Dissertations / Theses on the topic 'RNA metabolism'
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Stoppel, Rhea. "Chloroplast RNA metabolism." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-152718.
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Conrad, Heather Miller. "Nuclear and mitochondrial mutations affecting mitochondrial RNA metabolism." Connect to resource, 1987. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1230739011.
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Waters, Margaret Fiona. "Enzymes of RNA metabolism in Nostoc sp. MAC." Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.329409.
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LOFFREDA, ALESSIA. "RNA Metabolism alteration in amyotrophic lateral sclerosis models." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2014. http://hdl.handle.net/10281/81488.
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Project1: Unraveling the impact of microRNA on Amyotrophic Lateral Sclerosis pathogenesis.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that specifically affects upper and lower motor neurons leading to progressive paralysis and death. There is currently no effective treatment. Thus, identification of the signaling pathways and cellular mediators of ALS remains a major challenge in the search for novel therapeutics.
Recent studies have shown that microRNA have a significant impact on normal CNS development and onset and progression of neurological disorders. Based on this evidence, in this study we test the hypothesis that misregulation of miRNA expression play a role in the pathogenesis of ALS. Hence, we exploited human neuroblastoma cell lines expressing SOD(G93A) mutation as tools to investigate the role of miRNAs in familiar ALS. To this end, we initially checked the key molecules involved in miRNAs biogenesis and processing on these cells and we found a different protein expression pattern. Subsequently, we performed a genome-wide scale miRNA expression, using whole-genome small RNA deep-sequencing followed by quantitative real time validation (qPCR). This strategy allowed us to find a small group of up and down regulated miRNA, which are predicted to play a role in the motorneurons physiology and pathology. We measured this group of misregulated miRNA by qPCR on cDNA derived from (G93A) mice at different stage of disease and furthermore on cDNA derived from lymphocytes from a group of sporadic ALS patients. We found that mir-129-5p was up-regulated in cells, mice and in patients and we validated that HuD as mir129-5p target. It has been reported that ELAVL4/HuD plays a role in neuronal plasticity, in recovery from axonal injury and multiple neurological diseases. Furthermore, we generated stable cell line overespressing mir129-5p and we found a reduction in neurite outgrowth and in the expression of differentiation markers in compare to control cells. Taken together these data strongly suggest that microRNAs play a role in ALS pathogenesis and in particular that mir129-5p can affect neuronal plasticity by modulating ELAVL4/HuD level.
Project 2: FUS/TLS depletion leads an impairment of cell proliferation and DNA Damage Response.
FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, a ubiquitously expressed RNA-binding protein, has been linked to a variety of cellular processes, such as RNA metabolism, microRNA biogenesis and DNA repair. However, the precise role of FUS protein remains unclear. Recently, FUS has been linked to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disorder characterized by the dysfunction and death of motor neurons. Based on the observation that some mutations in the FUS gene induce cytoplasmic accumulation of FUS aggregates, we decided to explore a loss-of-function hypothesis (i.e. inhibition of FUS’ nuclear function) to unravel the role of this protein. To this purpose, we generated a SH-SY5Y human neuroblastoma cell line which expresses a doxycycline induced shRNA targeting FUS and that specifically depletes the protein. In order to characterize this cell line we performed growth proliferation and survival assays. From these experiments emerged that FUS-depleted cells display alterations in cell proliferation. In order to explain this observation, we tested different hypothesis (e.g. apoptosis, senescence or slow-down growth). We observed that FUS-depleted cells growth slower than control cells. Based on the notion that FUS interacts with the miRNA processing proteins (Morlando et al. 2012), to explain this phenotype, we looked at miRNAs expression and we found an up-regulation of mir-7. Interestingly, this up-regulation is also observed in cells that express the ALS-linked FUS R521C mutation. Finally, since an increasing number of work correlated FUS with DNA damage and repair we explored the effects of DNA damage in FUS-depleted cells by monitoring important components of DNA Damage Response (DDR). We found that FUS depletion had an effect on the initial level of DNA damage by inducing the phosphorylation of H2AX in basal condition and that it delayed DSB repair when acute DNA damage occurs. Interestingly, genotoxic treatment resulted in changes in the subcellular localization of FUS in normal cells. We are currently exploring on one hand the mechanism by which FUS depletion leads to DNA damage, and on the other the functional significance of FUS relocalization after genotoxic stress. Taken together, these studies may contribute to the knowledge of the role of FUS in these cellular processes and will allow us to draw a clearer picture of mechanisms of neurodegenerative diseases.
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Miller, Harvey. "The metabolism of tRNAAspargine in the friend erthroleukemia cell /." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60725.
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I have measured the metabolism of tRNA$ sp{ rm Asn}$ in Friend cells undergoing erythroid differentiation, using an assay exploiting the tRNA$ sp{ rm Asn}$ gene's ability to specifically hybridize tRNA$ sp{ rm Asn}$. The concentrations of tRNA$ sp{ rm Asn}$ measured as a proportion of the total tRNA population (relative concentration) decreased until day 3 post-induction, and then increased on day 4. The relative concentrations of tRNA$ sp{ rm Asn}$ are influenced by tRNA$ sp{ rm Asn}$ having lower turnover rate in comparison to the total tRNA population, and by the relative rate that labelled, newly synthesized tRNA$ sp{ rm Asn}$ accumulates in vivo. The rate of tRNA$ sp{ rm Asn}$ synthesis in nuclei isolated from Friend cells at different times during differentiation also fluctuates. This fluctuation may reflect changes in RNA polymerase III activity in these isolated nuclei.
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Bird, Gregory A. "Exploring the roles of the RNA Polymerase II CTD in pre-MRNA metabolism /." Connect to full text at ProQuest Digital Dissertations. IP filtered, 2005.
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Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005.Typescript. Includes bibliographical references (leaves 130-152). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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Goulet, Isabelle. "New Roles for Arginine Methylation in RNA Metabolism and Cancer." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20293.
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Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.
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Hong, Lingzi. "Act1-Mediated RNA Metabolism in IL-17-Driven Inflammatory Diseases." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case162673878106271.
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Smith, Richard Wilson. "RNA metabolism and the control of protein synthesis in fish." Thesis, University of Aberdeen, 1996. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU089891.
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This thesis examines the regulation of protein synthesis by pre - translational events; with particular reference to the means by which RNA is able to reduce the energetic cost of protein synthesis. Protein synthesis was measured by the application of a "flooding dose" of 3H-phenylalnine. Protein synthesis rates are then calculated from the "free intracellular" and "protein bound" specific radioactivity (dpm nmole-1 phenylalanine). A similar approach was used to investigate RNA synthesis: i.e. a flooding dose of 3H-uridine. As with protein synthesis RNA synthesis rates was assessed by the relating precursor and product (uridine nucleotide and RNA) radioactivity. Oxygen consumption was measured by monitoring the decline in partial pressure in calibrated respirometery chambers. In fish cells protein synthesis was regulated in terms of the amount (ie the "capacity" for protein synthesis) and the translational efficiency of the RNA. Translationally efficient RNA equated to RNA with an increased turnover. In order to minimise RNA production costs, rapidly synthesised RNA places more reliance on the salvage of exogenous nucleosides, as opposed to the relatively expensive alternative of intracellular synthesis. During yolk sac larval development of the African wels (Clarius gariepinus) protein synthesis rates decline whilst oxygen consumption and the amount of RNA (relative to protein) remains constant. Thus the increasing protein synthesis costs resulted from a reduction in RNA translational efficiency. This was mirrored by a declining RNA synthesis rate. Larval fish growth is primarily due to the repartitioning of yolk sac proteins since early life history stages are thought unable to sustain rapid rates of protein turnover. This pre - translational strategy optimises growth and regulates protein synthesis; whilst, at the same time maintaining the capacity for protein synthesis in anticipation of exogenous feeding.
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Benbahouche, Nour el Houda. "Investigating the role of extended CBC complexes in RNA metabolism." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS002.
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Le CBC intervient dans de nombreuses étapes du métabolisme des ARN, telle que l’épissage, la maturation de l’extrémité 3’, la dégradation, l’export et la traduction. Ainsi, le CBC constitue un complexe majeur qui peut orchestrer les différentes étapes de maturation des ARN. Récemment, nous avons identifié le complexe CBCAP, composé de CBC, ARS2 et PHAX. Nous avons montré que la protéine ARS2 stimule la formation des extrémités 3’ de plusieurs familles d’ARN dont les snARN. De plus, ARS2 stimule le recrutement de PHAX sur le CBC. Ainsi, nous proposons un modèle où CBC-ARS2 stimule la formation de l’extrémité 3’ des pré-snARN et recrute PHAX pour favoriser leur export. Une autre étude a identifié un autre complexe le CBCN, constitué de CBC, Ars2, et de ZC3H18-NEXT au lieu de PHAX. CBCN recrute l’exosome et stimule la dégradation de certains ARN, comme les PROMPTS et les transcrits «read-through » des snARN et des ARNm d’histone. Ainsi, PHAX et ZC3H18 destinent leur ARN cibles vers l’export ou la dégradation. Il a été montré que PHAX reconnait et lie spécialement les ARN de petite taille. D’une manière remarquable, nos données de CLIP-Seq et de RIP suivie par des analyses avec des puces « All genes » montrent que PHAX lie aussi d’autres familles d’ARN. En effet, PHAX lie les ARNm ainsi que des ARN non-codant avec une légère préférence pour les snARN (en comparaison avec ZC3H18). Afin de mieux comprendre le rôle de PHAX et ZC3H18, j’ai tout d’abord démontré si les deux protéines se lient simultanément au CBC. Pour ce faire, J’ai réalisé des tests de compétitions entre PHAX et ZC3H18, in vivo, et j’ai montré que la surexpression de ZC3H18 déplace PHAX du CBC et vice versa. Puis en utilisant la technique de « Tethering Assays » j’ai pu montrer que PHAX et ZC3H18 ont des effets opposés sur la biogénèse des ARNm. De plus PHAX semble avoir un effet positif sur la maturation des ARNm et ce, en empêchant ZC3H18 et l’exosome d’être recruter. Nous avons aussi montré que la déplétion de PHAX et ZC3H18 a des conséquences fonctionnelles sur le taux des formes matures des snARN. Dans le but de caractériser la protéine ZC3H18, j’ai réalisé un crible double-hybride et j’ai montré que ZC3H18 interagit avec plusieurs facteurs d’épissage. J’ai aussi identifié les domaines de ZC3H18 impliqués dans ses différentes interactions. D’une manière intéressante, l’interaction de ZC3H18 avec certains facteurs d’épissage peut être exclusive à son interaction avec NEXT. De plus, des expériences de protéomique réalisés sur un des facteurs d’épissage trouvé dans le crible, montrent qu’il co-purifie au sein d’un complexe qui pourrait faire le lien entre la coiffe et la machinerie d’épissage. En accord avec ces résultats, nos données de RNA-seq montrent que la déplétion de ZC3H18 engendre un défaut d’épissage pour des introns qui sont proches de la coiffe et ceci pour un nombre restreint de gènes. Ainsi, notre travail décode davantage le rôle de la coiffe dans les différentes étapes de maturation des ARN et suggère un modèle où la séquence des transcrits naissant stimule la formation d’un complexe spécifique à cet ARN parmi plusieurs autresThe cap binding complex (CBC) plays a key role in a number of gene expression pathways and has been proposed to participate in the discrimination of RNA families. It also enhances many RNA processing steps, including transcription, splicing, 3’end formation, degradation, export and translation.Recently, we identified the CBCAP complex, composed of CBC, Ars2 and PHAX. We showed that Ars2 stimulates snRNA 3'-end processing as well as PHAX binding to the CBC, hence coupling snRNA maturation with their export. Other studies showed that the CBC and ARS2 can form another complex that contains ZC3H18-NEXT instead of PHAX. This complex, named CBCN, is a cofactor of the RNA exosome and is involved in the degradation of cryptic RNAs such as PROMPTs and read-through transcripts at histone and snRNA genes. Thus, PHAX and ZC3H18 target specific families of capped RNA toward either export or degradation. Previous studies proposed that PHAX binds specifically to small RNAs and discriminates them over other RNA species. Surprisingly, our CLIP-Seq and RIP-microarrays data showed that in contrast to expectations, PHAX was not specific for snRNAs. It also binds mRNAs as well as other non-coding RNAs and has a weak preference for snRNAs comparing to ZC3H18. To better understand the role of PHAX and ZC3H18, Ifirst determined whether PHAX and ZC3H18 can bind simultaneously to the CBC. Competitive LUMIER IPs indicated that binding of these proteins is mutually exclusive. I then used tethering assays and could show that PHAX and ZC3H18 have opposite effect on mRNA biogenesis. These data go against a model where binding of PHAX or ZC3H18 discriminate RNA families, and instead suggest promiscuous binding for these proteins. In addition, PHAX may exert a positive effect on mRNA processing by preventing binding of ZC3H18 and recruitment of the RNA exosome. Last but not least, our RT-QPCR data show that PHAX and ZC3H18 depletions have functional consequences on the level of mature snRNA, and this is due to a competition between both proteins which occur on those snRNA read-through transcripts.To further explore the role of ZC3H18, I performed a two-hybrid screen and identified several splicing factors. I could validate these interactions, identify the domains involved and show that binding of some of these factors is exclusive with that of NEXT. Importantly, proteomic experiments with one of these factors identified a complex that makes the link between the cap and the splicing machinery. In agreement, RNA-Seq analysis of ZC3H18 knock-down cells showed alterations in splicing of cap-proximal introns, for a small set of genes.Altogether, this work reveals how the multiple roles of the RNA cap are achieved at the biochemical level, and suggests that the nascent RNA sequence triggers formation of one among several mutually exclusive complexes
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Cornella, Nicola. "Characterization of the hnRNP RALY in RNA transcription and metabolism." Doctoral thesis, Università degli studi di Trento, 2017. https://hdl.handle.net/11572/369300.
