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Hunt, Sarah Louise. "Cellular proteins required for rhinovirus RNA translation". Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313880.
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Chan, Annie Yee-Man. "Interactions between the influenza virus RNA polymerase and cellular RNA polymerase II". Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670083.
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Bailey, Daniel John. "Cellular proteins in picornavirus replication". Thesis, University of Reading, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298484.
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Vasale, Jessica J. "Roles of Cellular RNA-Dependent RNA Polymerases in Endogenous Small RNA Pathways in Caenorhabditis elegans: A Dissertation". eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/481.
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The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact with RdRP-dependent small RNAs; and 3) investigate the biological function of RdRP-dependent small RNA pathways in C. elegans.
In this thesis, I describe genetic, deep sequencing, and molecular studies, which identify 22G-RNAs as the most abundant class of endogenous small RNA in C. elegans. The 22G-RNAs resemble RdRP-dependent secondary siRNAs produced during exogenous RNAi, in that they possess a triphosphorylated 5’ guanine residue and exhibit a remarkable strand bias at target loci. Indeed, I show that 22G-RNAs are dependent on the activity of the RdRPs RRF-1 and EGO-1 and function in multiple distinct endogenous small RNA pathways. Interestingly, I have found that RRF-1 and EGO-1 function redundantly in the germline to generate 22G-RNAs that are dependent on and interact with members of an expanded family of worm-specific Argonaute (WAGO) proteins. The WAGO/22G-RNA pathway appears to be a transcriptome surveillance pathway that silences coding genes, pseudogenes, transposons, and non-annotated, or cryptic, transcripts. In contrast, I have found that EGO-1 alone is required for the biogenesis of a distinct class of 22G-RNAs that interact with the Argonaute CSR-1. Surprisingly, the CSR-1/22G-RNA pathway does not appear to silence its targets transcripts. Instead, the CSR-1/22G-RNA pathway is essential for the proper assembly of holocentric kinetochores and chromosome segregation.
Lastly, I show that a third endogenous small RNA pathway, the ERI pathway, is a two-step silencing pathway that requires the sequential activity of distinct RdRPs and Argonautes. In the first step of this pathway, the RdRP, RRF- 3, is required for the biogenesis of 26G-RNAs that associate with the Argonaute, ERGO-1. In the second step, RRF-1 and EGO-1 generate 22G-RNAs that associate with the WAGO Argonautes.
This work demonstrates how several C. elegans small RNAs pathways utilize RdRPs to generate abundant populations of small RNAs. These distinct categories of small RNAs function together with specific Argonaute proteins to affect gene expression, to play essential roles in development, and in the maintenance of genome and transcriptome integrity.
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Osborn, Maire. "Cellular RNA Targeting by Platinum (II) Anticancer Therapeutics". Thesis, University of Oregon, 2014. http://hdl.handle.net/1794/17920.
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Cis-diamminedichloroplatinum (II), or cisplatin, is a widely prescribed anticancer compound, currently one of only three platinum (II) complexes FDA approved for cancer treatment. Despite its widespread use, we lack a comprehensive picture of global drug targets, which would lend valuable insights into the molecular mechanisms of action and resistance in different tissues. Drug binding to genomic DNA is an accepted cause of downstream apoptotic signaling, but less than 10% of Pt (in the case of cisplatin) accumulates within genomic DNA. Non-genomic contributions to cisplatin's therapeutic action are also under active investigation. In particular, cisplatin treatment can disrupt RNA-based processes such as splicing and translation. Pt(II) targeting of non-DNA species such as RNA may contribute to or sensitize a cell to the downstream effects of this drug, including the induction of apoptosis.
Chapter I summarizes the activity profile of Pt(II) therapeutics, describing cellular uptake, cellular localization, incidences of Pt(II) accumulation within RNA, and RNA processes affected following drug treatment. Chapter II reports our thorough investigation of the distribution of Pt species throughout messenger and ribosomal RNA, with the discovery that Saccharomyces cerevisiae ribosomes act as a de facto cellular Pt sponge. In Chapter III, we report the synthesis of an azide-functionalized platinum (II) species, picazoplatin, for post-treatment click labeling and isolation of drug targets in vivo. Picazoplatin was designed to circumvent mislocalization and misprocessing of Pt typically encountered when trying to track small molecules tethered to large, charged fluorophores. This chapter contains several proof-of-principle studies validating the use of this class of reagents for future purification and sequencing of Pt-bound nucleic acids. Chapter IV describes the first application of the click-capable Pt reagent technology: the demonstration of significant in-gel fluorescent detection of Pt-bound ribosomal RNA and transfer RNA extracted from picazoplatin-treated S. cerevisiae and the first evidence that cellular tRNA is a platinum substrate. Chapter V summarizes these data, which suggest a potential ribotoxic mechanism for cisplatin cytotoxicity and broadly describe a convenient click chemistry methodology that can be applied to identify other metal or covalent modification-based drug targets.
This dissertation includes previously published and unpublished co-authored material.
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Brown, E. C. "Cellular proteins involved in translation of human rhinovirus RNA". Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596963.
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Translation of picornavirus RNA takes place by internal initiation, determined by the presence of an internal ribosome entry site (IRES) in the 5'-untranslated region of the genomic RNA. Efficient translation from the human rhinovirus-2 (HRV-2) IRES is dependent on host cell trans-acting factors. These include unr, p38 and polypyrimidine tract binding protein (PTB). This thesis details the investigation into how these factors act to promote translation from the HRV-2 IRES. Unr, an RNA-binding protein with five cold-shock domains (CSDs), binds to the HRV-2 IRES and this interaction was studied by crude and then fine mapping of the binding sites of unr on the IRES. The functions of the CSDs of unr were investigated by point mutation of each of the CSDs and testing the ability of these mutants to bind the IRES and stimulate translation from it. p38, a WD-motif protein with no RNA-binding activity, was expressed using recombinant baculovirus-infected insect cells. An in vivo interaction between unr and p38 was demonstrated, and the effect of p38 on unr's binding to the HRV-2 IRES was tested in vivo. After gaining insight into the complexes of unr and p38 that form on the IRES, the function of p38 in translation from the HRV-2 IRES was demonstrated. Unr and PTB were also used as tools to compare the factor requirements of the HRV-2 and poliovirus IRESs for efficient translation. Finally, an investigation was made into the cellular role of unr, in terms of the cellular mRNAs that unr binds, and those whose translation it stimulates.
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Todorova, Tanya (Tanya Todorova). "Function and regulation of PARP13 binding to cellular RNA". Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/97789.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
Poly(ADP-ribose) polymerase-13 (PARP13) is a member of the PARP family of proteins - enzymes that use NAD+ to synthesize a posttranslational protein modification called poly(ADP-ribose) (PAR). PARPs function in multiple cellular pathways, and recently several members of the family have been implicated in regulating various steps in RNA metabolism, from splicing to translation and decay. PARP1 3 is the best-understood RNA-regulatory PARP. Initially discovered as a host immune factor, PARP13 functions by binding viral transcripts via its four CCCH-type zinc fingers and targeting them for degradation. In the context of the immune response PARP1 3 can also inhibit the translation of its viral targets and enhance the activity of other RNA-binding viral receptors, such as RIG-1. More recently PARP13 was shown to also indirectly regulate the cellular transcriptome by inhibiting the activity of Argonaute 2 (Ago2), a member of the miRNA silencing pathway. While itself catalytically inactive, PARP13 is modified by PAR and can target Ago2 for modification by a yet unknown PARP. However, it remains unclear if RNA binding is required for this function of PARP1 3. Indeed, even though multiple viruses are known to be restricted by PARP13, cellular mRNA targets of PARP13 binding and regulation have not yet been identified. Here we show that PARP1 3 binds endogenous RNA and regulates the cellular transcriptome. We identify TRAILR4 mRNA as the first cellular target of PARP13 regulation and demonstrate that PARP13 represses TRAILR4 expression posttranscriptionally by binding to a specific region in the 3' untranslated region of the transcript and targeting it for degradation in a primarily 3'-5' decay mechanism. By inhibiting the expression of TRAILR4 - a decoy pro-survival receptor of the apoptotic ligand TRAIL, PARP1 3 regulates the cellular response to TRAIL and acts as a pro-apoptotic factor. We also examine possible mechanisms of regulation of PARP1 3 function. We identify the RNA-helicase DHX30 as a constitutive PARP1 3-interacting protein and show that the two proteins co-regulate a subset of cellular transcripts. We further demonstrate that the PAR-binding domain of PARP1 3 inhibits RNA binding, while PARP1 3 interaction with PARP5a and covalent modification with PAR appear to be mutually exclusive with RNA binding.
by Tanya Todorova.
