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Articles de revues sur le sujet "ARN Dependent ARN Polymerase"
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Blake, Timo, Anne Barnard, Stephen J. W. Busby et Jeffrey Green. « Transcription Activation by FNR : Evidence for a Functional Activating Region 2 ». Journal of Bacteriology 184, no 21 (1 novembre 2002) : 5855–61. http://dx.doi.org/10.1128/jb.184.21.5855-5861.2002.
Texte intégralRésumé :
ABSTRACT The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters. Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 [AR1 and AR3, respectively]) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription. Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2. Here we show that the substitution of two surface-exposed lysine residues (Lys49 and Lys50) of FNR impaired transcription from class II (FNR box centered at −41.5) but not class I (FNR box centered at −71.5) FNR-dependent promoters. The degree of impairment was greater when a negatively charged residue (Glu) replaced either Lys49 or Lys50 than when uncharged amino acid Ala was substituted. Oriented heterodimers were used to show that only the downstream subunit of the FNR dimer was affected by the Lys→Ala substitutions at a class II promoter. Site-directed mutagenesis of a negatively charged patch (162EEDE165) within the N-terminal domain of the RNAP α subunit that interacts with the positively charged AR2 of the cyclic AMP receptor protein suggested that Lys49 and Lys50 of FNR interact with this region of the α subunit of RNAP. Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR.
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Wetchapit, Pattarat, Usanee Tungsattayathitthan, Sutasinee Boonsopon, Nattaporn Tesavibul et Pitipol Choopong. « Acute Retinal Necrosis : A Review of Diagnosis and Management ». Siriraj Medical Journal 76, no 10 (1 octobre 2024) : 727–32. http://dx.doi.org/10.33192/smj.v76i10.268914.
Texte intégralRésumé :
Acute retinal necrosis (ARN) is a profound infection of the retina, marked by acute panuveitis, retinal periarteritis, and widespread necrotizing retinitis. The etiology of ARN involves human herpesviruses, such as herpes simplex virus (HSV) and varicella-zoster virus (VZV), which can lead to severe visual impairment or even blindness. A diagnosis of ARN is based on clinical characteristics and disease progression according to the standard diagnostic criteria established by the American Uveitis Society (AUS) in 1994. The polymerase chain reaction(PCR) of aqueous specimens can enable identification of the type of virus. Early initiation of antiviral medication is essential for treatment efficacy to stop lesion progression, accelerate the healing process, and prevent contralateral eye involvement. Ocular complications of ARN include atrophic retina, multiple retinal breaks, rhegmatogenous retinal detachment (RRD), tractional retinal detachment (TRD), optic atrophy, macular edema, epiretinal membrane (ERM), and retinal and optic disc neovascularization. This review summarizes the clinical features, diagnostic criteria, and recently recommended ARN management.
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Oe, Chiaki, Miki Hiraoka, Sachie Tanaka et Hiroshi Ohguro. « Acute Retinal Necrosis Associated with Epstein-Barr Virus in a Patient Undergoing Immunosuppressive Therapy ». Case Reports in Ophthalmology 7, no 1 (12 avril 2016) : 195–201. http://dx.doi.org/10.1159/000445372.
Texte intégralRésumé :
Acute retinal necrosis (ARN) is a rapidly progressive and severe retinitis resulting in a poor visual outcome. Infections caused by herpes viruses such as herpes simplex virus (HSV) types 1 and 2 or the varicella zoster virus (VZV) are known to be implicated in the development of ARN. In the present study, an 80-year-old female with ARN was examined. She had been affected with rheumatoid arthritis and had taken methotrexate for over 10 years. Her right eye showed clinical features of ARN, and her left eye showed mild retinitis. The genomic DNA in the aqueous humor and vitreous fluid from her right eye were analyzed by a comprehensive polymerase chain reaction (PCR) assay to screen infectious pathogens including viruses. The Epstein-Barr virus (EBV) was detected from both specimens, but neither HSV or VZV nor cytomegalovirus was detected. She underwent intraocular surgery following systemic corticosteroid and acyclovir applications. However, although the retinitis of her right eye was extinguished, the final visual outcome was blindness due to optic nerve atrophy. There are few reports indicating that EBV is associated with ARN development. The present findings suggest that EBV alone can be the causative agent of ARN.
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Chujo, Shinichiro, Hisashi Matsubara, Yoshitusgu Matsui, Kumiko Kato et Mineo Kondo. « Case of acute retinal necrosis with rapid progression to proliferative vitreoretinopathy : A case report ». Medicine 103, no 20 (17 mai 2024) : e38150. http://dx.doi.org/10.1097/md.0000000000038150.