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The heterogeneous nuclear ribonucleoproteins (hnRNPs) form a large family of RNA-binding proteins (RBPs) that exert numerous functions in RNA metabolism. For example, soluble hnRNPs bind to RNAs to mediate their maturation, processing, and shuttling from the nuclear compartment to the cytoplasm. Additionally, hnRNPs might interact with chromatin to regulate the transcription and the post-transcriptional modification of nascent transcripts.
RALY is a member of the hnRNP family that binds poly-U rich elements within several RNAs and regulates the expression of specific transcripts. RALY is upregulated in different types of cancer and its downregulation has been shown to impair cell proliferation. In my PhD project, I characterized RALY to interact with transcriptionally active chromatin in a transcription-dependent manner and to cause a global decrease of RNA Polymerase II (RNAPII)-mediated transcription when downregulated, without affecting RNAPII elongation rate. Through microarray analysis of RALY-downregulated HeLa cells, I detected an altered expression of numerous genes involved in transcription promotion and cell cycle regulation, including the E2F transcription factors family. Due to its relevant role in regulating the cell cycle, I focused on the proliferation-promoting factor E2F1. I demonstrated that the stability of E2F1 mRNA is reduced in cells lacking RALY expression, with a resulting reduction of E2F1 protein levels. As a consequence of RALY knock-out, HeLa cells present a slower cell proliferation compared to control cells. Finally, by crossing the list of RALY targets with the list of genes affected by RALY downregulation, I propose a positive role of RALY in regulating the fate of specific transcripts.
Taken together, my results highlight the importance of RALY expression for transcription and cell proliferation.
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Cornella, Nicola. "Characterization of the hnRNP RALY in RNA transcription and metabolism." Doctoral thesis, University of Trento, 2017. http://eprints-phd.biblio.unitn.it/2625/2/Disclaimer_Cornella.pdf.
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The heterogeneous nuclear ribonucleoproteins (hnRNPs) form a large family of RNA-binding proteins (RBPs) that exert numerous functions in RNA metabolism. For example, soluble hnRNPs bind to RNAs to mediate their maturation, processing, and shuttling from the nuclear compartment to the cytoplasm. Additionally, hnRNPs might interact with chromatin to regulate the transcription and the post-transcriptional modification of nascent transcripts.
RALY is a member of the hnRNP family that binds poly-U rich elements within several RNAs and regulates the expression of specific transcripts. RALY is upregulated in different types of cancer and its downregulation has been shown to impair cell proliferation. In my PhD project, I characterized RALY to interact with transcriptionally active chromatin in a transcription-dependent manner and to cause a global decrease of RNA Polymerase II (RNAPII)-mediated transcription when downregulated, without affecting RNAPII elongation rate. Through microarray analysis of RALY-downregulated HeLa cells, I detected an altered expression of numerous genes involved in transcription promotion and cell cycle regulation, including the E2F transcription factors family. Due to its relevant role in regulating the cell cycle, I focused on the proliferation-promoting factor E2F1. I demonstrated that the stability of E2F1 mRNA is reduced in cells lacking RALY expression, with a resulting reduction of E2F1 protein levels. As a consequence of RALY knock-out, HeLa cells present a slower cell proliferation compared to control cells. Finally, by crossing the list of RALY targets with the list of genes affected by RALY downregulation, I propose a positive role of RALY in regulating the fate of specific transcripts.
Taken together, my results highlight the importance of RALY expression for transcription and cell proliferation.
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Whitman, Samantha. "Fragile X Related Protein-1 (FXR1) Regulates RNA Metabolism in Striated Muscle." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/195153.
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Cardiac muscle function necessitates the meticulous assembly and interactions of several cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and transmission forces. Despite extensive studies identifying the protein components responsible for these important aspects of heart development, putative RNA based mechanisms remain poorly understood, even with their demonstrated importance in other tissues. Evidence suggests that post-transcriptional regulation is critical for muscle function, but the molecular players involved (RNA binding proteins and mRNA targets) have remained elusive. We investigated the molecular mechanisms and targets of the muscle-specific Fragile X Related protein-1 (FXR1), an RNA binding protein whose absence leads to perinatal lethality in mice. Loss of FXR1 results in global protein level alterations. Morphological and biochemical analyses of Fxr1^(-/-) mice revealed severe disruption of intercalated disc and costamere architecture and composition. We identified several candidate mRNAs specifically enriched in the FXR1 protein complex. Two targets that likely contribute to the architectural defects are desmoplakin (dsp) and talin2 (tln2). In vitro assays indicate that FXR1 binds to these mRNA targets directly and represses their translation. Additionally, we provide preliminary evidence that the Fxr1^(-/-) mice mimic a hypothyroid state of cardiac gene expression, with alterations in myosin heavy chain and troponin I isoforms. Our findings reveal the first mRNA targets of FXR1 in muscle and support translational repression as a novel mechanism for cardiac muscle development and function.
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Beauchamp, Pascal. "The functional role of the RNA-binding protein HuR in the regulation of muscle cell differentiation /." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111586.
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Muscle tissue development (myogenesis) involves the formation of specific fibers (myotubes) from muscle cells (myoblasts). For this to occur, the sequential expression of Myogenic Regulatory Factors (MRFs), such as MyoD and myogenin, is required. The expression of these MRFs is regulated posttranscriptionally by the RNA-binding protein HuR, whereby HuR associates with the 3'-untranslated regions of MyoD and myogenin mRNA, leading to a significant increase in their half-lives. Here we show that the cleavage of HuR by caspases at the aspartate (D) 226 residue is one of the main regulators of its pro-myogenic function. This proteolytic activity generates two cleavage products (CPs), HuR-CP1 and HuR-CP2, that differentially affect the myogenic process. Myoblasts overexpressing HuR-CP1 or the non-cleavable mutant of HuR, HuRD226A, are not able to engage myogenesis, while overexpressing HuR-CP2 enhances myotube formation. HuR-CP2 but not -CP1 promotes myogenesis by stabilizing the MyoD and myogenin mRNAs to the same levels as wt-HuR. Conversely, the inhibitory effects of HuR-CP1 and HuRD226A depend on their abilities to associate during myogenesis with the HuR import receptor, Trn2, leading to HuR accumulation in the cytoplasm. Therefore, we propose a model whereby the caspase-mediated cleavage of HuR generates two CPs that collaborate to regulate myogenesis; HuR-CP1 by interfering with the Trn2-mediated import of HuR and HuR-CP2 by participating in the stabilization of mRNAs encoding key MRFs.
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Greenhalgh, Duncan Alan. "Effects of 5-fluorouracil on RNA metabolism in human tumour cells." Thesis, University of Leeds, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236315.
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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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Audano, M. "THE RNA BINDING PROTEIN ZC3H10 COUPLES MITOCHONDRIAL FUNCTION AND IRON METABOLISM." Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/481986.
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Mitochondria play a crucial role in energy metabolism. Mitochondria have their own genome (mtDNA), whose replication and transcription are mainly regulated by the mitochondrial transcription factor A (Tfam). Recent researches demonstrate how mitochondria participate to a large number of cellular processes like cell cycle and differentiation. Our goal is to identify new mitochondrial regulators to light up the molecular mechanisms underlying mitochondrial function biology.
We used a high throughput screening in 293 cells in order to identify positive mitochondrial regulators. By these means, we identified Zinc Finger CCCH-type containing 10 (Zc3h10) as the best hit. Following experiments demonstrated that Zc3h10 knockdown decreased mitochondrial function and differentiation in myotubes. RNA immunoprecipitation assay indicates that Zc3h10 is able to bind 410 transcripts. Several target genes are involved in energy metabolism and iron balance. Notably, Zc3h10 downregulation in C2C12 leads to iron overload while its overexpression restores ferric ion content to control levels.
Collectively, our findings annotate Zc3h10 as a new mitochondrial regulator in skeletal muscle.
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Figueiredo, Cardoso Tainã. "Analysis of the genetic basis of porcine meat quality and coat color by using genomic and transcriptomic tools." Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/662617.