Ph. D.
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Mullani, Nowsheen. "An RNA Signature Links Oxidative Stress To Cellular Senescence". Electronic Thesis or Diss., Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2019SORUS560.pdf.
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Le stress oxydatif est l’une des voies menant à la sénescence cellulaire. Bien que les dommages causés par les espèces réactives de l'oxygène aux protéines et à l'ADN soient bien décrits, notre compréhension de la manière dont la transcription peut participer à l'apparition de la sénescence est encore limitée. Au niveau de la transcription, le stress oxydatif entraîne l’accumulation d’ARN promoteurs (ARNAu) et d’ARN amplificateur (ARNs), conséquence de la libération défectueuse du RNAPII de la chromatine, un phénomène connu sous le nom de RNAPII crawling. Nous avons observé que l'exploration de RNAPII était également détectée en aval d'une petite série de gènes connus pour être régulés par HP1Υ au niveau de leur terminaison. L'exploration de ce phénomène a donné un résultat inattendu, en ce sens qu'il a révélé un effet inhibiteur du peroxyde d'hydrogène sur le complexe exosome d'ARN impliqué dans la dégradation des ARN polyadénylés. Le RNAPII rampant a pour résultat la transcription de séquences d’ALU situées au voisinage des promoteurs et amplificateurs et en aval de gènes sans intron et de petites séries de gènes contenant un intron. Comme les séquences ALU contiennent des séquences A codées par le génome, elles doivent normalement être dégradées par l’exosome de l’ARN. Cependant, comme le stress oxydatif inhibe également cette activité d'ARNase, les ARNm contenant des séquences d'ALU transcrites par hasard se stabilisent et sont détectés dans le cytoplasme et même dans les fractions de polysomes. Ce phénomène peut participer à l'apparition de la réponse à l'interféron associée au stress oxydatifOxidative Stress is one of the routes leading to cellular senescence. While the damages that reactive oxygen species inflict on proteins and DNA are well described, our insight on how transcription may participate in the onset of senescence is still limited. At a transcriptional level, oxidative stress results in accumulation of promoter RNAs (uaRNAs) and enhancer RNAs (eRNAs) as a consequence of defective release of the RNAPII from the chromatin a phenomenon known as RNAPII crawling. We observed that RNAPII crawling was also detected downstream of a small series of genes known to be regulated by HP1Υ at the level of their termination. Exploring this phenomenon yielded an unexpected result in the sense that it revealed an inhibiting effect of hydrogen peroxide on the RNA exosome complex involved in degradation of polyadenylated RNAs. The crawling RNAPII results in the transcription of ALU sequences located in the neighborhood of promoters and enhancers and downstream of intron-less genes and of small series of intron-containing genes. As ALU sequences contain genome encoded A tracts, they should normally be degraded by the RNA exosome. Yet, as oxidative stress also inhibits this RNAse activity, mRNAs containing serendipitously transcribed ALU sequences get stabilized and are detected in the cytoplasm and even polysome fractions. This phenomenon may participate in the onset of the interferon response associated with oxidative stress
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Stassinopoulos, Ioannis A. "Interactions of picornavirus internal ribosome entry sites with cellular proteins". Thesis, University of Sussex, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322940.
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Schmier, Brad J. "The Molecular Machinery Critical to the Degradation of Cellular RNA". Scholarly Repository, 2012. http://scholarlyrepository.miami.edu/oa_dissertations/714.
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Exoribonucleases are indispensable for cellular RNA metabolism. RNA processing, end-turnover, and degradation all require the concerted action of exoribonucleases. In this thesis, two families of exoribonucleases that act in the final steps of RNA decay pathways are explored. The first of these is the RNR superfamily of processive 3’→5’ RNases with major roles in both mRNA and stable RNA degradation. The initial focus of this work is the structural and enzymatic characterization of an unusual RNR family enzyme from the radiation-resistant bacterium Deinococcus radiodurans. This enzyme is demonstrated biochemically to be an RNase II-type enzyme (DrII), based on its sensitivity to secondary structure. Analysis of the DrII X-ray structure reveals that a novel, winged-HTH domain has replaced the canonical RNA binding clamp typical of RNR family proteins. The exposed architecture of DrII’s RNA binding surface offers an explanation for the nuclease’s ability to approach within 3-5 nt of a duplex, an important mechanistic difference from the well-studied E. coli RNase II. The open, clamp architecture of DrII may have broader relevance to mechanisms of duplex RNA recognition in the RNR superfamily. RNA decay by processive exonucleases such as RNR family proteins leaves 2-5 nt nanoRNA limit products that are further degraded to mononucleotides by nanoRNases. In E. coli, the DEDD family enzyme Oligoribonuclease (ORN) executes nanoRNA decay and represents the first major family of nanoRNases, with homologs widely conserved in eubacteria and eukaryotes. The B. subtilis NanoRNase A (NrnA), a DHH family phosphoesterase, represents a second major class of nanoRNases, with broad phylogenetic distribution in organisms that lack orn homologs. The second major focus of this thesis is a structural and mechanistic study of this nanoRNase machinery. The atomic structure of the B. subtillis nanoRNase NrnA is described, and unveils a bi-lobal architecture similar to the 5’→3’ DNase RecJ, where the catalytic DHH domain is linked via a partially helical connector to the C-terminal RNA binding domain. NrnA is a highly dynamic molecule, adopting both open and closed conformations. Co-crystallization with several substrates shows that NrnA has a nanoRNA specific substrate-binding patch that offers a structural explanation for its 3’→5’ nanoRNase activity. This RNA binding site feeds substrate to the DHH active site in an orientation opposite to the 5’→3’ path proposed for RecJ. Surprisingly, NrnA also maintains a weak 5’→3’ activity on certain substrates, and thus possesses both 5’→3’ and 3’→5’ exonuclease activities. In conclusion, an overall model is presented for how DHH family exonucleaess can degrade nucleic acids from both the 5’→3’ and 3’→5’ directions. Thus, the studies described in this thesis offer both an atomic and a biochemical view of the macromolecular machinery critical to the degradation of RNA.
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Tedeschi, Frank A. Tedeschi. "IDENTIFICATION OF CELLULAR RNA BINDING SITES OF DEAD-BOX HELICASES". Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1531217057171378.
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Idris, Jalilah. "Investigating novel roles of RNA binding proteins SAFB1 and SAFB2 in RNA processing and in cellular stress". Thesis, University of Bristol, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.738206.
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Cordiner, Ross Andrew Alex. "The cellular functions of the microprocessor complex". Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25877.
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DGCR8 (DiGeorge critical region 8) protein constitutes part of the Microprocessor complex together with Drosha, and is involved in the nuclear phase of microRNA (miRNA) biogenesis. DGCR8 recognises the hairpin RNA substrates of precursor miRNAs through two double-stranded RNA (dsRNA) binding motifs and acts as a molecular anchor to direct Drosha cleavage at the base of the pri-miRNA hairpin. Recent characterisation of the RNA targets of the Microprocessor by HITSCLIP of DGCR8 protein revealed that this complex also binds and regulates the stability of several types of transcripts, including mRNAs, lncRNAs and retrotransposons. Of particular interest is the binding of DGCR8 to mature small nucleolar RNA (snoRNA) transcripts, since the stability of these transcripts is dependent on DGCR8, but independent of Drosha. This raises the interesting possibility that there could be alternative DGCR8 complex/es using different nucleases to process a variety of cellular RNAs. We performed mass spectrometry experiments and revealed that DGCR8 copurifies with subunits of the nucleolar exosome, which contains the exonuclease RRP6. We demonstrated DGCR8 and the exosome form a nucleolar complex, which degrade the mature snoRNAs tested within this study. Interestingly, we also show that DGCR8/exosome complex controls the stability of the human telomerase RNA component (hTR/TERC), and absence of DGCR8 creates a concomitant telomere phenotype. In order to identify the RNA targets of the DGCR8/Exosome complex on a global scale we performed iCLIP of endogenous and overexpressed RRP6 (wild-type and a catalytically inactive form). Thus, intersection of CLIP datasets from DGCR8 and RRP6 identified common substrates; accordingly snoRNAs were the most represented. In addition, we identified the cellular RNA targets of the RRP6 associated human exosome. The use of a catalytically inactive form of RRP6 stabilised important in vivo interactions that are highly dynamic and transient and also highlighted the role of RRP6-mediated trimming of 3’flanks of immature non-coding RNAs. We will present a global view of the RNA-binding capacity of the RRP6-associated exosome. In sum, we identified a novel function for DGCR8, acting as an adaptor to recruit the exosome to structured RNAs and induce their degradation. Moreover, we have identified DGCR8-depenedent substrates of the exosome and have demonstrated the requirement of RRP6 for 3’ processing of ncRNAs.
