèse s’assemble progressivement avec des facteurs de synthèse ainsi que des RP pour former le « SSU processome » ou « pré-ribosome 90S », tout premier précurseur de la petite sous-unité 40S. Le SSU processome/pré-ribosome 90S est localisé dans le nucléole et est consisté du pré-ARNr 35S naissant, du petit ARN nucléolaire (snoRNA) U3, d’une douzaine de RP de la petite sous-unité 40S et de plus de 40 facteurs de synthèse. Le snoRNA U3 et ces facteurs de synthèse sont tous impliqués dans les clivages du pré-ARNr 35S aux sites A0, A1 et A2, et donc dans la biogenèse de l’ARNr 18S. L’association du snoRNA U3 avec le pré-ARNr 35S naissant est importante pour l’assemblage du SSU processome/pré-ribosome 90S. Par ailleurs, sa dissociation après les clivages aux sites A0-A2 permet un remodelage structural général de l’ARNr 18S et la formation du « pré-ribosome 40S » à partir de la particule précoce 90S.
Au cours de cette thèse, nous avons identifié et caractérisé fonctionnelement chez la levure deux nouveaux facteurs de synthèse de la petite sous-unité 40S et composants du SSU processome/pré-ribosome 90S: Esf2 et Bfr2. Ces deux protéines sont localisées dans le nucléole. Leur déplétion entraîne une incapacité à produire la sous-unité ribosomique 40S. En l’absence d’Esf2 ou Bfr2, le pré-ARNr 35S n’est plus clivé aux sites A0-A2 et l’ARNr 18S mature n’est plus produit. L’assemblage des pré-ribosomes est aussi affecté, notamment la formation du pré-ribosome 40S. De manière importante, en l’absence de l’un ou l’autre de ces facteurs, le snoRNA U3 reste associé au pré-ARNr 35S non clivé au sein des pré-ribosomes, indiquant qu’Esf2 et Bfr2 sont requises pour la dissociation d’U3 des pré-ribosomes. Ce processus implique aussi Dbp8, une hélicase à ARN présumée.
Doctorat en sciences, Spécialisation biologie moléculaire
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De, Leeuw Frédéric. "Etude de la protéine CIRP et sa fonction dans le métabolisme des ARN messagers." Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210577.
Full textAbstract:
La protéine CIRP (Cold Induced RNA binding Protein) est une petite protéine de liaison à l’ARN de 172 acides aminés, qui est constituée du côté amino-terminal d’un domaine de liaison à l’ARN de type RRM (RNA recognition motif), et d’une partie carboxy-terminale riche en glycine et arginine qui comprend plusieurs motifs RGG. Elle a été identifiée comme étant inductible par hypothermie mais aussi par irradiation aux UV et par hypoxie. Nous avons analysé son expression et sa localisation en réponse à différents stress cellulaires. Nous avons montré qu’un traitement à l’arsénite qui induit un stress oxydant n’altère pas l’expression de CIRP provoque sa localisation dans les granules de stress (SG). Les SG sont des structures ribonucléoprotéiques cytoplasmiques contenant des complexes de pré-initiation incompétents pour la traduction, et qui s’accumulent dans les cellules exposées à un stress. Ces structures constituent des sites de triages des ARNm, dans lesquels les ARNm sont soit stockés en attente d’une réinitiation de la traduction une fois le stress surmonté, soit destinés à être dégradés. La protéine CIRP se localise dans les SG que ce soit suite à un stress cytoplasmique ou du réticulum endoplasmique. Nous avons montré également que la localisation de la protéine CIRP dans les SG se déroule indépendamment de la présence de la protéine TIA-1 qui a été décrite comme responsable de l’assemblage des SG. De plus la surexpression de la protéine CIRP conduit à la formation de SG. Nous suggérons donc qu’il existe plusieurs voies qui mènent à l’assemblage de ces structures. En outre, nous avons analysé la localisation de mutants par délétion de la protéine CIRP et avons montré que le domaine RRM et le domaine RGG peuvent indépendamment localiser la protéine dans les SG. Par contre, la méthylation des résidus arginine du domaine RGG est une modification nécessaire à la localisation de CIRP dans les SG. Ensuite, nous avons étudié la fonction de la protéine CIRP dans le métabolisme des ARN messagers. Nous avons montré par une méthode d’adressage, que CIRP est un répresseur de la traduction des ARNm et que le domaine carboxy-terminal est nécessaire et suffisant à cette fonction.Doctorat en Sciences
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Ziegler, Véronique. "La rhizomanie, une maladie virale de la betterave a sucre : contribution a l'etude du mecanisme d'expression et des proprietes biologiques du genome du virus des nervures jaunes et necrotiques de la betterave." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13017.