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Los principales objetivos de esta Tesis fueron investigar la base genética de la composición y deposito de la grasa en cerdos, e identificar los factores genéticos involucrados en el establecimiento de los patrones de pigmentación rubia vs roja en cerdos Mangalitza, mediante el uso de herramientas genómicas y transcriptómicas. En el primer estudio comparamos los patrones de expresión del músculo esquelético en dos grupos de cerdos Duroc, con diferentes perfiles de crecimiento y engrasamiento (HIGH: elevado espesor del tocino dorsal, grasa intramuscular, contenido de ácidos grasos saturados e insaturados y lípidos séricos vs LOW: fenotipos opuestos). Mediante el uso de la técnica RNA-seq, hemos encontrado que 96 genes se expresan diferencialmente en el músculo gluteus medius de cerdos HIGH y LOW. Varios de estos genes están relacionados con el metabolismo lipídico (p.ej, SLC27A4, SFRP5, y CES1) y el factor de transcripción PPARG parece ser un regulador clave del engrasamiento en porcino. También hemos observado que muy pocos RNAs no codificantes se expresan diferencialmente en estos dos grupos de cerdos, lo que sugiere que el transcriptoma no codificante tiene un efecto limitado sobre el establecimiento de los fenotipos HIGH y LOW. En el segundo estudio, analizamos la expresión de isoformas de mRNA en cerdos HIGH y LOW y demostramos la expresión diferencial de isoformas específicas de cuatro genes muy relacionados con la obesidad (ITGA5, LITAF, TIMP1 y ANXA2). La expresión diferencial de estas isoformas podría tener efectos sobre la estructura del transcrito, así como sobre la secuencia de la proteína. En el tercer estudio, hemos analizado la expresión diferencial de genes que codifican mRNA en respuesta a la ingestión de alimentos. Este objetivo se ha logrado al comparar los patrones de expresión muscular de cerdas Duroc antes de comer (T0), 5 h. (T1) y 7 h. (T2) después de comer. Además de los genes con un papel bien conocido en la homeostasis energética (p.ej, PFKFB3 y G0S2), hemos identificado varios genes con un rol plausible pero mal caracterizado en el metabolismo (p.ej, MIGA2, SDC4 y CSRNP1). También hemos observado un enriquecimiento de un conjunto de genes expresados diferencialmente antes y después de comer que engloba diversos factores de transcripción así como genes implicados en el estrés oxidativo, la angiogénesis y los ritmos circadianos. Teniendo en cuenta estos resultados, en el cuarto estudio hemos desarrollado un experimento basado en RT-qPCR para descubrir cómo la expresión de 8 genes circadianos (ARNTL, BHLHE40, CRY2, NPAS2, NR1D1, PER1, PER2 y SIK1) se modifica en respuesta a la ingestión de alimentos en cinco tejidos porcinos (músculo esquelético, hipotálamo, hígado, intestino y grasa dorsal). Nuestros resultados indican que la expresión de los genes circadianos no cambia en el hipotálamo, el tejido que contiene el reloj central influenciado por la luz. Por el contrario, dicha expresión sí que presenta fuertes variaciones en los otros cuatro tejidos. Este hallazgo demuestra que la nutrición cambia la expresión de los genes circadianos integrados en los relojes periféricos. Finalmente, en el quinto estudio, hemos analizado, en colaboración con investigadores del Research Institute for Animal Breeding and Nutrition (Hungría) y la Universidad de Cluj-Napoca (Rumanía), la base genética del color de la capa (rojo vs rubio) en cerdos Mangalitza. Combinando un barrido de selección y un estudio de asociación del genoma completo, hemos encontrado que el gen SLC45A2 probablemente esté involucrado en la determinación genética de la pigmentación roja y rubia de los cerdos Mangalitza, un resultado que concuerda bien con estudios previos que demuestran la implicación de este locus en los patrones de color de múltiples especies de mamíferos, incluyendo la especie humana. Más específicamente, dos SNP con efecto no-sinónimo, c.806G>A (p.Gly269Glu) y c.956G>A (p.Arg319His), situados en el gen SLC45A2, están fuertemente asociados con los colores rojo y rubio, no obstante dicha asociación no es completa por lo que cabe deducir la existencia de factores genéticos adicionales en la pigmentación de los cerdos Mangalitza.The main objectives of this Thesis were to investigate the genetic basis of fatness in pigs and to identify the genetic factors involved in the establishment of blond vs red pigmentation patterns in Mangalitza pigs by using genomic and transcriptomic tools. In the first study of the Thesis (Chapter 3), we compare the skeletal muscle expression patterns of two groups of Duroc pigs with different growth and fatness profiles (HIGH: high backfat thickness, intramuscular fat, saturated and unsaturated fatty acid content and serum lipids vs LOW: opposite phenotypes). By using a RNA-Seq technology, we identified 96 genes differentially expressed. Several of these genes are related to lipid metabolism (e.g. SLC27A4, SFRP5 and CES1) and the transcription factor PPARG appears to be a key regulator of porcine fatness. We have also observed that very few non-coding RNAs are differentially expressed in these two groups of pigs, suggesting that the non-coding transcriptome has a limited effect on the establishment of the HIGH and LOW phenotypes. In the second study of the Thesis, we demonstrate the differential expression of specific mRNA isoforms of four genes with a known role in obesity (ITGA5, LITAF, TIMP1 and ANXA2) in HIGH vs LOW pigs. The differential expression of these isoforms may have effects on transcript structure as well as on the protein sequence. In the third study, we aimed to investigate the differential expression of mRNA encoding genes in response to food ingestion. This goal has been achieved by comparing the muscle mRNA expression patterns of Duroc sows before feeding (T0) and 5 h. (T1) and 7 h. (T2) after feeding. Besides genes with a well-known role in energy homeostasis (e.g. PFKFB3 and G0S2), we have identified several genes with a plausible but poorly characterized role in metabolism (e.g. MIGA2, SDC4, and CSRNP1). We have also observed that the set of genes differentially expressed before and after feeding is enriched in transcription factors and pathways related to oxidative stress, angiogenesis, and circadian rhythms. Considering these results, in the fourth study we use quantitative RT-qPCR technique to find out how the expression of 8 circadian genes (ARNTL, BHLHE40, CRY2, NPAS2, NR1D1, PER1, PER2 and SIK1) changes in response to food ingestion in five porcine tissues i.e. skeletal muscle, hypothalamus, liver, intestine and dorsal fat. Our results indicate that the expression of the Clock genes does not change in the hypothalamus, the tissue containing the central clock entrained by light, but in contrast, it is strongly modified in the other four tissues. This finding demonstrates that nutrition changes the expression of circadian genes integrated in peripheral clocks. Finally, in the fifth study, we have analysed, in collaboration with researchers of the Research Institute for Animal Breeding and Nutrition (Hungary) and the University of Cluj-Napoca (Romania), the genetic basis of coat color (red vs blond) of Mangalitza pigs. By combining a selection scan and a genome-wide association study, we have found that the SLC45A2 gene is probably involved in the genetic determination of pigmentation in Mangalitza pigs, a result that agrees well with previous studies demonstrating the implication of this locus on the color patterns of multiple mammalian species including humans. More specifically, two missense SNPs c.806G>A (p.Gly269Glu) and c.956G>A (p.Arg319His) in the SLC45A2 locus appear to be strongly but not fully associated with the red and blond coat colors of Mangalitza pigs. This finding suggests the existence of addiitonal genetic factors regulating the pigmentation of Mangalitza pigs.
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Ajamian, Lara. "Up-frameshift proteins and their distinct roles in HIV-1 RNA metabolism." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116866.
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HIV-1 co-opts host cell proteins at every step of its replication cycle to ensure proper replication. Our work identified that the HIV-1 genomic RNA is not a substrate for nonsense-mediated mRNA decay (NMD) even though it has multiple open reading frames as well as a long 3'UTR. We demonstrate that Up-frameshift protein 1 (UPF1) is involved in HIV-1 genomic RNA stability such that overexpression of UPF1 increases HIV-1 genomic RNA levels and Gag translation. Moreover, the role of UPF1 in HIV-1 is NMD-independent, is observed in both nuclear and cytoplasmic compartments and does not require binding to UPF2. Furthermore, the shuttling function of UPF1 is required for HIV-1 genomic RNA export since a UPF1 nuclear export mutant sequesters the genomic RNA in the nucleus and a nuclear localization mutant does not immunoprecipitate with the HIV-1 genomic RNA. UPF1's role in HIV-1 genomic RNA export is observed in both Rev-dependent and -independent conditions. In addition, UPF1 is found in complex with Rev, CRM1, Nup62 and DDX3, cellular proteins with already characterized roles in HIV-1 genomic RNA export. Lastly, we also identified UPF2 as a negative regulator, such that its binding to UPF1 results in the nuclear sequestration of the HIV-1 genomic RNA. We have identified a possible mechanism to explain how HIV-1 escapes the RNA quality control mechanism of NMD by co-opting UPF1 function for efficient HIV-1 genomic RNA export, stability and translation.Le VIH-1 requiert plusieurs protéines cellulaires à chaque étape de son cycle de réplication pour assurer une réplication efficace. Notre travail a mené à l'identification que l'ARN génomique du VIH-1 n'est pas un substrat pour la dégradation des ARNm aberrants (NMD), même si elle a plusieurs cadres de lecture ainsi qu'une longue 3'UTR. Nous avons démontré que UPF1 est impliqué dans la stabilité de l'ARN génomique du VIH-1 car la surexpression de UPF1 a engendré une augmentation des niveaux d'ARN du VIH-1 et de Gag. Par ailleurs, le rôle de UPF1 est distinct de son rôle dans le mécanisme NMD, il est observé dans les compartiments nucléaires et cytoplasmiques et ne nécessite pas sa liaison à UPF2. De plus, la fonction navette de UPF1 est requise pour assurer l'exportation de l'ARN génomique du VIH-1 car le mutant NES d'UPF1 bloque son export et le mutant NLS n'immunoprécipite pas avec celui-ci. Ce nouveau rôle d'UPF1 est observé dans la présence et l'absence de Rev. De plus, UPF1 se trouve en complexe avec Rev, CRM1, Nup62 et DDX3, les protéines cellulaires déjà caractérisées comme étant impliquées dans l'exportation de l'ARN génomique du VIH-1. Enfin, nous avons également identifié UPF2 comme étant un régulateur négatif, de telle sorte que sa liaison à UPF1 engendre un blocage nucléaire de l'ARN génomique du VIH-1. Nous avons identifié un mécanisme possible démontrant comment le VIH-1 s'évade du mécanisme NMD en cooptant UPF1 pour faciliter l'exportation, la stabilité et la traduction de l'ARN génomique du VIH-1.
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Kew, Chun [Verfasser], and Adam [Gutachter] Antebi. "Control of Innate Immunity by RNA Metabolism / Chun Kew ; Gutachter: Adam Antebi." Köln : Universitäts- und Stadtbibliothek Köln, 2018. http://d-nb.info/1180601556/34.
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Leech, M. J. "Studies on the metabolism of poly(A)'+RNA in the imbibing wheat embryo." Thesis, University of Manchester, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.383174.
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Ma, Jing 1978. "The requirement of the DEAD-box protein DDX24 for the packaging of human immunodeficiency virus type 1 RNA /." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116084.
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Human immunodeficiency virus (HIV) is the causing agent of the acquired immune deficiency syndrome (AIDS). Like all retroviruses, HIV carries two copies of viral genomic RNA in each virion. HIV genome encodes three structural genes, including gag, pol and env, as well as two regulatory genes (rev and tat) and four accessory genes (vif, vpr, vpu and nef). It is noted that none of these nine viral proteins bears the helicase activity. Helicases are able to unwind RNA duplex and remodel the structure of RNA-protein (RNP) complexes using energy derived from hydrolysis of nucleotide triphosphates (NTPs). They are involved in every step of cellular RNA metabolisms. It is conceivable that HIV needs to exploit cellular RNA helicases to promote the replication of its RNA at various steps such as transcription, folding and transport.In this study, we found that a DEAD-box protein named DDX24 associates with HIV-1 Gag in an RNA-dependent manner but is not found within virus particles. Knockdown of DDX24 inhibits the packaging of HIV-1 RNA and thus diminishes viral infectivity. The decreased viral RNA packaging as a result of DDX24-knockdown is observed only in the context of the Rev/RRE (Rev response element)-dependent but not the CTE (constitutive transport element)-mediated nuclear export of viral RNA, which is explained by the specific interaction of DDX24 with the Rev protein. We propose that DDX24 acts at the early phase of HIV-1 RNA metabolism prior to nuclear export and the consequence of this action extends to the viral RNA packaging stage during virus assembly.
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Séguin, Béatrice. "Control of HIV-1 RNA metabolism, the role of splice sites and intron sequences in unspliced viral RNA subcellular distribution." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0002/MQ40793.pdf.
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Voss, Jarrod Edwin. "Structural studies of bacterial and macromolecular machines of RNA metabolism and drug transport." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708564.