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Ozes, Ali Rayet. "Targeting the long non coding RNA HOTAIR in cancer". Thesis, Indiana University, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10154781.
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Ovarian cancer (OC) takes the lives of nearly 14,000 US women every year. Although platinum is one of the most effective drugs in treating ovarian cancer, the development of platinum resistance is one of the biggest challenges facing patients. I have shown that the long non-coding RNA HOTAIR contributes to platinum-resistant OC and determined the regulators and targets of HOTAIR during the platinum-induced DNA damage response. My published data supports the role of HOTAIR in contributing to DNA damage induced cellular senescence and secretion of pro-inflammatory cytokines leading to cisplatin resistance. My unpublished work (under review) analyzed the interaction of HOTAIR with the PRC2, its known interacting partner. In this study, I developed a novel strategy blocking HOTAIR-PRC2 interaction and resensitized ovarian tumors to platinum in mouse studies. The results offer a pre-clinical proof of concept for targeting long non-coding RNAs as a therapeutic approach and may represent a strategy to overcome chemotherapy resistance in tumors exhibiting high expression of HOTAIR, a frequent observation in high grade serous OC.
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Babendure, Jeremy R. "Utilizing RNA structure as a tool in molecular and cellular biology". Diss., Connected to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3190000.
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Thesis (Ph. D.)--University of California, San Diego, 2005.Title from first page of PDF file (viewed Mar. 6, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 122-131).
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Nurmohamed, Salima. "Communication between the Escherichia coli RNA degradative machineries and cellular metabolism". Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611286.
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Öhrmalm, Christina. "Functional characterization of the cellular protein p32 : a protein regulating adenovirus transcription and splicing through targeting of phosphorylation /". Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6794.
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McNally, Beth Anne. "A role for cytoplasmic PML in the cellular antiviral response". Columbus, Ohio : Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1133377007.
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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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Jeyaraj, Selvi Chrysolyte. "A role for the mRNA-stabilizing protein HuR in protection from cellular ATP depletion". Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1186773861.
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Wong, Tsz-lo, i 黃子璐. "Cellular role of miR-143 in cervical cancer". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48274045.
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Cervical cancer is a largely preventable malignancy due to the availability of cytology screening and vaccination against the essential initiation factor of cervical carcinogenesis, human papillomavirus (HPV). However, cervical cancer remains a significant medical burden worldwide, particularly in developing countries where large scale screening or vaccination programs are not financially feasible. Molecular tests such as HPV DNA tests have the potential to improve the speed and sensitivity of cervical cancer screening but suffer from limited specificity. Additional adjunct molecular markers are therefore desirable for enhancing molecular tests. Our previous research has revealed miR-143, a microRNA downregulated in a number of cancers, could be detected in liquid based cytology samples and is significantly reduced in cervical cancer samples and cell lines. Cellular role of miR-143 and mechanism behind its downregulation remain an unknown in cervical carcinogenesis. To explore the cellular roles of miR-143 in cervical cancer, a construct expressing miR-143 was transfected into cervical cancer cell lines HeLa, SiHa and C33A. miR- 143 overexpression was verified by qPCR. The miR-143 overexpressing cell lines were used to conduct a number of cellular function assays. It has been reported that miR-143 is able to suppress cell growth in HPV-positive HeLa. We followed up the findings and revealed miR-143 overexpression in HPV-negative C33A did not suppress cell growth in an MTT cell proliferation assay. ERK5 and KRAS, two targets of miR-143, are downregulated in colon cancer and bladder cancer to suppress cell grwoth. However, mRNA level of ERK5 and KRAS were not altered in all three miR-143 overexpressed cervical cancer cell lines, suggesting that miR-143 may not target ERK5 and KRAS transcriptionally in cervical cancer. Ability of miR-143 in regulating cell differentiation was evaluated by the expression of K10, an early keratinocyte differentiation marker. K10 was upregulated only in miR-143 overexpressed HeLa and SiHa as revealed by qPCR. A parallel increase in hSkn-1a mRNA, a transcription factor of K10, was also observed specifically in the two miR-overexpressed HPV-positive cell lines. miR-143 level is inversely correlated with cytology grading and progression of cervical disease, hinting its role in mediating cell migration and invasion during cancer progression and metastasis. A reduction of cell migration as demonstrated in wound healing assay and in vitro transwell migration assay was observed exclusively in miR-143 overexpressed HeLa and SiHa. miR-143 overexpression in C33A did not introduce any effect in cell migration. A reduction of cell invasion was also observed merely in miR-143 overexpressed HeLa and SiHa as revealed in a transwell invasion assay. Apart from studying the cellular roles of miR-143 in cervical cancer, this study has also explored mechanisms behind miR-143 downregulation in cervical cancer owing to the fact that certain miR-143 mediated cellular functions were observed only in HPV-positive cervical cancer cell lines. We hypothesized that HPV E6 and E7 oncoprotein may downregulate miR-143 in cervical cancer. The hypothesis was supported by our findings where normal cervical epithelial cell line immortalized by E6 and E7 had an undetectable level of endogenous miR-143 level. The same primary cells immortalized by shp16-hTERT expressed residual amounts of miR-143 as revealed by qPCR. Owing to the low miR-143 expression in shp16-hTERTimmortalized normal cervical epithelial cell line, downregulation of miR-143 in cervical cancer cell lines may also be contributed to hTERT overexpression and p16 silencing. Overall, miR-143 plays an important role in suppressing cell proliferation, enhancing keratinocyte differentiation marker expression, reducing migration and invasion in HPV-positive cervical cancer. Downregulation of miR-143 level may be an effect as manifested by E6 and E7 in HPV-positive cervical cancer. Differential cellular effects in miR-143 overexpressed HPV-positive and HPV-negative cervical cancer cell lines suggest that HPV oncoprotein mediates miR-143 cellular functions.published_or_final_version
Pathology
Master
Master of Medical Sciences
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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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Mahmoudi, Massoud. "Induction of Interferon Messenger RNA and Expression of Cellular Oncogenes in Human Lymphoblastoid Cells". Thesis, North Texas State University, 1986. https://digital.library.unt.edu/ark:/67531/metadc798199/.
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The purposes of this study was to demonstrate the induction of alpha interferon mRNA in Sendai virus-induced Namalava cells, to follow the level of alpha interferon mRNA synthesis at the transcriptional level, and to determine whether the Namalava cell line expresses the c-myc oncogene and to what degree. The amount of c-myc message deteted in Namalva cell RNA was about one-tenth that of Daudi cell RNA, whereas no difference in the amount of the c-Ha-ras message was observed between the two cell lines.
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Mina, Ibarra Leonardo Bruno. "Cellular mRNA decay factors involved in the hepatitis C virus life cycle". Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7215.