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Choquet, Yves. "Contribution a l'etude de la structure et de la biogenese des centres photochimiques des vegetaux superieurs et de l'algue verte chlamydomonas reinhardtii." Paris 6, 1987. http://www.theses.fr/1987PA066307.
Full textAbstract:
Les techniques de spectrometrie en lumiere polarisee et de preparation de complexes photosynthetiques isoles de vegetaux superieurs et de l'algue verte chlamydomonas reinhardtii ont permis de mettre en evidence l'existence d'une profonde similitude structurale entre les centres photochimiques des vegetaux superieurs et ceux de l'algue. La biogenese du cpi shez c. Reinhardtii, la sequence des genes chloroplastiques psa a1/2 et psa a2 codant pour les apoproteines du cpi ont ete determinees. Mise en evidence d'un mecanisme pour la maturation du message de ce gene : assemblage en trans de precurseurs transcrits independemment. Caracterisation biochimique et moleculaire de mutants nucleaires ou chloroplastiques deficients pour la synthese des apoproteines du cpi
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"RNA Aptamer-Based Systems for Pathogen Detection and Biomolecule Synthesis." Doctoral diss., 2020. http://hdl.handle.net/2286/R.I.63037.
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abstract: RNA aptamers adopt tertiary structures that enable them to bind to specific ligands. This capability has enabled aptamers to be used for a variety of diagnostic, therapeutic, and regulatory applications. This dissertation focuses on the use RNA aptamers in two biological applications: (1) nucleic acid diagnostic assays and (2) scaffolding of enzymatic pathways. First, sensors for detecting arbitrary target RNAs based the fluorogenic RNA aptamer Broccoli are designed and validated. Studies of three different sensor designs reveal that toehold-initiated Broccoli-based aptasensors provide the lowest signal leakage and highest signal intensity in absence and in presence of the target RNA, respectively. This toehold-initiated design is used for developing aptasensors targeting pathogens. Diagnostic assays for detecting pathogen nucleic acids are implemented by integrating Broccoli-based aptasensors with isothermal amplification methods. When coupling with recombinase polymerase amplification (RPA), aptasensors enable detection of synthetic valley fever DNA down to concentrations of 2 fM. Integration of Broccoli-based aptasensors with nucleic acid sequence-based amplification (NASBA) enables as few as 120 copies/mL of synthetic dengue RNA to be detected in reactions taking less than three hours. Moreover, the aptasensor-NASBA assay successfully detects dengue RNA in clinical samples. Second, RNA scaffolds containing peptide-binding RNA aptamers are employed for programming the synthesis of nonribosomal peptides (NRPs). Using the NRP enterobactin pathway as a model, RNA scaffolds are developed to direct the assembly of the enzymes entE, entB, and entF from E. coli, along with the aryl-carrier protein dhbB from B. subtilis. These scaffolds employ X-shaped RNA motifs from bacteriophage packaging motors, kissing loop interactions from HIV, and peptide-binding RNA aptamers to position peptide-modified NRP enzymes. The resulting RNA scaffolds functionalized with different aptamers are designed and evaluated for in vitro production of enterobactin. The best RNA scaffold provides a 418% increase in enterobactin production compared with the system in absence of the RNA scaffold. Moreover, the chimeric scaffold, with E. coli and B. subtilis enzymes, reaches approximately 56% of the activity of the wild-type enzyme assembly. The studies presented in this dissertation will be helpful for future development of nucleic acid-based assays and for controlling protein interaction for NRPs biosynthesis.Dissertation/Thesis
Doctoral Dissertation Biochemistry 2020
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Davidson, Eric Alan. "Synthetic transcription systems." Thesis, 2010. http://hdl.handle.net/2152/ETD-UT-2010-05-948.
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In this work, we seek to expand synthetic in vitro biological systems by using water-in-oil emulsions to provide an environment conducive to directed evolution. We approach this primarily by utilizing a model transcription system, the T7 RNA polymerase and promoter, which is orthogonal to both bacterial and eukaryotic transcription systems and is highly functional in vitro. First, we develop a method to identify functional promoter sequences completely in vitro. This method is tested using the T7 RNA polymerase-promoter model system. We then configure the T7 transcription system as an ‘autogene’ and investigate how this positive feedback circuit (whereby a T7 promoter expresses a T7 RNA polymerase gene) functions across various in vitro platforms, including while compartmentalized. The T7 autogene can be envisioned as a self-replicating system when compartmentalized, and its use for directed evolution is examined. Finally, we look towards future uses for these in vitro systems. One interesting application is to expand the utilization of unnatural base pairs within the context of a synthetic system. We investigate the ability of T7 RNA polymerase to recognize and utilize unnatural base pairs within the T7 promoter, complementing existing work on the utilization of unnatural base pairs for in vitro replication and transcription with an investigation of more complex protein-dependent regulatory function. We envision this work as a foundation for future in vitro synthetic biology efforts.text