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Giunta, Michele. "Exosomal protein deficiencies : how abnormal RNA metabolism results in childhood-onset neurological diseases." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3669.
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RNA metabolism is of critical importance for normal cellular functions and needs to be finely tuned in order to maintain stable conditions within the cell. The exosome complex is the most important RNA processing machinery, responsible for the correct processing of many different types of RNAs and interacting with different co-factors which bind and carry specific subtypes of RNA for degradation to the complex. Mutations in exosome complex subunits (EXOSC3, EXOSC8) were reported to cause severe childhood onset complex neurological disorders presenting with pontocerebellar hypoplasia type 1 (PCH1), spinal muscular atrophy (SMA) and central nervous system hypomyelination. We have recently identified a homozygous pathogenic mutation in RNA Binding Motif Protein 7 RBM7, a subunit of the nuclear exosome targeting (NEXT) complex in a single patient with SMA-like phenotype and proved that RBM7 is a novel human disease gene related to the exosome complex. In order to understand the disease mechanism in RBM7 deficiency and to explore the role of exosome complex in neurodevelopment, we performed gene expression studies (RT-PCR, RNA sequencing) in human cells of patients carrying mutations in EXOSC8 and RBM7. Furthermore we performed functional studies in zebrafish (D. rerio) by morpholino oligonucleotide mediated knock-down of rbm7, exosc8 and exosc3 and also by introducing pathogenic mutations in exosomal protein genes in zebrafish embryos by the CRISPR/Cas9 system. We showed that mutations in RBM7 and EXOSC8 mutant fibroblasts cause differential expression of several different transcripts, 62 of them being shared between the two cell lines. Altered gene expression of some AU-rich element containing genes may potentially contribute to the clinical presentation. Knock-down of rbm7, exosc8 and exosc3 caused impaired neurodevelopment in zebrafish, illustrated by abnormal growth of motor neuron axons and failure to differentiate cerebellar Purkinje cells. RT-PCR analysis in zebrafish showed a dramatic increase in expression of atxn1b (an AU-rich element containing homolog of the human ATXN1 gene) in rbm7, exosc8 and exosc3 downregulated fish, which may be responsible for the cerebellar defects. We have successfully introduced several germline mutations with CRISPR/Cas9 technology in rbm7. Phenotype of the F1 mutants is milder than what observed with the morpholino oligonucleotide injected fish. Mutants at a closer look do not show any morphological defect but further experiment may indicate similar characteristics to the morphants, although more iv subtle. Further studies on the CRISPR/Cas9 generated zebrafish models will extend our knowledge on the disease mechanisms caused by defective RNA metabolism.
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Sun, Xin. "Molecular structure and intracellular location of five RNA-associated proteins in the salivary glands of the dipteran Chironomus tentans /." Stockholm, 1998. http://diss.kib.ki.se/1998/91-628-3291-3/.
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Prichard, Lisa. "The role of the IQ motif, a protein kinase C and calmodulin regulatory domain, in neuroplasticity, RNA processing, and RNA metabolism /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/6302.
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Zhang, Tong. "Characterization of the shuttling properties of RNA-binding TIA proteins." Doctoral thesis, Universite Libre de Bruxelles, 2005. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210999.
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Kafaie, Jafar. "Role of the NC protein of human immunodeficiency virus type 1 in viral RNA dimerization and packaging, as well as in virus replication and stability." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111914.
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In the past three decades, various steps of the human immunodeficiency virus type 1 (HIV-1) life cycle have been thoroughly studied. Many of these steps, such as viral entry, reverse transcription and proteolysis have been targets of antiretroviral therapy. Retroviral genomic RNA (gRNA) dimerization appears essential for viral infectivity and this process appears to be chaperoned by the nucleocapsid (NC) protein of HIV-1. In this dissertation, the role of NC in genome dimerization and other aspects of the viral life cycle have been thoroughly studied. Various positions of the NC protein have been mutated through site-directed mutagenesis and relevant and dispensable positions of NC have been identified through this method. 34 of its 55 residues were mutated, individually or in small groups, in a panel of 40 HIV-1 mutants. It was found that the amino-terminus, the proximal zinc finger, the linker, and the distal zinc finger of NC each contributed roughly equally to efficient HIV-1 gRNA dimerization. The various mutations introduced into NC show the first evidence that gRNA dimerization can be inhibited by: 1) mutations in the N-terminus or the linker of retroviral NC; 2) mutations in the proximal or distal zinc finger of lentiviral NC; 3) mutations in the hydrophobic patch (plateau) or the conserved glycines of the proximal or the distal retroviral zinc finger. Some NC mutations impaired gRNA dimerization more than mutations inactivating the viral protease, indicating that gRNA dimerization may be stimulated by the NC component of the Gag polyprotein (Pr55gag). In the second section of my work, I studied the effect of Pr55gag processing on gRNA dimerization by introducing rate alternating mutants into Pr55gag protein cleavage sites. I showed that Maturation ofNCp15 into NCp9 is essential for fast rates of genomic RNA dimerization and maturation of NCp9 into NCp7 has no incidence on genomic RNA dimerization but is essential for viral replication. In order to delineate the amount of viral protease activity needed to produce mature virus 48 hours post transfection, we also studied, by cotransfection studies, the effect of various ratios of wild-type (BH10) and protease-inactive (PR- ) plasmids and found that HIV-1 reaches its full genomic RNA dimerization despite 75% unprocessed Pr55gag polyproteins. We have also shown that wild type BH10 plasmid can rescue those mutations in NCp7 protein that have an effect on gRNA dimerization through rescue experiments. Overall, this thesis sheds light on the role of NC in HIV-1 genome dimerization and other aspects of the viral life cycle and identifies the importance of each component of NC during these processes.
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Eser, Philipp [Verfasser], and Patrick [Akademischer Betreuer] Cramer. "Quantitative genome-wide studies of RNA metabolism in yeast / Philipp Eser. Betreuer: Patrick Cramer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1104128985/34.
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Stoppel, Rhea [Verfasser], and Jörg [Akademischer Betreuer] Meurer. "Chloroplast RNA metabolism : global players and site specific factors / Rhea Stoppel. Betreuer: Jörg Meurer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1030475458/34.
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Kiss, Daniel L. "The Exozyme Model: A New Paradigm of Exosome Subunit Activity Revealed by Diverse and Distinct Substrate Specificities of Exosome Subunits In Vivo." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1263237977.
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Dantas, Ive Maíra de Carvalho. "Perfil lipídico na leishmaniose visceral em hamster e expressão de mRNA de genes relacionados ao metabolismo liprotéico." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/99/99131/tde-03082015-103941/.
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Na fase ativa da leishmaniose visceral (LV) ocorrem alterações no metabolismo de lipoproteínas com redução dos níveis de HDL e aumento de triglicérides. A partir desses dados, focamos neste projeto essas alterações na progressão da infecção e apontamos alguns elementos como seus possíveis desencadeantes. Como essas alterações poderiam resultar de redução de atividade e expressão da lipoproteína lipase (LPL), do receptor alfa do proliferador ativado de peroxissoma (PPAR?) e da proteína transferidora de ésteres de colesteril (CETP), a sua expressão foi avaliada durante a progressão da LV em hamster. Em hamsteres infectados com 2 x 107 amastigotas de L. (L.) infantum observamos aumento de triglicérides nos hamsteres com 55 dias (mediana = 294,0 mg/dL) e 90 dias (303,0 mg/dL ) de infecção comparados aos controles de 55 dias (119,0 mg/dL) e de 90 dias (117,0 mg/dL) (p <= 0,05). Os níveis de colesterol total e de HDL não apresentaram diferença significante entre controles e infectados com 30, 55 e 90 dias de infecção. A expressão de mRNA de PPAR? no fígado com 55 e 90 dias de infecção apresentou tendência de redução nos infectados. Já de CETP no fígado dos hamsteres com 55 dias de infecção, a expressão relativa (CT) estava reduzida nos infectados (0,08) comparados aos controles (1,69) (p <= 0,05) e de LPL no coração dos hamsteres com 90 dias de infecção também estava reduzida (1,43) com relação aos controles (2,61) (p <= 0,05). Há dados na literatura sugerindo a importância de lipídios para o desenvolvimento de amastigotas no hospedeiro vertebrado e é possível que as alterações dos níveis de lipoproteínas contribuam na progressão da infecção. Assim, avaliamos neste estudo o efeito da droga hipolipemiante ciprofibrato no controle do parasitismo na LV em hamster, sabendo-se que ciprofibratos atuam aumentando a expressão de PPAR? e a produção e atividade de LPL. O tratamento com ciprofibrato nos hamsteres com 55 dias de infecção gerou redução de triglicérides (123,0 mg/dL) em relação aos infectados não tratados (294,0 g/dL) (p <= 0,05), além dos níveis de triglicérides nos animais infectados não tratados terem aumentado quando comparados aos controles não tratados (119,0 mg/dL) (p <= 0,05). Houve também, redução de triglicérides nos animais não infectados tratados com ciprofibrato (89,0 mg/dL) comparando-se aos infectados não tratados (p <= 0,05). Os níveis de colesterol nos hamsteres não infectados tratados com ciprofibrato reduziram (53,5 mg/dL) em comparação aos infectados não tratados (93,0 mg/dL) (p <= 0,05). Já naqueles que foram infectados e tratados com ciprofibrato, constatamos redução de colesterol (53,5 mg/dL) quando comparados aos infectados não tratados (p <= 0,05). Os níveis de HDL não aumentaram com ciprofibrato e foram similares entre os hamsteres infectados não tratados e os controles não tratados. A carga parasitária no baço e no fígado não foi reduzida com ciprofibrato. Na leishmaniose visceral em hamster ocorrem alterações do metabolismo lipídico com aumento de triglicérides e redução da expressão da mRNA de LPL e CETP. O tratamento com ciprofibrato foi eficaz no controle das alterações de níveis de lipoproteínas.In the active phase of visceral leishmaniasis (VL) changes occur in lipoprotein me-tabolism with reduction in HDL and increase in triglyceride (TG) levels. From these data, in this project we focused these changes during the progression of the infection and we approached some elements as their underlying factors. Since these changes may result from the reduction of the activity and the expression of the lipoprotein lipase (LPL), of the peroxisome proliferator-activated receptor alpha (PPAR?) and of the cholesteryl ester transfer protein (CETP), their expression were evaluated during VL progression in hamster. In 2 x 107 L. (L.) infantum amastigote-infected hamsters we observed an increase in the triglycerides in hamsters with 55 days (median = 294.0 mg/dL) and 90 days (303.0 mg/dL) of infection compared with controls of 55 days (119.0 mg/dL) and of 90 days (117.0 mg/dL) (p <= 0.05). The total cholesterol and the HDL levels did not present significant differences between control and in-fected groups at 30, 55 and 90 days of infection. The expression of mRNA of the PPAR in the liver with 55 and 90 days of infection tended to be reduced in infected animals. However the relative expression (CT) of CETP in the liver of hamsters with 55 days of infection was signicantly reduced in infected (0.08) compared with control animals (1.69) (p <= 0.05). The relative expression (CT) of LPL in the heart of hamsters with 90 days of infection was also reduced (1.43) in relation to controls (2.61) (p <= 0.05). There are data in the literature suggesting the importance of lipids for the development of amastigotes in vertebrate host and it is possible that the changes in the lipoprotein levels contribute for the infection progression. Therefore, we evaluated in this study the effect of the lipid-lowering drug ciprofibrate in the control of parasitism in VL in the hamster, knowing that ciprofibrate acts increasing the expression of the PPAR? and of the LPL production and activity. The treatment with ciprofibrate in infected hamsters at 55 days lead to the reduction of triglyceride level (123.0 mg/dL) in relation to non-treated infected animals (294.0 g/dL) (p <= 0.05). Further the triglyceride levels in the non-treated infected animals were in-creased when compared with untreated controls (119.0 mg/dL) (p <= 0.05). There was also reduction of triglyceride in ciprofibrate treated-non infected animals (89.0 mg/dL) compared with non-treated infected animals (p <= 0.05). The cholesterol lev-els were reduced in the ciprofibrate-treated non-infected hamsters (53.5 mg/dL) in comparison to the non-treated infected ones (93.0 mg/dL) (p <= 0.05). In the ciprofibrate-treated infected ones we found a reduction of cholesterol level (53.5 mg/dL) when compared with non treated infected animals (p <= 0.05). The HDL lev-els did not increase with ciprofibrate and they were similar between the non-treated infected hamsters and non-treated controls. The parasite load in the spleen and liver were not reduced with ciprofibrate. In the visceral leishmaniasis in hamster changes occur in the lipid metabolism with increase in the triglyceride level and the reduction of expression of mRNA of LPL and CETP. The treatment with ciprofibrate was ef-fective in the control of changes in the lipoprotein levels.