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The group of positive strand RNA ((+)RNA) viruses includes numerous plant, animal and human pathogens such as the hepatitis C virus (HCV). Their viral genomes mimic cellular mRNAs, however, besides acting as messengers for translation of viral proteins, they also act as templates for viral replication. Since these two functions are mutually exclusive, a key step in the replication of all (+) RNA viruses is the regulated exit of the genomic RNAs from the cellular translation machinery to the viral replication complexes. By using a model system that allows the replication of the plant (+) RNA Brome Mosaic Virus in yeast, it was shown by our group that the cellular decapping activators Dhh1, LSm1 and PatL1 play a key role in such transition. The LSm1 protein is a subunit of the LSm1-7 complexes. We have recently shown that these complexes interact directly with sequences in the BMV genome and that these interactions regulate the translation and replication of BMV. Interestingly, the LSm1 homolog in bacteria, Hfq, is required for the replication of the (+) RNA bacteriophage Qβ in E. coli.Since all (+)RNA viruses need to regulate the exit of the viral genome from the cellular translation machinery to replication and these proteins are conserved from yeast to humans, We hypothesized that the human homologues of Lsm1-7/Dhh1/Pat1 are required for the replication of human (+) RNA viruses. In this work we proved this hypothesis by showing that HCV translation and replication depend on these cellular proteins. Moreover, the requirement of these factors for efficient HCV RNA translation was linked exclusively to the 5´ and 3´ nontranslated regions (NTRs) of the viral genome. Furthermore, the LSm1-7 complex specifically interacts in vitro with essential cis-acting HCV RNA elements located in the NTRs.
El grupo de virus de RNA de cadena de polaridad positiva ((+)RNA) incluye numerosos patógenos de plantas, animales y humanos, tales como el virus de la Hepatitis C (VHC). Sus genomas virales imitan a los RNAm celulares, sin embargo, además de actuar como mensajeros para la traducción de proteínas virales, también actúan como moldes para la replicación viral. Debido a que estas dos funciones son mutuamente excluyentes, un paso clave en la replicación de todos los virus (+) RNA es la salida regulada del RNA genómico desde la maquinaria de traducción celular hacia las complejos de replicación virales. Utilizando un sistema modelo que permite la replicación del virus del mosaico del bromo (VMB), un virus (+) RNA de plantas, en levaduras, nuestro grupo ha demostrado que los activadores de decapping celulares Dhh1, LSm1 y Pat1 juegan un papel clave en este paso. La proteína LSm1 es una subunidad del complejo celular LSm1-7. Hemos demostrado recientemente que dichos complejos interactúan directamente con secuencias específicas en el genoma del VMB y que dichas interacciones regulan la traducción y replicación del VMB. De manera interesante, la proteína homologa de LSm1 en bacterias, Hfq, es necesaria para la replicación del bacteriófago (+) RNA Qβ en E. coli.
Debido a que todos los virus (+) RNA necesitan regular la salida del genoma viral desde la maquinaria traduccional de la célula hacia los complejos de replicación virales, y que las proteínas Dhh1, LSm1-7 y Pat1 están conservadas de levaduras a humanos, nuestra hipótesis es que lo homólogos humanos de Lsm1-7/Dhh1/Pat1 son requeridos para la replicación de virus (+)RNA de humanos. En este trabajo, hemos demostrado esta hipótesis mostrando que la traducción y la replicación del virus de la hepatitis C (VHC) dependen de estas proteínas celulares. Más aún, el requerimiento de estos factores para una eficiente traducción del genoma del VHC está vinculado de manera exclusiva a las regiones no traducidas (RNT) 5´ y 3´ del genome viral. Además, el complejo cellular LSm1-7 interactúa in vitro de manera específica con elementos esenciales del RNA del VHC que actúan en cis localizados en las RNTs.
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Plakos, Kory. "Platinum-seq: High-throughput mapping of small-molecule platinum adducts on cellular RNA". Thesis, University of Oregon, 2017. http://hdl.handle.net/1794/22269.
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Methods to map small-molecule interactions with cellular RNAs are important for understanding endogenous activation, such as in riboswitches, as well as the potential for exogenous compounds to target RNA. Cisplatin is one of the most widely used of the platinum anticancer drugs that are prescribed in approximately 40-50% of all chemotherapy treatments (Dyson and Sava, 2006; Harper et al., 2010). Despite nearly 40 years of experience with this class of drugs, we still lack a comprehensive understanding of the targets of Pt compounds and their effects on cells. Pt(II) compounds are well-known DNA and RNA crosslinking agents, but the latter area is under-studied. In order to better understand the impacts of cisplatin and other platinum(II)-derived small molecules on cellular RNA, we have developed a technique we call “Platinum-seq,” which couples reverse transcription mapping of platinated RNAs to high-throughput sequencing. Chapter 1 is a study of cisplatin and a novel click-functionalized platinum compound (2-ADAP Pt) binding to the HDV ribozyme, a small catalytic RNA. Chapter 2 moves our platinum mapping approaches from low-throughput, sequencing gel based methods into next-generation sequencing for high-throughput analysis of all platinum sites in cellular RNA, a method we have named “Platinum-seq.” Chapter 3 is a study of differential gene expression of Saccharomyces cerevisiae treated with cisplatin and a second novel platinum(II) compound (azaplatin), using data acquired from the work in Chapter 2. Chapter 4 describes recent efforts to implement pre-enrichment of sequencing targets using click chemistry followed by DNA hybridization, in order to enrich for platinated fragments before sequencing library construction. Together, this work represents a significant step forward in advancing analysis of Pt(II) binding to cellular RNA, a potentially important target for this widely used class of anticancer compounds. Methods developed here are broadly applicable to genome-wide identification of platinum accumulation on DNA as well, which has not been pursued despite the extensive use of these compounds.
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Chambers, A. "RNA 3' cleavage and polyadenylation in oocytes, eggs and embryos of Xenopus laevis". Thesis, University of Warwick, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380275.
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Rubilar, Guzman Paulina. "Cellular host factors involved in the translation of the HIV-1 genomic RNA". Thesis, Lyon, École normale supérieure, 2015. http://www.theses.fr/2015ENSL1009/document.
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Le virus de l’immunodéficience humaine de type 1 (VIH-1) est un virus à simple brin positif qui appartient au genre Lentivirus dans la famille retroviridae et qui constitue l’agent étiologique du SIDA pandémique.Pendant le cycle réplicatif du VIH-1, la traduction de protéines virales dépend exclusivement de la machinerie traductionnelle cellulaire. Pour cette raison, nous avons cherché à comprendre le rôle de quelques facteurs cellulaires qui pourraient contrôler la traduction du VIH-1 à différents nivaux. Nous avons centré nos recherches sur la traduction de l’ARN génomique (ARNg) du virus qui sert en même temps de génome pour être encapsidé et comme ARN messager pour la traduction des protéines virales Gag et Gag-Pol. 1) Le rôle de l’hélicase d’ARN DDX3 dans la traduction du VIH-1. L’ARNg du VIH-1 possède une région 5’ non traduite très structurée, raison pour laquelle nous avons spéculé sur un possible rôle de DDX3 dans la traduction du VIH-1. Nous avons utilisé une combinaison de techniques in vitro et ex vivo afin de pouvoir démontrer que DDX3 était capable de lier et faire des complexes avec l’ARN de la région 5’ non traduite pour promouvoir l’initiation de la traduction. Nous avons aussi pu démontrer que DDX3 formait des complexes avec les facteurs d’initiation de la traduction PABP, eIF4G et eIF4E. 2) Le changement programmé du cadre de lecture (PRF) dans l’ARN génomique du VIH-1. La traduction de la polyprotéine Gag-Pol du VIH-1 nécessite un décalage de phase de 1 nucléotide en arrière. Ce mécanisme permet la synthèse des protéines Gag et Gag-Pol avec des ratios de 95 et 5% respectivement à partir du même ARN. Cette proportion doit être conservée pour assurer la réplication du virus. Nous avons utilisé un système de double gène rapporteurs et un système de réplication complète du provirus pour montrer que la protéine associé aux granules de stress TIAR pouvait contrôler la réplication viral en régulant la proportion de ribosome qui assurentHuman Immunodeficiency virus type 1 (HIV-1) is a positive strand RNA virus belonging to the lentivirus genus of the retroviridae family and it is the etiological agent of the pandemic AIDS, which is a major health concern worldwide. Throughout HIV-1 replication cycle, the production of viral proteins depends exclusively on the cellular translational machinery. This is the reason why we have explored the role of some cellular factors that could control HIV-1 translation at different stages. We have focused our studies on the translation of the full length genomic RNA (gRNA), which serves both as genome for viral encapsidation and as a messenger for translation of Gag and Gag-Pol viral polyproteins.1) The role of the RNA helicase DDX3 in HIV-1 translation Initiation The fact that HIV-1 possesses a highly structured 5’ untranslated region (5’UTR) prompted us to speculate that DDX3 may be involved in HIV-1 translation. We used a combination of in vitro and ex-vivo approaches to show that DDX3 was able to bind and form complexes with the 5’-UTR of HIV-1 to assist translation initiation. We also demonstrated that DDX3 can form a complex with initiation factors such as PABP, eIF4G and eIF4E. 2) Programmed Ribosomal Frameshift (PRF) in the genomic RNA of HIV-1Translation of HIV-1 Gag-Pol polyprotein requires a -1 PRF. This mechanism allows the synthesis of Gag and Gag-Pol polyproteins, using the same mRNA template, at ratios of 95 and 5% respectively. Keeping the -1PRF ratio is important as any change leads to reduction in virus infectivity.By means of a dual reporter construct and full provirus replication system we were able to demonstrate that the stress granules associated protein TIAR, controls HIV-1 infectious progeny by regulating the ratio of the HIV-1 PRF
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Win, Maung Nyan Parker Carl Stevens Smolke Christina D. "Engineering RNA devices for gene regulation, biosensing, and higher-order cellular information processing /". Diss., Pasadena, Calif. : Caltech, 2008. http://resolver.caltech.edu/CaltechETD:etd-05282008-142750.