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Jenkins, Kristin Perth 1965. "Identification and partial characterization of two chloroplast-encoded potential RNA metabolism genes roaA and mat1." Diss., The University of Arizona, 1996. http://hdl.handle.net/10150/282114.
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Although group I and group II introns are capable of self-splicing in vitro, there is growing evidence that trans acting factors facilitate splicing of group I and group II introns in vivo. For group II introns, these trans acting factors include maturases and RNA chaperones. Maturases are intron-encoded enzymes which splice the intron which encodes them and in some cases mobilize the intron. RNA chaperones are a group of general RNA binding proteins which facilitate formation of correctly folded catalytic RNAs. The Euglena gracilis chloroplast genome is an ideal system in which to study RNA metabolism, especially splicing. The genome is composed of 38% introns, including group II, group III and twintrons. Although Euglena gracilis chloroplast pre-mRNAs must undergo extensive processing and splicing before they are translated, no chloroplast encoded maturases or RNA chaperones have been described. I have characterized two potential RNA metabolism genes. The roaA gene is unique to the Euglena chloroplast and encodes a potential general RNA binding protein. The roaA gene and mRNA are defined in this dissertation. The roaA protein product and the protein product of a potential maturase, mat1, have also been identified. Identification of these two protein products lays the groundwork for future studies regarding the role of these two proteins in chloroplast RNA metabolism.
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CESENA, DANIELE. "The RNA processing proteins Xrn1 and Rrp6 regulate DNA damage checkpoint activation and telomere metabolism." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/158272.
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L’instabilità genomica è una delle caratteristiche principali delle cellule tumorali e può essere causata da difetti nella riparazione del DNA, dal mancato arresto del ciclo cellulare e dalla perdita della protezione telomerica all’estremità dei cromosomi, che porta alla degradazione e alla fusione delle estremità. Tra i vari tipi di danno al DNA, le rotture della doppia elica del DNA (Double-Strand Break o DSB) rappresentano una delle lesioni più pericolose, poiché possono causare mutazioni o riarrangiamenti cromosomici. In presenza di DSBs, le cellule eucariotiche attivano un checkpoint, dipendente dalle protein chinasi Tel1/ATM e Mec1/ATR, che arresta il ciclo cellulare finché il danno non è stato riparato. Mec1/ATR è attivata dal DNA a singolo filamento (ssDNA) ricoperto da RPA che si forma dopo il processamento nucleolitico (resection) delle estremità del DSB.
Una simile risposta è attivata anche quando le estremità naturali dei cromosomi eucariotici perdono la loro protezione, generando delle estremità simili ad un DSB che vengono riconosciute dal checkpoint e dai meccanismi di riparazione. Questa protezione è fornita da complessi nucleoproteici specializzati, chiamati telomeri. Il DNA telomerico è costituito da sequenze ripetute ricche in G che terminano con una coda a singolo filamento sporgente in 3’ (detta coda G), la quale è importante per l’estensione dei telomeri ad opera della telomerasi. Diverse proteine, tra cui il complesso CST, sono necessarie al mantenimento della struttura e della lunghezza dei telomeri sia in lievito che nei mammiferi.
Recenti dati sperimentali indicano che i fattori che processano l’RNA hanno un ruolo fondamentale nella stabilità del genoma e nel metabolismo telomerico, anche se il meccanismo è ancora poco compreso. In questa tesi abbiamo dimostrato che in Saccharomyces cerevisiae le proteine che degradano l’RNA Xrn1, Rrp6 e Trf4 promuovono l’attivazione di Mec1/ATR facilitando la formazione di DNA a singolo filamento ricoperto da RPA ai DSB. Inoltre, Xrn1 e Rrp6 sono necessarie per attivare il checkpoint anche ai telomeri deprotetti a causa del malfunzionamento di Cdc13, una delle subunità del complesso CST coinvolto nella protezione dei telomeri. Xrn1 facilita la formazione di DNA a singolo filamento ai DSBs promuovendo il caricamento del complesso MRX, mentre come svolga questa funzione ai telomeri deprotetti rimane ancora da chiarire. Al contrario, la generazione di ssDNA ai DSBs non è influenzata dalla mancanza di Rrp6 o Trf4, ma la loro assenza ostacola il reclutamento di RPA e quindi di Mec1 al sito di danno. L’inattivazione di Rrp6 e Trf4 non influenza né l’associazione di Rad51/Rad52 ai DSB né la riparazione della rottura attraverso la ricombinazione omologa (Homologous Recombination o HR), suggerendo che la piena attivazione di Mec1 richieda più DNA a singolo filamento ricoperto da RPA di quanto ne sia richiesto per la riparazione attraverso la ricombinazione omologa. Infine, Xrn1, regolando negativamente il trascritto di RIF1, è coinvolto nel mantenimento della lunghezza dei telomeri promuovendo l’associazione di Cdc13 indipendentemente dalla formazione di DNA a singolo filamento.
In conclusione, i nostri risultati forniscono un nuovo collegamento tra il processamento dell’RNA e il mantenimento della stabilità del genoma.Genome instability is one of the most pervasive characteristics of cancer cells. It can be due to DNA repair defects, failure to arrest the cell cycle and loss of telomere-end protection that lead to end-to-end fusion and degradation. Among the many types of DNA damage, the DNA Double Strand Break (DSB) is one of the most severe, because it can cause mutations and chromosomal rearrangements. Eukaryotic cells respond to DSBs by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR, in order to arrest the cell cycle until DSBs are repaired. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA) that arises upon nucleolytic degradation (resection) of the DSB. A similar checkpoint response is triggered when the natural ends of eukaryotic chromosomes lose their protection, resembling and being recognized as DSBs. This protection is provided by specialized nucleoprotein complexes called telomeres. Telomeric DNA consists of repetitive G-rich sequences that terminate with a 3’-ended single-stranded overhang (G-tail), which is important for telomere extension by telomerase. Several proteins, including the CST complex, are necessary to maintain telomere structure and length in both yeast and mammals. Emerging evidences indicate that RNA processing proteins play critical, yet poorly understood, roles in genomic stability and telomere metabolism. We provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 facilitate activation of Mec1/ATR by promoting the generation of RPA-coated ssDNA at intrachromosomal DSBs. Xrn1 and Rrp6 are also required to activate a Mec1/ATR-dependent checkpoint at uncapped telomeres due to loss of the CST component Cdc13. Xrn1 promotes checkpoint activation by facilitating the generation of ssDNA at both DSBs and uncapped telomeres. Xrn1 exerts this function at DSBs by promoting the loading of the MRX complex, whereas how it does at uncapped telomeres remains to be determined. By contrast, DSB resection is not affected by the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Finally, we demonstrate that Xrn1 maintains telomere length by promoting the association of Cdc13 to telomeres independently of ssDNA generation and exerts this function by downregulating the RIF1 transcript. Our results provide novel links between RNA processing and genome stability.
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Liang, Chao. "Aptamer-functionalized lipid nanoparticles targeting osteoblasts as a novel RNA Interference-based bone anabolic strategy." HKBU Institutional Repository, 2016. https://repository.hkbu.edu.hk/etd_oa/325.
Full textAbstract:
Osteoporosis remain major clinical challenges. RNA interference (RNAi) provides a promising approach for promoting osteoblastic bone formation to settle the challenges. However, the major bottleneck for translating RNAi with efficacy and safety to clinical bone anabolic strategy is lack of osteoblast-specific delivery systems for osteogenic siRNAs. Previously, we developed a targeting system involving DOTAP-based cationic liposomes attached to oligopeptides (AspSerSer)6, (also known as (DSS)6), which had good affinity for bone formation surface. Using this system, osteogenic Pleckstrin Homology Domain Containing, Family O Member 1 (Plekho1) siRNA could be specifically delivered to bone formation surface at tissue level and promoted bone formation in osteopenic rodents. However, concerns still exist regarding indirect osteoblast-specific delivery, detrimental retention in hepatocytes, mononuclear phagocyte system (MPS)-induced dose reduction and inefficient nanoparticle extravasation. Aptamers, selected by cell-based Systematic evolution of ligands by exponential enrichment (cell-SELEX), are single-stranded DNA (ssDNA) or RNA which binds to target cells specifically by distinct tertiary structures. By performing positive selection with osteoblasts and negative selection with hepatocytes and peripheral blood mononuclear cells (PBMCs), we aimed to screen an aptamer that could achieve direct osteoblast-specific delivery and minimal hepatocyte and PBMCs accumulation of Plekho1 siRNAs. In addition, lipid nanoparticles (LNPs) have been widely used as nanomaterials encapsulating siRNA due to their small particle size below 90 nm. Polyethylene glycol¡(PEG) as the mostly used hydrophilic polymer, could efficiently prevent LNPs from MPS uptake. So, LNPs with PEG shielding could serve as siRNA carriers to realize efficient extravasation from fenestrated capillaries to osteoblasts and help reduce MPS uptake of the siRNAs. Recently, we screened an aptamer (CH6) by cell-SELEX specifically targeting both rat and human osteoblasts and developed the aptamer-functionalized LNPs encapsulating osteogenic Plekho1 siRNA, i.e., CH6-LNPs-siRNA. Our results demonstrated that CH6-LNPs-siRNA had an average particle size below 90 nm and no significant cytotoxicity in vitro. CH6 aptamer facilitated osteoblast-selective uptake of Plekho1 siRNA and gene silencing in vitro. In this study, we further found that CH6 aptamer facilitated the bone-specific distribution of siRNA by biophotonic imaging and quantitative analysis. Immunohistochemistry results showed that CH6 achieved in vivo osteoblast-specific delivery of Plekho1 siRNA. Dose-response experiment indicated that CH6-LNPs-siRNA achieved almost 80% gene knockdown at the siRNA dose of 1.0 mg/kg and maintained 12 days for over 50% gene silencing. microCT, bone histomorphometry and mechanical testing confirmed that CH6 facilitated bone formation, leading to improved bone micro-architecture, increased bone mass and enhanced mechanical properties in osteoporotic rodents. Furthermore, CH6-LNPs-siRNA achieved better bone anabolic action when compared to the previously developed (AspSerSer)6-liposome-siRNA. There was no obvious toxicity in rats injected with CH6-LNPs-siRNA. All these results indicated that osteoblast-specific aptamer-functionalized LNPs could act as a novel RNAi-based bone anabolic strategy and advance selectivity of targeted delivery for osteogenic siRNAs from tissue level toward cellular level. In addition, the generation of ssDNA from double-stranded PCR products is an essential step in selection of aptamers in SELEX. We found that the size separation derived from unequal primers with chemical modification could be a satisfactory alternative to the classic magnetic separation.
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Beick, Susanne. "Funktion und Evolution chloroplastidärer PPR-Proteine." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16343.
Full textAbstract:
PPR-Proteine bilden die größte Familie von RNA-Bindeproteinen in Pflanzen und sie werden fast ausschließlich in die Mitochondrien oder Plastiden importiert, wo sie eine wesentliche Rolle im RNA-Metabolismus spielen (Lurin et al., 2004). Doch die Funktionsweise der Proteine ist noch weitgehend unbekannt. In dieser Arbeit wurde das plastidäre PPR-Protein PPR5 in Zea mays funktionell charakterisiert, dessen Ortholog in Arabidopsis thaliana essentiell für die Embryogenese ist (Cushing et al., 2005). Mittels PPR5-Immunopräzipitation und einer Analyse der kopräzipitierten RNA konnte in vivo eine spezifische Assoziation mit der ungespleißten tRNA-Glycin (UCC) nachgewiesen werden. Analysen von ppr5-Mais-Mutanten offenbarten einen Stabilitätsverlust dieser RNA. Es wurde gefolgert, dass PPR5 das Transkript vor einem endonukleolytischen Abbau schützt. Die weiteren Projekte der Arbeit widmeten sich der Evolution der Familie. Um Erkenntnisse zur Funktion und Spezifität nahe verwandter PPR-Proteine zu erhalten, wurden die drei nächsten Verwandten von PPR5 identifiziert und Mais-Mutanten isoliert. Weiterhin wurde PPR54 untersucht. Es konnte gezeigt werden, dass PPR54 in Mais – wie in Arabidopsis (Tillich, nicht publiziert) – für das Spleißen des ndhA-Introns benötigt wird. Damit wurden erstmalig orthologe PPR-Proteine in einer Mono- und einer Dikotylen funktionell analysiert. Die vorgelegten Analysen mündeten in drei allgemeingültigen Schlussfolgerungen zur Funktion der PPR-Proteine. 1) Plastidäre PPR-Proteine, die in Dikotylen wie Arabidopsis für die Embryogenese notwendig sind, üben eine Funktion in der plastidären Translation aus. 2) Die vorgeschlagene Funktionsweise von PPR5 erfordert nicht die Rekrutierung anderer, katalytisch aktiver Proteine, sondern ihr liegt ein passiver, auf der Bindung einer RNA beruhender Mechanismus zugrunde. 3) Die Funktion orthologer PPR-Proteine ist in Mono- und Dikotylen konserviert, wie am Beispiel von PPR54 experimentell nachgewiesen wurde.PPR proteins are the largest family of RNA binding proteins in plants and the vast majority of them is localized to mitochondria or chloroplasts, where they are major players in the RNA metabolism of defined transcripts (Lurin et al., 2004). However, the mechanistic function of these proteins is still not clear. In this study, the plastid PPR protein PPR5, whose ortholog in Arabidopsis thaliana is embryo-essential (Cushing et al., 2005), was functionally characterized in Zea mays. By PPR5 immunoprecipitation and analyses of the coimmunoprecipitated RNA, a specific association to the unspliced tRNA glycine (UCC) was shown in vivo. The analysis of ppr5 maize mutants demonstrated a loss of stability of the tRNA precursor in mutants. It was concluded that the interaction with PPR5 protects the unspliced tRNA from endonucleolytic decay. In addition, close relatives of PPR5 were identified in maize (PPR2, PPR50, and PPR51) by phylogenetic means and maize mutants were isolated. A future characterization of four paralogous PPR proteins might answer whether closely related PPR proteins have similar functions or RNA targets. The analysis of PPR54 in maize demonstrated that PPR5 is needed for the splicing of the ndhA intron in maize as it is in Arabidopsis (Tillich, not published). Three important conclusions concerning the function of PPR proteins in general were drawn from the studies of chosen PPR proteins presented here. First, plastid PPR proteins that are essential in embryo development in eudicots like Arabidopsis should be necessary for plastid translation in most cases. Second, the characterization of PPR5 revealed a possibly ancient functional mechanism of PPR proteins which does not invoke the recruitment of additional catalytic factors but relies on the passive binding of RNA elements. Last, the conservation of function of orthologous PPR proteins in monocots and eudicots, which was shown in the case of PPR54, was demonstrated experimentally for the first time.
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Silva, Cátia Cláudia Bárria da. "The role of ribonuclease R in bacterial adaptation to cold shock." Master's thesis, Faculdade de Ciências Médicas, 2011. http://hdl.handle.net/10362/6634.
Full textAbstract:
RESUMO:Os microrganismos reagem à súbita descida de temperatura através de uma
resposta adaptativa específica que assegura a sua sobrevivência em condições desfavoráveis. Esta adaptação inclui alterações na composição da membrana, na
maquinaria de tradução e transcrição. A resposta ao choque térmico pelo frio induz uma repressão da transcrição. No entanto, a descida de temperatura induz a produção de um
grupo de proteínas específicas que ajudam a ajustar/re-ajustar o metabolismo celular às
novas condições ambientais. Em E. coli o processo de adaptação demora apenas quatro
horas, no qual um grupo de proteínas específicas são induzidas. Depois desde período recomeça lentamente a produção de proteínas.A ribonuclease R, uma das proteínas induzidas durante o choque térmico pelo frio, é uma das principais ribonucleases em E. coli envolvidas na degradação do RNA.
É uma exoribonuclease que degrada RNA de cadeia dupla, possui funções importantes
na maturação e “turnover” do RNA, libertação de ribossomas e controlo de qualidade de
proteínas e RNAs. O nível celular desta enzima aumenta até dez vezes após exposição
ao frio e estabiliza em células na fase estacionária. A capacidade de degradar RNA de
dupla cadeia é importante a baixas temperaturas quando as estruturas de RNA estão mais estáveis. No entanto, este mecanismo é desconhecido.
Embora a resposta específica ao “cold shock” tenha sido descoberta há mais de
duas décadas e o número de proteínas envolvidas sugerirem que esta adaptação é rápida e simples, continuamos longe de compreender este processo.
No nosso trabalho pretendemos descobrir proteínas que interactuem com a
RNase R em condições ambientais diferentes através do método “TAP-tag” e
espectrometria de massa. A informação obtida pode ser utilizada para deduzir algumas
das novas funções da RNase R durante a adaptação bacteriana ao frio e durante a fase
estacionária. Mais importante ainda, RNase R poderá ser recrutada para um complexo
de proteínas de elevado peso molecular durante o “cold-shock”.------------ABSTRACT:Microorganisms react to the rapid temperature downshift with a specific adaptative response that ensures their survival in unfavorable conditions. Adaptation includes changes in membrane composition, in translation and transcription machinery.
Cold shock response leads to overall repression of translation. However, temperature downshift induces production of a set of specific proteins that help to tune cell metabolism and readjust it to the new environmental conditions. For Escherichia coli the adaptation process takes only about four hours with a relatively small set of
specifically induced proteins involved. After this time, protein production resumes,
although at a slower rate.
One of the cold inducible proteins is RNase R, one of the main E. coli ribonucleases involved in RNA degradation. RNase R is an exoribonuclease that digest double stranded RNA, serves important functions in RNA maturation and turnover, release of stalled ribosomes by trans-translation, and RNA and protein quality control. The level of this enzyme increases about ten-fold after cold induction, and it is also stabilised in cells growing in stationary phase. The RNase R ability to digest structured RNA is important at low temperatures where RNA structures are stabilized but the exact
role of this mechanism remains unclear.
Although specific bacterial cold shock response was discovered over two
decades ago and the number of proteins involved suggests that this adaptation is fast and simple, we are still far from understanding this process.
In our work we aimed to discover the proteins interacting with RNase R in
different environmental conditions using TAP tag method and mass spectrometry analysis. The information obtained can be used to deduce some of the new functions of
RNase R during adaptation of bacteria to cold and in stationary growth phase. Most
importantly RNase R can be recruited into a high molecular mass complex of protein in
cold shock.
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Borisova, Marina E. [Verfasser]. "p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage / Marina E. Borisova." Mainz : Universitätsbibliothek Mainz, 2019. http://d-nb.info/1178732967/34.
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Makwana, Kuldeep Makwana. "CIRCADIAN MECHANISMS OF CALORIE RESTRICTION IN DELAYING AGING." Cleveland State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=csu1543844626233009.
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Gandhi, Minakshi [Verfasser], and Peter [Akademischer Betreuer] Angel. "Role of long non-coding RNA lincNMR in nucleotide metabolism in cancer / Minakshi Gandhi ; Betreuer: Peter Angel." Heidelberg : Universitätsbibliothek Heidelberg, 2019. http://d-nb.info/1195143826/34.
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Laberge, MacDonald Tammy. "Molecular Aspects of Nitrogen Metabolism in Fishes." Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/668.
Full textAbstract:
Molecular aspects of nitrogen metabolism in vertebrates is an interesting area of physiology and evolution to explore due to the different ways in which animals excrete nitrogenous waste as they transition from an aquatic to a terrestrial lifestyle. Two main products of nitrogen metabolism in fishes are ammonia and urea. Ammonia is produced during protein catabolism and build up of ammonia is toxic. Some aquatic vertebrates convert ammonia into a less toxic compound urea via de novo synthesis through the ornithine-urea cycle (O-UC). Five enzymes are involved in the O-UC: carbamoyl phosphate synthetase (CPS), ornithine carbamoyl transferase (OCT), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (ARG). An accessory enzyme, glutamine synthetase (GS) also participates in the "fish-type" O-UC. Teleosts excrete ammonia passively over their gills into the aquatic environment. The teleost, Opsanus beta, has been shown to increase urea production after 48 hours of crowding. This thesis explored how crowding stress affected nitrogen metabolite levels of ammonia and urea and O-UC gene expression and enzyme activity in O. beta. Lungfishes while in an aquatic environment avoid ammonia toxicity by releasing excess ammonia across their gills, but when stranded on land they produce urea through the O-UC. Urea production via the O-UC has a metabolic cost of at least four ATP molecules. This thesis explored the response of a lungfish, Protopterus annectens, to six days of aerial exposure and re-immersion conditions by measuring concentrations of O-UC mRNA expression and enzyme activity and nitrogen metabolites ammonia and urea. CPS acts as the entry point to the O-UC and based on enzymatic studies, most aquatic vertebrates utilize one isoform of this enzyme (CPSIII) while terrestrial vertebrates utilize a different isoform of this enzyme (CPSI). Lungfishes are a particularly interesting group of air-breathing fishes, not only because of their link to the origins of tetrapods, but also because CPS I may have originated within this group. Both CPS III and CPS I have been enzymatically described within this group. This thesis uses phylogenetics to investigate how CPS nucleotide sequences in lungfishes evolved compared to other vertebrates.
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Pfennig, Juliane [Verfasser], Tomas [Akademischer Betreuer] Pieler, and Michael [Akademischer Betreuer] Kessel. "XDazl function in RNA metabolism in Xenopus laevis / Juliane Pfennig. Betreuer: Tomas Pieler. Gutachter: Tomas Pieler ; Michael Kessel." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/1079384626/34.
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Stirling, Susan Renee, and n/a. "The Roles of RasGAP SH3 Domain Binding Proteins (G3BPs) in RNA Metabolism, the Cellular Stress Response and Tumorigenesis." Griffith University. School of Biomolecular and Biomedical Science, 2006. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20070705.175459.