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Palsule, Geeta. "Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron". The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555628225881656.
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Kheimar, Ahmed Mahmoud Osman [Verfasser]. "Tumor promoting functions of cellular telomerase RNA and viral RNAs in herpesvirus-induced cancer formation / Ahmed Mahmoud Osman Kheimar". Berlin : Freie Universität Berlin, 2017. http://d-nb.info/1147758247/34.
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Ostertag, Derek Glenn. "Novel dsRNA-dependent activation of a cellular antiviral response to vesicular stomatitis virus /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3167840.
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Childs, April Celeste. "The Effects of EIF5A and of the Polyamines on RNA Processing, Translation, and P-Body Formation". Diss., Tucson, Ariz. : University of Arizona, 2005. http://etd.library.arizona.edu/etd/GetFileServlet?file=file:///data1/pdf/etd/azu%5Fetd%5F1326%5F1%5Fm.pdf&type=application/pdf.
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Guo, Bing. "Positive correlation between cellular levels of storage compounds and RNA in activated sludge bacteria". Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107874.
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In order to implement rational solutions to solids separation problems such as bulking and foaming and to rationally design new processes for the production of value-added products, it is necessary to understand the ecological functions and dynamics of heterotrophic bacterial populations inhabiting activated sludge wastewater treatment plants. Although phylogenetic (DNA- and RNA-based) characterization of these microbial communities demonstrated their wide diversity, the link between heterotrophic populations and specific functional niches is still tenuous. Because RNA levels are related to the growth rates and storage compounds are accumulated in response to specific growth dynamics, the abundance of these cellular constituents may serve as functional biomarkers. A flow cytometry method was developed to measure them on a cell-by-cell basis using fluorescent dyes: RNASelect for RNA, Nile Red for polyhydroxyalkanoates (PHA) and 7-AAD for DNA. The method was validated against five strains representing a wide range of bacterial diversity: Escherichia coli K-12, Rhodococcus jostii RHA1, Bacillus subtilis, Cupriavidus necator DSM428 and DSM 541 (a non-PHA producing mutant of DSM428). For these strains together, the RNA/DNA ratios determined by flow cytometry and by nucleic acid extraction were generally correlated.The method was then used to study samples obtained from three independent activated sludge reactors located at a wastewater treatment plant near Montréal: a full-scale reactor, a pilot-scale reactor using ozone to reduce sludge production, and a pilot-scale control reactor. In all cases, it was found that high RNA/DNA fluorescence signals ratios correlated with high fluorescence signals associated to PHA. Furthermore, when the PHA-associated signals of biomass sampled from the pilot-scale reactors were compared, it was found that the PHA signal was higher for the ozone-treated reactor samples. The increased PHA probably resulted from the production of extra readily degradable substrates by the ozone treatment of sludge. We suggest that this correlation between RNA/DNA ratios and PHA abundance represents a functional characterization of heterotrophs. Populations consuming readily degradable substrates would have a high RNA/DNA ratio and accumulate storage compounds, while it would be the opposite for populations consuming slowly degradable substrates.Pour mettre en œuvre des solutions rationnelles aux problèmes de la séparation des solides tels que la prolifération de bactéries filamenteuses causant le foisonnement et le moussage et afin de concevoir rationnellement de nouveaux procédés pour la production de produits à valeur ajoutée, il est nécessaire de comprendre les fonctions écologiques et la dynamique des populations de bactéries hétérotrophes qui résident dans les installations de traitement des eaux usées par boues activées. Bien que la caractérisation phylogénétique (baseée sur l'ADN et l'ARN) de ces communautés microbiennes ait démontré leur grande diversité, les liens entre les populations hétérotrophes et des niches fonctionnelles spécifiques sont encore ténus. Parce que les niveaux d'ARN sont liés aux taux de croissance et que les composés de stockage sont accumulés en réponse à des dynamiques de croissance spécifiques, l'abondance de ces constituants cellulaires peut servir de marqueurs biologiques fonctionnels. Une méthode de cytométrie en flux a été développée pour les mesurer sur une base cellule par cellule en utilisant des colorants fluorescents : RNASelect pour l'ARN, le rouge du nil pour les polyhydroxyalcanoates (PHA) et le 7-AAD pour l'ADN. La méthode a été validée contre cinq souches représentant un large éventail de diversité bactérienne : Escherichia coli K-12, Rhodococcus jostii RHA1, Bacillus subtilis, Cupriavidus necator DSM428 and DSM 541 (une souche mutante de DSM428 incapable d'accumuler des PHA). La comparaison des ratios ARN/ADN déterminés par cytométrie en flux et par extraction d'acide nucléique entre ces souches a démontré une corrélation générale acceptable.La méthode a ensuite été utilisée pour étudier les échantillons obtenus à partir de trois réacteurs par boues activées indépendants situés dans une usine de traitement des eaux usées près de Montréal : un réacteur à pleine échelle, un réacteur à l'échelle pilote utilisant de l'ozone pour réduire la production de boues, et un réacteur de contrôle à l'échelle pilote. Dans tous les cas, on a constaté qu'un niveau élevé des ratios des signaux fluorescents d'ARN/ADN était corrélé avec des signaux élevés de fluorescence associée au PHA. Par ailleurs, lorsque les signaux associés au PHA de la biomasse échantillonnée à partir des réacteurs à l'échelle pilote ont été comparés, on a constaté que le signal PHA était plus élevé pour les échantillons du réacteur traité à l'ozone. L'augmentation du PHA résulte probablement de la production de substrats supplémentaires facilement dégradables par le traitement par ozone des boues. Nous suggérons que cette corrélation entre les ratios d'ARN/ADN et l'abondance de PHA représente une caractérisation fonctionnelle des hétérotrophes. Les populations consommant des substrats facilement dégradables auraient un ratio ARN/ADN élevé et elles accumulent des composés de stockage, tandis que ce serait l'inverse pour les populations consommant des substrats lentement dégradables.
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Wang, Dong 1975. "Cisplatin-induced nucleosome and RNA polymerase II modification mediate cellular response to the drug". Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/28693.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2004.Vita.
Includes bibliographical references.
(cont.) with recombinant material. The in vitro system established in this study will facilitate the investigation of platinum-DNA damage by DNA repair processes and help elucidate the role of specific post-translational modification in NER of platinum-DNA adducts at the physiologically relevant nucleosome level. Chapter 3: Nucleotide Excision Repair of Site-Specifically Platinum-Modified Tetrasomes. The nucleosome, the basic structural unit of chromatin, is composed of a histone (H3/H4)₂ tetramer flanked by two H2A/H2B dimers, around which is wrapped 146 base pairs (bp) of DNA. The (H3/H4)₂ tetramer plays a central role in the structural integrity and positioning of the nucleosome core particle. Site- specifically platinated tetrasomes were prepared to investigate the modulating effects of histone tetramers on excision repair of cisplatin-intrastrand cross-links. In addition, (Pt(DACH))²⁺-modified tetrasome was prepared to investigate the effect of spectator ligands on excision repair of platinated tetrasomes. The NER results reveal that the (H3/H4)₂ tetramer is sufficient to block excision of both cisplatin-DNA and Pt(DACH))²⁺-DNA adduct in vitro. The efficiency of excision of cisplatin-modified tetrasomal DNA is about half (53%) that of the [Pt(DACH)]²⁺-modified tetrasome. Chapter 4: Cisplatin Adducts Change the Rotational Positioning of DNA on the Nucleosome ...