Full textAbstract:
G3BP1 and G3BP2 are members of a highly conserved family of multi-functional RNA binding proteins, which appear to co-ordinate signal transduction and post-transcriptional gene regulation. Both proteins are over-expressed in cancer, and G3BP1 promotes cell proliferation and survival. Aberrant expression of various RNA binding proteins is common in cancer, and several of these proteins influence tumorigenesis. Therefore, detailed examination of RNA binding proteins, such as G3BPs, may provide insights into the post-transcriptional mechanisms underlying tumorigenesis. Tumours arise as a consequence of genetic mutation or alteration, which often result from stress-induced DNA damage. Cancer progression is facilitated by various epigenetic stress adaptation mechanisms. Stressful stimuli induce transitory translational shut-off, mediated by phosphorylation of eukaryotic initiation factor alpha;(eIF2alpha;). This phosphorylation event leads to formation of discrete cytoplasmic foci known as stress granules (SGs), which are translationally-silent sites of mRNA sorting. It was initially thought that an RNA-binding protein, T-cell internal antigen 1 (TIA-1), was instrumental in both the formation and functioning of SGs, because over-expression of TIA-1 induces spontaneous SGs and concomitantly causes a decrease in reporter gene expression. It is now clear that SG content can change depending on the type of stress, and that various proteins, including G3BP1, can induce spontaneous SGs. In vitro evidence previously implicated both G3BP1 and G3BP2 as endoribonucleases, so it was suggested that G3BPs act to target mRNA for decay at the SG. This project sought to further investigate this proposal, and in this way gain insight into the specific function of G3BPs in post-transcriptional regulation during tumorigenesis. Characterisation of G3BP1 and G3BP2 expression and localisation patterns in human cells and cancer was necessary before functional analyses in human cell systems could be undertaken. Both proteins were found to be over-expressed in breast cancer, irrespective of cancer stage or grade. G3BP1 and G3BP2 were also expressed in all human cell lines tested, despite previously observed tissue-specific expression. These results support the notion that G3BP expression is switched on in parallel with cell proliferation, and as such, may influence tumorigenesis. The results of further analyses suggested that the diverse functions attributed to G3BP1 and G3BP2 may be facilitated by isoform-specific expression, various post-translational modifications and sub-cellular localisation. Despite the absence of a canonical endoribonuclease domain, it was previously reported that site-specific phosphorylation of G3BP1 enables the protein to degrade a synthetic c-myc RNA substrate in vitro. This finding implicated G3BP in the specific regulation of a proto-oncogene. Tailored reporter assays were thus designed in order to address the in vivo consequences of G3BP's putative endoribonuclease activity. Contrary to expectations, all G3BP family members increased or maintained the expression of a range of reporters, at both the mRNA and protein level, irrespective of the presence of any particular cis-acting element, coding sequence or promoter. These results support the emerging notion that G3BPs positively affect the expression of at least some of their target mRNAs, and may also indirectly promote transcription. In contrast to the theory that G3BPs degrade proto-oncogenic mRNA/s, these findings are consistent with a role for G3BP in promoting cell proliferation and survival. Further analyses showed that G3BP1 and G3BP2 simultaneously increased reporter gene expression and induced SG formation. These findings highlighted the fact that SGs are dynamic sorting stations for mRNAs, and not merely sites of stalled translation. This result also supports the notion that a variety of proteins may be recruited to the SG to facilitate a multitude of mRNA fates. Although the precise role of the SG in stress adapation is not known, it is clear that an appropriate integrated stress response (ISR) is required for cells to survive in sub-optimal conditions. It was found that specific G3BP1 knockdown inhibited SG formation and cell survival, and this appeared to occur downstream of eIF2alpha; phosphorylation. The phosphorylation of eIFalpha; is the only factor known to be necessary for SG formation and cell survival. This data is the first to implicate SG formation itself, downstream of eIF2alpha; phosphorylation, in the survival phase of the ISR. The results also suggest that G3BP1 plays a pivotal role in the post-transcriptional mechanisms underlying stress adaptation. To facilitate future analysis of G3BP roles in the regulation of specific transcripts and in SG biology, a pilot study to identify G3BP RNA ligands was undertaken. Immunoprecipitation of epitope-tagged G3BP1 from stable cell lines facilitated purification and isolation of RNA in association with G3BP1. Specific RNA transcripts were subsequently detected and identified by microarray. Many genes were enriched in the G3BP1 immunoprecipitate. Transcript enrichment in the control immunoprecipitate was comparatively weak and seemingly random, suggesting that several replicates will enable generation of a reliable target list. This work forms a promising basis for further investigations into G3BP functionality, and also provides a platform for broader and more large-scale analyses of the mechanisms of post-transcriptional gene regulation. The work presented in this thesis addressed the potential post-transcriptional mechanisms by which the G3BP family of proteins mediate cell proliferation and survival. Both G3BP1 and G3BP2 were shown to be over-expressed in tumours and each appeared to promote reporter gene expression. G3BP1 was also found to play a pivotal role in stress adaptation. A technique to identify novel RNA ligands was assessed, and it was found that G3BP1 may interact with various mRNA transcripts. It is hypothesised that the G3BP family of proteins, and in particular G3BP1, function to determine the fate of specific RNAs in response to cellular stress and other stimuli. In this way, G3BP proteins may facilitate appropriate responses to extra-cellular stimuli which allow for cell proliferation and survival.
45
Stirling, Susan Renee. "The Roles of RasGAP SH3 Domain Binding Proteins (G3BPs) in RNA Metabolism, the Cellular Stress Response and Tumorigenesis." Thesis, Griffith University, 2006. http://hdl.handle.net/10072/366889.
Full textAbstract:
G3BP1 and G3BP2 are members of a highly conserved family of multi-functional RNA binding proteins, which appear to co-ordinate signal transduction and post-transcriptional gene regulation. Both proteins are over-expressed in cancer, and G3BP1 promotes cell proliferation and survival. Aberrant expression of various RNA binding proteins is common in cancer, and several of these proteins influence tumorigenesis. Therefore, detailed examination of RNA binding proteins, such as G3BPs, may provide insights into the post-transcriptional mechanisms underlying tumorigenesis. Tumours arise as a consequence of genetic mutation or alteration, which often result from stress-induced DNA damage. Cancer progression is facilitated by various epigenetic stress adaptation mechanisms. Stressful stimuli induce transitory translational shut-off, mediated by phosphorylation of eukaryotic initiation factor alpha;(eIF2alpha;). This phosphorylation event leads to formation of discrete cytoplasmic foci known as stress granules (SGs), which are translationally-silent sites of mRNA sorting. It was initially thought that an RNA-binding protein, T-cell internal antigen 1 (TIA-1), was instrumental in both the formation and functioning of SGs, because over-expression of TIA-1 induces spontaneous SGs and concomitantly causes a decrease in reporter gene expression. It is now clear that SG content can change depending on the type of stress, and that various proteins, including G3BP1, can induce spontaneous SGs. In vitro evidence previously implicated both G3BP1 and G3BP2 as endoribonucleases, so it was suggested that G3BPs act to target mRNA for decay at the SG. This project sought to further investigate this proposal, and in this way gain insight into the specific function of G3BPs in post-transcriptional regulation during tumorigenesis. Characterisation of G3BP1 and G3BP2 expression and localisation patterns in human cells and cancer was necessary before functional analyses in human cell systems could be undertaken. Both proteins were found to be over-expressed in breast cancer, irrespective of cancer stage or grade. G3BP1 and G3BP2 were also expressed in all human cell lines tested, despite previously observed tissue-specific expression. These results support the notion that G3BP expression is switched on in parallel with cell proliferation, and as such, may influence tumorigenesis. The results of further analyses suggested that the diverse functions attributed to G3BP1 and G3BP2 may be facilitated by isoform-specific expression, various post-translational modifications and sub-cellular localisation. Despite the absence of a canonical endoribonuclease domain, it was previously reported that site-specific phosphorylation of G3BP1 enables the protein to degrade a synthetic c-myc RNA substrate in vitro. This finding implicated G3BP in the specific regulation of a proto-oncogene. Tailored reporter assays were thus designed in order to address the in vivo consequences of G3BP's putative endoribonuclease activity. Contrary to expectations, all G3BP family members increased or maintained the expression of a range of reporters, at both the mRNA and protein level, irrespective of the presence of any particular cis-acting element, coding sequence or promoter. These results support the emerging notion that G3BPs positively affect the expression of at least some of their target mRNAs, and may also indirectly promote transcription. In contrast to the theory that G3BPs degrade proto-oncogenic mRNA/s, these findings are consistent with a role for G3BP in promoting cell proliferation and survival. Further analyses showed that G3BP1 and G3BP2 simultaneously increased reporter gene expression and induced SG formation. These findings highlighted the fact that SGs are dynamic sorting stations for mRNAs, and not merely sites of stalled translation. This result also supports the notion that a variety of proteins may be recruited to the SG to facilitate a multitude of mRNA fates. Although the precise role of the SG in stress adapation is not known, it is clear that an appropriate integrated stress response (ISR) is required for cells to survive in sub-optimal conditions. It was found that specific G3BP1 knockdown inhibited SG formation and cell survival, and this appeared to occur downstream of eIF2alpha; phosphorylation. The phosphorylation of eIFalpha; is the only factor known to be necessary for SG formation and cell survival. This data is the first to implicate SG formation itself, downstream of eIF2alpha; phosphorylation, in the survival phase of the ISR. The results also suggest that G3BP1 plays a pivotal role in the post-transcriptional mechanisms underlying stress adaptation. To facilitate future analysis of G3BP roles in the regulation of specific transcripts and in SG biology, a pilot study to identify G3BP RNA ligands was undertaken. Immunoprecipitation of epitope-tagged G3BP1 from stable cell lines facilitated purification and isolation of RNA in association with G3BP1. Specific RNA transcripts were subsequently detected and identified by microarray. Many genes were enriched in the G3BP1 immunoprecipitate. Transcript enrichment in the control immunoprecipitate was comparatively weak and seemingly random, suggesting that several replicates will enable generation of a reliable target list. This work forms a promising basis for further investigations into G3BP functionality, and also provides a platform for broader and more large-scale analyses of the mechanisms of post-transcriptional gene regulation. The work presented in this thesis addressed the potential post-transcriptional mechanisms by which the G3BP family of proteins mediate cell proliferation and survival. Both G3BP1 and G3BP2 were shown to be over-expressed in tumours and each appeared to promote reporter gene expression. G3BP1 was also found to play a pivotal role in stress adaptation. A technique to identify novel RNA ligands was assessed, and it was found that G3BP1 may interact with various mRNA transcripts. It is hypothesised that the G3BP family of proteins, and in particular G3BP1, function to determine the fate of specific RNAs in response to cellular stress and other stimuli. In this way, G3BP proteins may facilitate appropriate responses to extra-cellular stimuli which allow for cell proliferation and survival.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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Boone, Lindsey R. "Thyroid Hormone Regulation of Cholesterol Metabolism." [Tampa, Fla] : University of South Florida, 2009. http://purl.fcla.edu/usf/dc/et/SFE0003089.
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Zanardi, Geila Maria. "Detecção da expressão gênica de enzimas da gliconeogênese na placenta de fêmeas bovinas." Universidade do Estado de Santa Catarina, 2008. http://tede.udesc.br/handle/handle/810.
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A singular feature during mammalian development is the provision of nutrients from the maternal system to the conceptus through the placenta. To the fetus, the placenta consists of a multi-functional organ with an intense metabolism, characterized in an adult as separated functions in multiple organs. This study aimed to verify the presence of key enzymes in the bovine placenta responsible for glyconeogenesis, which is a metabolic pathway used for Dglucose synthesis from non-carbohydrate compounds. Such pathway is common to the liver metabolism used to maintain glucose homeostasis in plasma, especially during fasting or postprandial absorption. No studies on the occurrence of glyconeogenesis in the bovine placenta have been reported thus far. In this study, a qualitative gene expression analysis was carried out for three key glyconeogenic enzymes: fructose-1,6-bisphosphatase (F1,6B), glucose-6- phosphatase (G6-P) e phosphoenolpyruvate carboxikinase (PEP-CK) in the bovine placental tissue at three distinct gestation periods. For that, placentome fragments from 5 pregnant females from each period were used for the detection of transcripts for the three enzymes above by reverse transcription-polymerase chain reaction (RT-PCR). Amplified PCR products obtained for each enzyme from the placenta and adult liver (positive control) were analyzed in 1% agarose gel, followed by purification, DNA cloning e sequencing for confirmation of amplification specificity. The RT-PCR analysis followed by DNA sequencing revealed the presence of all three glyconeogenic enzymes in the bovine placenta from all three gestational periods. Based on our results, the glyconeogenic pathway may occur in the placental tissue in cattle during pregnancy. However, more studies are still needed for the confirmation of the occurrence and temporal importance of the glyconeogenesis in the bovine placenta
Uma característica singular do desenvolvimento dos mamíferos é a provisão de nutrientes do organismo materno por intermédio da placenta. Para o feto, a placenta consiste na combinação, em apenas um órgão, de muitas atividades funcionais, que no adulto são separadas e, além disso, possui um intenso metabolismo. Este trabalho teve como objetivo verificar a expressão de enzimas chave na placenta bovina para a gliconeogênese, que consiste em uma via metabólica capaz de sintetizar D-glicose, utilizando-se compostos que não são carboidratos. Essa via se processa no fígado e é utilizada pelo organismo para manter os níveis glicêmicos normais em condições de jejum e de pós-absorsão. Atualmente não existem estudos sobre a gliconeogênese na placenta bovina. Neste estudo foi avaliada a expressão gênica na placenta bovina de três enzimas gliconeogênicas: frutose-1,6-bisfosfatase (F1,6B), glicose-6-fosfatase (G6-P) e fosfoenolpiruvato carboxiquinase (PEP-CK) nos três trimestres gestacionais. Para tanto, foram utilizados fragmentos de placentônios de 5 fêmeas bovinas para cada terço de gestação para a avaliação da expressão gênica das três enzimas gliconeogênicas após a transcripção reversa e reação em cadeia da polimerase (RT-PCR). Os produtos de PCR amplificados obtidos para cada enzima na placenta bem como para o fígado de animal adulto, que foi utilizado como controle positivo, foram analisados em gel de agarose a 1%, purificados, clonados e seqüenciados para a confirmação da especificidade de amplificação. A análise da expressão gênica através do RT-PCR e o sequenciamento demonstrou a presença de mRNA para as três enzimas gliconeogênicas na placenta bovina nos três períodos gestacionais. Baseado em nossos resultados, é possível que a rota gliconeogênica ocorra no tecido placentário de bovinos durante a gestação. Porém, ainda são necessários mais estudos para a confirmação da ocorrência e importância temporal da gliconeogênese na placenta bovina
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Presnyak, Vladimir. "Effects of Codon Usage on mRNA Translation and Decay." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1427387336.
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Gkatza, Nikoletta A. "RNA modifications and processing in cell homeostasis and in response to oxidative stress." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277276.
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RNA modifications and processing events are important modulators of global gene expression. Genomic mutations in the RNA methylase NSun2 and the alternative splicing factor Srsf2 are linked to neurological disorders and cancer in humans, respectively. NSun2 methylates cytosine-5 in most tRNAs and, to a lesser extent, other ncRNAs and mRNAs. Srsf2 is a critical component of the spliceosome and interacts with abundant ncRNAs that are methylated by NSun2. However, how precisely these processes effect homeostasis is largely unexplored. Therefore, the main aims of my PhD were (1) to dissect the molecular mechanisms of NSun2-mediated RNA methylation pathways that regulate cell survival under normal conditions and in response to oxidative stress, and (2) to investigate the importance of Srsf2 in stem cells using skin as a model system. In the context of RNA modifications, firstly I described how NSun2-expressing cells enrich for transcripts related to enhanced cell survival. Subsequently, by metabolically profiling wildtype and patient-derived dermal fibroblasts carrying loss-of-function mutations in the NSUN2 gene, I showed that the absence of NSun2 is synonymous to an energy-saving, low-translating and stressed cellular state. I further confirmed that lack of NSun2 was sufficient to instigate a cellular stress response, by monitoring BIRC5, a member of the inhibitor of apoptosis family. To further answer whether lack of NSun2 enhanced the susceptibility of patient cells to external stress stimuli, I next exposed them to oxidative stress and measured transcriptional and translational changes. I discovered that NSun2 is required to adapt global protein synthesis to the stress response, while NSun2-depleted cells failed to do so. This was concurrent with NSun2-depleted cells enriching for transcripts related to mRNA degradation and negative regulators of protein translation in response to stress. Generally, since loss of NSun2-driven methylation in tRNAs triggers their cleavage into small ncRNA fragments by angiogenin, I asked how angiogenin or tRNA-derived ncRNAs affect translation levels. In the presence of NSun2, angiogenin alone did not reduce global protein synthesis, yet tRNA fragmentation was required to modulate translation levels. Finally, to uncover how the lack of NSun2 influenced tRNA cleavage and methylation patterns in response to stress, I exposed wildtype and patient cells to sodium arsenite and measured the abundance of tRNA-derived fragments and occurrence of methylation events. With this I discovered unique tRNA fragmentation patterns and global RNA methylation profiles for wildtype and NSun2-depleted cells, that can account for the underlying molecular and phenotypical differences in response to stress. In the context of alternative splicing, and since the cellular functions of Srsf2 are largely unknown, I explored its role in cellular survival and differentiation. By conditionally deleting SRSF2 in two different stem cell populations of the mouse epidermis, I observed significant thickening of the epidermis, altered expression of cell proliferation and stem cell differentiation markers, and distorted hair follicle structures. Moreover, I demonstrated that lack of Srsf2 promotes skin regeneration following injury, thus strongly indicating that Srsf2 is required for normal skin development and regeneration after injury. In summary, my research suggests that NSun2-mediated RNA methylation pathways orchestrate transcriptional and translational programmes in response to external stress stimuli, and my studies are the first to show that the alternative splicing factor Srsf2 is required for stem cell differentiation in skin.
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Carvalho, Ana Elisa Teófilo Saturi de. "Metabolismo energético mitocondrial e cardiomiogênese para regeneração cardíaca." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/5/5131/tde-25102016-161531/.
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Apesar dos avanços dos últimos anos, a reposição de cardiomiócitos permanece como um dos maiores desafios da medicina regenerativa. A comprovação da existência de mecanismos endógenos de proliferação cardíaca nos impulsionou a buscar o entendimento dos eventos moleculares envolvidos na proliferação de cardiomiócitos na vida pós-natal. Neste trabalho foi testada a hipótese da influência do metabolismo energético mitocondrial na cardiomiogênese, e seu impacto na regeneração cardíaca. No primeiro momento, foi descrito pela primeira vez o modelo de ressecção apical cardíaca em ratos neonatos. Demonstrou-se que há um período restrito as primeiras 24 horas de vida em que o animal é capaz de regenerar o tecido cardíaco, formando novos cardiomiócitos e permitindo a manutenção da função cardíaca na vida adulta. Esta capacidade é perdida 7 dias após o nascimento, havendo apenas reparo com tecido fibroso e prejuízo à função cardíaca. De maneira interessante, os dados apontaram para hipoperfusão da região apical em ambos os animais ressectados. Isso possivelmente acarretou em dano mitocondrial na vida adulta, sem influenciar a função cardíaca. De maneira a investigar os eventos moleculares da regeneração cardíaca neonatal foi realizado o sequenciamento de RNA dos corações de ratos neonatos de 1 e 7 dias de vida, ressectados e sham, pela técnica de RNASeq, que apontou a relevância da idade nas diferenças de expressão de genes relacionados ao metabolismo, sendo que a intervenção da ressecção pouco influenciou o perfil de expressão gênica. Os resultados mostraram a troca de expressão de isoformas da via glicolítica com a maturação pós-natal, e a hiper-regulação da expressão de genes das vias da ?-oxidação, fosforilação oxidativa e ciclo do ácido tricarboxílico durante o mesmo período. Entretanto, os dados funcionais da atividade metabólica do tecido cardíaco e cultura de cardiomiócitos neonatais mostraram que tanto a glicólise anaeróbia quanto o consumo de oxigênio relacionado à oxidação mitocondrial estiveram elevados no neonato de 1 dia, e foram reduzidos com o desenvolvimento cardíaco. As elevadas taxas de consumo de oxigênio nas culturas de cardiomiócitos de 1 dia de vida foram relacionadas principalmente à produção de ATP. Esses cardiomiócitos foram capazes de proliferar em cultura na presença de soro como estimulador. Assim sendo, as análises de expressão gênica sozinhas pareceram ser indicadores parciais do estado funcional do metabolismo. A inibição não letal da fosforilação oxidativa evidenciou a importância do metabolismo mitocondrial na capacidade proliferativa dos cardiomiócitos na vida pós-natal. Os dados sugerem que o primeiro dia após o nascimento abrange uma alta demanda energética tanto para a diferenciação terminal quanto para a última fase robusta de proliferação de cardiomiócitos na vida pós-natal, e assim evidenciam a importância do metabolismo mitocondrial no processo regenerativoDespite advances in recent years, the replacement of cardiomyocytes remains one of the biggest challenges in regenerative medicine. The existence of endogenous mechanisms of cardiac proliferation prompted us to seek the understanding of molecular events involved in cardiomyocyte proliferation in postnatal life. In this study, we investigated the influence of mitochondrial energy metabolism in cardiomyogenesis, and its impact on cardiac regeneration. At first, it was described for the first time the model of heart apical resection in neonatal rats, where there is a limited period the first 24 hours of life that animal is able to regenerate cardiac tissue, forming new cardiomyocytes and allowing the maintenance cardiac function in adulthood. This ability is lost seven days after birth, when repair is basically by fibrotic tissue and consequent impairment for heart function. Interestingly, data showed hypoperfusion of the apical region in both resected animals, which possibly resulted in mitochondrial damage in adulthood without affecting heart function. In order to investigate the molecular events of neonatal cardiac regeneration was performed RNA sequencing of hearts from newborn rats with 1 and 7 days of life, resected and sham, which pointed out the importance of age in the different expression of genes related to metabolism, and the intervention of resection had little influence on this. The results showed exchange of expression of enzymes isoforms from glycolytic pathway and hyperregulation of genes from beta-oxidation, oxidative phosphorylation and tricarboxylic acid cycle pathways, during postnatal maturation. However, the functional data of the metabolic activity of cardiac tissue and culture of neonatal cardiomyocytes showed that both anaerobic glycolysis and oxygen consumption related to mitochondrial oxidation were higher in 1-day-old newborns, and were reduced with cardiac development. The high rates of oxygen consumption in 1-day-old cardiomyocytes were related mainly to ATP production. These 1-day-old cardiomyocytes were able to proliferate in culture by serum stimulation. Therefore, the analysis of gene expression alone appeared to be a partial indicator of functional state of metabolism. The non-lethal inhibition of oxidative phosphorylation highlighted the importance of mitochondrial metabolism in the proliferative capacity of cardiomyocytes in postnatal life. Data suggest that the first day after birth covers a high energy demand for both terminal differentiation of cardiac cells and last robust phase of cardiomyocyte proliferation in postnatal life, and show the importance of mitochondrial metabolism in the regenerative process