Chapter 1: Cellular Processing of Platinum Anti-Cancer Drugs --Identifying Pathways for Chemogenotherapeutic Drug Design. Cisplatin, carboplatin, and oxaliplatin are widely used in cancer chemotherapy. Platinum-DNA adducts, formed following uptake of the drug into the nucleus of cells, activate several cellular processes that mediate the cytotoxicity of these platinum drugs. This review focuses on recently discovered cellular pathways that are activated in response to cisplatin, including those involved in regulating drug uptake, the signaling of DNA damage, cell cycle checkpoints and arrest, DNA repair, and cell death. Such knowledge of the cellular processing of cisplatin adducts with DNA provides valuable clues for the rational design of more efficient platinum-based drugs as well as the development of new therapeutic strategies. Chapter 2: Nucleotide Excision Repair from a Site-Specifically Platinum-Modified Nucleosome. Nucleotide excision repair is a major cellular defense mechanism against the toxic effects of the anticancer drug cisplatin and other platinum based chemotherapeutic agents. In this study, mononucleosomes were prepared containing either a d(GpG) or a d(GpTpG) intrastrand cross-link. Comparison of the extent of repair by mammalian cell extracts of free and nucleosomal DNA containing the same platinum-DNA adduct reveals that the nucleosome significantly inhibits nucleotide excision repair. The effects of post-translational modification of histones on excision of platinum damage from nucleosomes were investigated by comparing native and recombinant nucleosomes containing the same intrastrand d(GpTpG) cross-link. Excision from native nucleosomal DNA is [approximately] 2-fold higher than the level observed
by Dong Wang.
Ph.D.
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Ribeiro, Diogo. "Discovery of the role of protein-RNA interactions in protein multifunctionality and cellular complexity". Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0449/document.
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Au fil du temps, la vie a évolué pour produire des organismes remarquablement complexes. Pour faire face à cette complexité, les organismes ont développé une pléthore de mécanismes régulateurs. Par exemple, les mammifères transcrivent des milliers d'ARN longs non codants (ARNlnc), accroissant ainsi la capacité régulatrice de leurs cellules. Un concept émergent est que les ARNlnc peuvent servir d'échafaudages aux complexes protéiques, mais la prévalence de ce mécanisme n'a pas encore été démontrée. De plus, pour chaque ARN messager, plusieurs régions 3’ non traduites (3’UTRs) sont souvent présentes. Ces 3’UTRs pourraient réguler la fonction de la protéine en cours de traduction, en participant à la formation des complexes protéiques dans lesquels elle est impliquée. Néanmoins, la fréquence et l’importance ce mécanisme reste à aborder.Cette thèse a pour objectif de découvrir et comprendre systématiquement ces deux mécanismes de régulation méconnus. Concrètement, l'assemblage de complexes protéiques promus par les ARNlnc et les 3'UTRs est étudié avec des données d’interactions protéines-protéines et protéines-ARN à grande échelle. Ceci a permis (i) de prédire le rôle de plusieurs centaines d'ARNlnc comme molécules d'échafaudage pour plus de la moitié des complexes protéiques connus, ainsi que (ii) d’inférer plus d’un millier de complexes 3'UTR-protéines, dont certains cas pourraient réguler post-traductionnellement des protéines moonlighting aux fonctions multiples et distinctes. Ces résultats indiquent qu'une proportion élevée d'ARNlnc et de 3'UTRs pourrait réguler la fonction des protéines en augmentant ainsi la complexité du vivantOver time, life has evolved to produce remarkably complex organisms. To cope with this complexity, organisms have evolved a plethora of regulatory mechanisms. For instance, thousands of long non-coding RNAs (lncRNAs) are transcribed by mammalian genomes, presumably expanding their regulatory capacity. An emerging concept is that lncRNAs can serve as protein scaffolds, bringing proteins in proximity, but the prevalence of this mechanism is yet to be demonstrated. In addition, for every messenger RNA encoding a protein, regulatory 3’ untranslated regions (3’UTRs) are also present. Recently, 3’UTRs were shown to form protein complexes during translation, affecting the function of the protein under synthesis. However, the extent and importance of these 3’UTR-protein complexes in cells remains to be assessed.This thesis aims to systematically discover and provide insights into two ill-known regulatory mechanisms involving the non-coding portion of the human transcriptome. Concretely, the assembly of protein complexes promoted by lncRNAs and 3’UTRs is investigated using large-scale datasets of protein-protein and protein-RNA interactions. This enabled to (i) predict hundreds of lncRNAs as possible scaffolding molecules for more than half of the known protein complexes, as well as (ii) infer more than a thousand distinct 3’UTR-protein complexes, including cases likely to post-translationally regulate moonlighting proteins, proteins that perform multiple unrelated functions. These results indicate that a high proportion of lncRNAs and 3’UTRs may be employed in regulating protein function, potentially playing a role both as regulators and as components of complexity
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Saini, Harleen. "Intron and Small RNA Localization in Mammalian Neurons". eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1044.
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RNA molecules are diverse in form and function. They include messenger RNAs (mRNAs) that are templates for proteins, splice products such as introns that can generate functional noncoding RNAs, and a slew of smaller RNAs such as transfer RNAs (tRNAs) that help decode mRNAs into proteins. RNAs can show distinct patterns of subcellular localization that play an important role in protein localization. However, RNA distribution in cells is incompletely understood, with prior studies focusing primarily on RNAs that are long (>200 nucleotides), fully processed, and polyadenylated. We examined the distribution of RNAs in neurons. Neuronal compartments can be separated by long distances and play distinct roles, raising the possibility that RNA localization is especially overt and functionally meaningful in these cells. In our exploration, we physically dissected projections from cell bodies of neurons from the rat brain and sequenced total RNA. We describe two main findings. First, we identified excised introns that are enriched in neuronal projections and confirmed their localization by single- molecule fluorescence in situ hybridization. These are a previously unknown set of circular RNAs in neuronal projections: tailless lariats that possess a non- canonical C branchpoint. Second, we observed a highly abundant population of small (20-150 nucleotide) RNAs in neuronal projections, most of which are tRNAs. For both circular introns and tRNAs, we did not observe known RNA localization signals. Thus, many types of RNA, if sufficiently stable, appear free to diffuse to distant locations, their localization perhaps aided by the movement of large organelles in the confines of neuronal projections. Our survey of RNA molecules across subcellular compartments provides a foundation for investigating the function of these molecules and the mechanisms that localize them.
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Svensson, Valentine. "Probabilistic modelling of cellular development from single-cell gene expression". Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267937.
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The recent technology of single-cell RNA sequencing can be used to investigate molecular, transcriptional, changes in cells as they develop. I reviewed the literature on the technology, and made a large scale quantitative comparison of the different implementations of single cell RNA sequencing to identify their technical limitations. I investigate how to model transcriptional changes during cellular development. The general forms of expression changes with respect to development leads to nonparametric regression models, in the forms of Gaussian Processes. I used Gaussian process models to investigate expression patterns in early embryonic development, and compared the development of mice and humans. When using in vivo systems, ground truth time for each cell cannot be known. Only a snapshot of cells, all being in different stages of development can be obtained. In an experiment measuring the transcriptome of zebrafish blood precursor cells undergoing the development from hematopoietic stem cells to thrombocytes, I used a Gaussian Process Latent Variable model to align the cells according to the developmental trajectory. This way I could investigate which genes were driving the development, and characterise the different patterns of expression. With the latent variable strategy in mind, I designed an experiment to study a rare event of murine embryonic stem cells entering a state similar to very early embryos. The GPLVM can take advantage of the nonlinear expression patterns involved with this process. The results showed multiple activation events of genes as cells progress towards the rare state. An essential feature of cellular biology is that precursor cells can give rise to multiple types of progenitor cells through differentiation. In the immune system, naive T-helper cells differentiate to different sub-types depending on the infection. For an experiment where mice were infected by malaria, the T-helper cells develop into two cell types, Th1 and Tfh. I model this branching development using an Overlapping Mixture of Gaussian Processes, which let me identify both which cells belong to which branch, and learn which genes are involved with the different branches. Researchers have now started performing high-throughput experiments where spatial context of gene expression is recorded. Similar to how I identify temporal expression patterns, spatial expression patterns can be identified nonparametrically. To enable researchers to make use of this technique, I developed a very fast method to perform a statistical test for spatial dependence, and illustrate the result on multiple data sets.
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Kolisnichenko, Marina. "The role of the polyadenylation site of the melanocortin 1 receptor in generating MC1R-TUBB3 chimeras and attenuation of TORC1 delays the onset of replicative and RAS-induced cellular senescience". Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711654.
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Cooper, Samuel J. "Proteomic investigation of factors binding Gurken RNA in Drosphila and Hepatitis C virus cellular interactions". Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.540148.
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Burns, David M. "Post-Transcriptional Control of Human Cellular Senescence: A Dissertation". eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/491.
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The central dogma of biology asserts that DNA is transcribed into RNA and RNA is translated into protein. However, this overtly simplistic assertion fails to portray the highly orchestrated and regulated mechanisms of transcription and translation. During the process of transcription, RNA provides the template for translation and protein synthesis as well as the structural and sequence specificity of many RNA and protein-based machines. While only 1-5% of the genome will escape the nucleus to be translated as mRNAs, complex, parallel, highly-conserved mechanisms have evolved to regulate specific mRNAs. Trans-acting factors bind cis-elements in both the 5" and 3" untranslated regions of mRNA to regulate their stability, localization, and translation. While a few salient examples have been elucidated over the last few decades, mRNA translation can be reversibly regulated by the shortening and lengthening of the 3" polyadenylate tail of mRNA. CPEB, an important factor that nucleates a complex of proteins to regulate the polyadenylate tail of mRNA, exemplifies a major paradigm of translational control during oocyte maturation and early development. CPEB function is also conserved in neurons and somatic foreskin fibroblasts where it plays an important role in protein synthesis dependent synaptic plasticity and senescence respectively. Focusing on the function of CPEB and its role in mRNA polyadenylation during human cellular senescence, the following dissertation documents the important finding that CPEB is required for the normal polyadenylation of p53 mRNA necessary for its normal translation and onset of senescence. Cells that lack CPEB have abnormal levels of mitochondria and ROS production, which are demonstrated to arise from the direct result of hypomorphic p53 levels. Finally, in an attempt to recapitulate the model of CPEB complex polyadenylation in human somatic cells, I unexpectedly find that Gld-2, a poly(A) polymerase required for CPEB-mediated polyadenylation in Xenopus laevis oocytes, is not required for p53 polyadenylation, but instead regulates the stability of a microRNA that in turn regulates CPEB mRNA translation. Furthermore, I demonstrate that CPEB requires Gld-4 for the normal polyadenylation and translation of p53 mRNA.
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Smith, Gregory Robert. "Unraveling the Role of Cellular Factors in Viral Capsid Formation". Research Showcase @ CMU, 2015. http://repository.cmu.edu/dissertations/475.
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Understanding the mechanisms of virus capsid assembly has been an important research objective over the past few decades. Determining critical points along the pathways by which virus capsids form could prove extremely beneficial in producing more stable DNA vectors or pinpointing targets for antiviral therapy. The inability of current experimental technology to address this objective has resulted in a need for alternative approaches. Theoretical and computational studies offer an unprecedented opportunity for detailed examination of capsid assembly. The Schwartz Lab has previously developed a discrete event stochastic simulator to model virus assembly based upon local rules detailing the geometry and interaction kinetics of individual capsid subunits. Applying numerical optimization methods to learn kinetic rate parameters that fit simulation output to in vitro static light scattering data has been a successful avenue to understand the details of virus assembly systems; however, information describing in vitro assembly processes does not necessarily translate to real virus assembly pathways in vivo. There are a number of important distinctions between experimental and realistic assembly environments that must be addressed to produce an accurate model. This thesis will describe work expanding upon previous parameter estimation algorithms for more complex data over three model icosahedral virus systems: human papillomavirus (HPV), hepatitis B virus (HBV) and cowpea chlorotic mottle virus (CCMV). Then it will consider two important modifications to assembly environment to more accurately reflect in vivo conditions: macromolecular crowding and the presence of nucleic acid about which viruses may assemble. The results of this work led to a number of surprising revelations about the variability in potential assembly rates and mechanisms discovered and insight into how assembly mechanisms are affected by changes in concentration, fluctuations in kinetic rates and adjustments to the assembly environment.
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Zhang, Wei. "Functional elucidation of BS69 /". View abstract or full-text, 2008. http://library.ust.hk/cgi/db/thesis.pl?BICH%202008%20ZHANG.
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Bryant, Helen Elizabeth. "Analysis of cellular and viral proteins that interact with the IE63 protein of herpes simplex virus type 1". Thesis, University of Glasgow, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312135.
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Zangl, Maximiliane Sophie [Verfasser]. "Influence of Viral Proteins and Cellular Exoribonuclease Xrn1 on RNA Recombination in Pestiviruses / Maximiliane Sophie Zangl". Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2015. http://d-nb.info/1073954560/34.
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Vreeland, Amanda C. "Cellular Retinoic Acid-Binding Protein 2 Cooperates with HuR to Stabilize RNA and Inhibit Tumor Growth". Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1409933949.
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Xu, Kai. "KEY ROLES OF SUB-CELLULAR MEMBRANES AND CO-CHAPERONE IN TOMBUSVIRUS REPLICATION". UKnowledge, 2014. http://uknowledge.uky.edu/plantpath_etds/15.
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Positive strand RNA viruses, inculding tombusviruses, are known to utilize cellular membranes to assemble their replicase complexes (VRCs). Two tombusviruses , Tomato bushy stunt virus (TBSV) and Carnation Italian ringspot virus (CIRV), replicate on different organellar membranes, peroxisomes or endoplasmic reticulum (ER) for TBSV and mitochodria outer membranes in case of CIRV. I showed that both TBSV and CIRV replicase proteins could assemble VRCs and replicate viral RNA on purified microsomes (ER) and mitochondria. Different efficiencies of assembly was shown determined by multiple domains on TBSV or CIRV replication proteins.
To study why VRC assembly could occur on an alternative organellar membranes, I focused on the phospholipids, key lipid components in ER or mitochondria membranes. Phospholipids directly interact with viral replicases, however, their specific roles during (+)RNA virus replication are far less understood. I used TBSV as a model (+) RNA virus, and established a cell-free TBSV replication system using artificial membranes prepared from different phospholipids. I showed that phosphatidylethanolamine (PE) is required for full cycle replication of the viral RNA.Moreover, PE is enriched at the sites of TBSV replication in plant and yeast cells, and was up-regulated during TBSV replication. Furthermore, up-regulation of total cellular PE content in yeast due to deletion of CHO2 leads to dramatically stimulated TBSV replication. Overall, I identified PE as the key lipid component of membranes required for TBSV replication, and my data highlighted that PE, an abundant phospholipid in all eukaryotic cells, not only serves as a structural component of membrane bilayers, its interaction with the viral replication proteins also stimulates (+)RNA virus replication. Further experiments indicated both early secretory pathway and endocytic pathway are involved in PE re-distribution to site of replication.
In addition to lipids and subcellular membranes, certain host proteins are also involved in (+) RNA virus replication and VRC assembly. I identified Hop-like stress- inducible protein 1 (Sti1p), which interacts with heat shock protein 70, is required for the inhibition of CIRV replication. My findings indicate that Hop/Sti1 co-chaperone could act as a virus restriction factor in case of mitochondrial CIRV, but not against peroxisomal tombusvirus.
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TenOever, Benjamin R. "Cellular recognition of RNA virus infection leading to activation of interferon regulatory factors three and seven and establishment of the antiviral state". Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84849.
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Virus infection represents an intracellular invasion of host cells for the sole purpose of multiplication. Successful virus replication requires entry into tropic cells, usurping of the cellular machinery, and the production of progeny virions to initiate further rounds of infection. Establishment of the antiviral state in response to virus begins at the site of infection and requires the initiation of a preexisting signaling network designed to inhibit virus replication and aid in the establishment of this coordinated immune response. Integral components of this network include the IRF-3 and IRF-7 transcription factors that play essential roles in the cellular response to infection through virus induced phosphorylation by an unknown virus activated kinase. The objective of this research was to elucidate the viral antigen(s) required to induce the phosphorylation and subsequent activation of IRF-3 and IRF-7, to identify the molecular component(s) required in this activation process, and to decipher the mechanism(s) by which cellular recognition of virus infection initiates this antiviral response. We demonstrate that activation of this signaling network, following RNA virus infection, is dependent on viral entry, viral transcription, and viral translation and propose that the molecular requirements governing activation of IRF-3 and IRF-7 are the result of viral byproducts formed during the process of cytoplasmic RNA self replication; including both the formation of double stranded RNA and ribonucleoprotein complexes. Following cellular recognition of these motifs, signal transduction networks converge onto an IKK-related kinase structure composed of adaptor proteins bound to two kinase subunits, TBK-1 and IKKepsilon, which we propose to be essential components of the virus activated kinase. We demonstrate that TBK-1 or IKKepsilon activation results in the establishment of an antiviral state that renders cells non permissive to viral replic
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Nitin, Nitin. "Optical and MR Molecular Imaging Probes and Peptide-based Cellular Delivery for RNA Detection in Living Cells". Diss., Available online, Georgia Institute of Technology, 2005, 2005. http://etd.gatech.edu/theses/available/etd-08102005-120350/.
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Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006.Dr. X. Hu, Committee Member ; Dr. Al Merrill, Committee Member ; Dr. Niren Murthy, Committee Member ; Dr. Gang Bao, Committee Chair ; Dr. Nicholas Hud, Committee Member. Includes bibliographical references.
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Galão, Rui Pedro Ribeiro. "Role of the cellular decapping activator LSM1-7 complex in the replication of positive-strand RNA viruses". Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7222.
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By using the ability of the positive-strand RNA ((+)RNA) virus BMV to replicate in yeast it was previously shown that subunits of the LSm1-7 ring, as well as Pat1 and Dhh1 play an essential role in the transit of the BMV genome from translation to replication. In non-infected cells, these proteins mediate the transition of cellular mRNAs from a translational to a non-translational state by activating decapping in the 5'-3' - deadenylation-dependent mRNA decay pathway. Given the conservation of this pathway from yeast to humans and the common need of all (+)RNA viruses to regulate the transition of their genomes from active translation to a translationally inactive state to allow replication, an exciting possibility, and our working hypothesis, was that LSm1-7, Dhh1 and Pat1 are used not only by BMV to replicate in yeast but also by human (+) RNA viruses, such as HCV, to replicate in mammalian cells. Furthermore, given the key role of these proteins in a common step to all (+)RNA viruses, it is essential to characterize the not yet defined molecular mechanisms associated with such function. In this regard, we also hypothesized that the LSm1-7 complex, as member of the Sm family of proteins, would directly interact with viral genomes of (+)RNA viruses in order to play their role in the virus life cycle in a similar way that other family counterparts directly interact with their RNA targets in order to achieve their different cellular functions. In this work we were able to confirm both hypothesis showing that human homologues of the upper mentioned proteins LSm1-7, Rck/p54 and PatL1, are required for HCV RNA translation and replication. Additionally, we also showed that reconstituted LSm1-7 complexes specifically recognize important signals, either in BMV or HCV genomes, that regulate their translation and/or replication. These observations constitute the first evidence that the LSm1-7 complex is able to directly interact with viral genomes representing also novel LSm1-7 interaction sites. Given the common replication strategies of (+)RNA viruses and the conserved cellular functions of LSm1-7, Pat1 and Dhh1 from yeast to humans, our findings pinpoint a weak spot that may be exploited to generate broad-spectrum antiviral drugs.Utilizando la capacidad del BMV, un virus de ARN de cadena positiva (ARN(+)), para replicar en levaduras se ha demostrado previamente que las subunidades del anillo LSm1-7, así como Pat1 y Dhh1, desempeñan un papel esencial en la transición del genoma del virus de BMV desde traducción a replicación. En células no infectadas, estas proteínas median la transición de ARNm celulares de la traducción a un estado de no-traducción mediante la activación del proceso de decapping en la via 5'-3' de degradación de los ARNs celulares dependiente de deadenilación. Teniendo en cuenta la conservación de esta vía desde levaduras a humanos y la necesidad común de todos los virus ARN(+) para regular la transición de sus genomas desde un estado activo de traducción a otro no activo para permitir la replicación, una posibilidad interesante, y nuestra hipótesis de trabajo, es que LSm1-7, Dhh1 y Pat1 son utilizadas no solo por BMV para replicar en levaduras, sino también por otros virus ARN(+) que infectan a humanos, como el virus de la hepatitis C, para replicar en células de mamíferos. Por otra parte, dado el papel clave de estas proteínas en un paso común en todos los virus de ARN(+), es esencial caracterizar los mecanismos moleculares aun no conocidos y asociados a dicha función. En este sentido, también estudiamos la hipótesis de que el complejo LSm1-7, como miembro de la familia de proteínas Sm, pueda interactuar directamente con los genomas virales de virus de ARN(+) con el fin de desempeñar su papel en el ciclo de vida del virus de una manera similar a la que otros miembros de su familia interactúan con sus ARN con el fin de lograr sus diferentes funciones celulares. En este trabajo hemos podido confirmar ambas hipótesis demostrando que los homólogos humanos de las proteínas anteriormente mencionadas, LSm1-7, Rck/p54 y PatL1, son necesarios para la traducción y replicación del ARN del virus de la Hepatitis C. Por otra parte, los anillos reconstituidos de LSm1-7 reconocen específicamente señales importantes, tanto en el genoma de BMV como en el de la Hepatitis C que regulan su traducción y/o replicación. Estas observaciones constituyen la primera evidencia de que el complejo LSm1-7 es capaz de interactuar directamente con genomas virales y representan también novedosos patrones de interacción de este complejo con ARN. Teniendo en cuenta las estrategias de replicación en común de los virus de ARN de cadena positiva y las funciones celulares conservadas de LSm1-7, Pat1 y Dhh1 de levaduras a humanos, nuestros resultados señalan la posibilidad de explotar estas proteínas para la generación de medicamentos antivirales de amplio espectro.
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Hopkins, M. J. "Characterisation of the human intestinal microbiota based on community cellular fatty acid analysis and ribosomal RNA measurements". Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604222.
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Chemostat studies were undertaken to validate the correlation between bacterial growth rate and 16S ribosomal RNA content for intestinal isolates, and also to identify potential 'signature' cellular fatty acids (CFA) for use as markers of bacterial community structure. These two methods were then used, in conjunction with traditional techniques, to investigate changes in the human colonic microflora associated with carbohydrate metabolism, antibiotic administration, and the ageing process. Short chain carbohydrates (SCC) were used to manipulate the composition and activities of the intestinal microbiota. They showed considerable potential as prophylactic agents in the prevention C. difficile infection. Viable count and 16S rRNA methodologies indicated that these carbohydrates predominantly stimulated bifidobacteria, although the metabolism of other bacterial genera could be affected, and the suppressive effect they induced against C. difficile was not limited to a specific species or genus. However, administration of SCC to patients undergoing antibiotic therapy could result in further impairment of colonisation resistance, with more fermentable carbohydrates, and broad spectrum antibiotics being of highest risk. Bifidobacteria were found to exhibit SCC substrate preferences when growing as part of the normal colonic microflora, which could have important consequences if a particular bacterial strain is to be targeted, such as during probiotic or prebiotic therapy. In conclusion, CFA analysis was effective in detecting differences between environmental samples, although careful consideration is needed to attribute these changes to an altered bacterial community structure rather than modification of the composition of bacterial membranes in stable populations. Viable count methodology proved advantageous for analysing inhibition of bacterial populations, whilst 16S rRNA analysis was the technique of choice for investigating metabolically active samples since it precludes the subjective nature of bacterial identification.