Texte intégralRésumé :
Rationale: Acute retinal necrosis (ARN) was first reported in 1971 by Urayama et al as an acute uveitis accompanied by retinal arteritis and white retinal lesions in the peripheral retina that can progress to a rhegmatogenous retinal detachment (RRD). We have experienced a case of ARN that, unlike the common developmental course to an RRD associated with ARN, progressed to proliferative vitreoretinopathy (PVR) involving the entire retina in 2 days. The purpose of this report is to present our findings in the case of ARN with an atypical rapid time course. Patient concerns: The patient was a 56-year-old woman who was treated for uveitis of unknown origin by her primary care physician. She was referred to our hospital because of a worsening of the fundus findings. Diagnosis: Fundus examination in our hospital revealed vitreous opacities in the right eye, yellowish-white lesions extending around the retina, and some retinal hemorrhages. Because the retinal changes suggested ARN, we performed a polymerase chain reaction of the anterior atrial fluid and detected varicella-zoster virus. Then, the diagnosis of ARN was confirmed, and treatment was begun. At 1 month and a half after beginning the treatment, focal retinal traction was observed in the right fundus. Two days later, a circumferential PVR and a total retinal detachment were detected. Interventions: We then performed vitrectomy with an encircling buckle and a silicone oil tamponade. Outcomes: Our examination 6 months postoperatively showed that the retina was attached and the BCVA was 20/200. Lessons: Our findings of a case of ARN showed that the progression from a local vitreous traction to a full circumferential PVR can develop in 2 days.
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Yip, Siew Hoong, Ulrich Boehm, Allan E. Herbison et Rebecca E. Campbell. « Conditional Viral Tract Tracing Delineates the Projections of the Distinct Kisspeptin Neuron Populations to Gonadotropin-Releasing Hormone (GnRH) Neurons in the Mouse ». Endocrinology 156, no 7 (9 avril 2015) : 2582–94. http://dx.doi.org/10.1210/en.2015-1131.
Texte intégralRésumé :
Kisspeptin neurons play an essential role in the regulation of fertility through direct regulation of the GnRH neurons. However, the relative contributions of the two functionally distinct kisspeptin neuron subpopulations to this critical regulation are not fully understood. Here we analyzed the specific projection patterns of kisspeptin neurons originating from either the rostral periventricular nucleus of the third ventricle (RP3V) or the arcuate nucleus (ARN) using a cell-specific, viral-mediated tract-tracing approach. We stereotaxically injected a Cre-dependent recombinant adenovirus encoding farnesylated enhanced green fluorescent protein into the ARN or RP3V of adult male and female mice expressing Cre recombinase in kisspeptin neurons. Fibers from ARN kisspeptin neurons projected widely; however, we did not find any evidence for direct contact with GnRH neuron somata or proximal dendrites in either sex. In contrast, we identified RP3V kisspeptin fibers in close contact with GnRH neuron somata and dendrites in both sexes. Fibers originating from both the RP3V and ARN were observed in close contact with distal GnRH neuron processes in the ARN and in the lateral and internal aspects of the median eminence. Furthermore, GnRH nerve terminals were found in close contact with the proximal dendrites of ARN kisspeptin neurons in the ARN, and ARN kisspeptin fibers were found contacting RP3V kisspeptin neurons in both sexes. Together these data delineate selective zones of kisspeptin neuron inputs to GnRH neurons and demonstrate complex interconnections between the distinct kisspeptin populations and GnRH neurons.
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Liu, Xinhuai, et Allan Herbison. « Kisspeptin Regulation of Arcuate Neuron Excitability in Kisspeptin Receptor Knockout Mice ». Endocrinology 156, no 5 (10 mars 2015) : 1815–27. http://dx.doi.org/10.1210/en.2014-1845.
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The G protein-coupled receptor 54 (GPR54) is critical for kisspeptin to activate GnRH neurons to modulate fertility. However, the often mismatching distribution of kisspeptin and GPR54 in the brain suggests that kisspeptin may also act on other receptors. The arcuate nucleus (ARN) is one brain region with a very high density of kisspeptin fibers but only limited evidence for the expression of GPR54. Using acute brain slice electrophysiology in combination with Gpr54 knockout (GPR54KO) mouse models, we examined whether actions of kisspeptin in the ARN were dependent upon GPR54. Cell-attached recordings from unidentified ARN neurons in wild-type mice revealed that approximately one third of neurons were either excited or inhibited by kisspeptin in a dose-dependent manner. The responses of ARN neurons to kisspeptin were exactly the same in GPR54KO mice despite effects of kisspeptin on GnRH neurons being abolished. To evaluate whether kisspeptin may be acting through neuropeptide FF receptors, the effects of an agonist RFamide-related peptide 3 (RFRP-3) and antagonists RF9 and BIBP-3226 were evaluated. Both the excitatory and inhibitory effects of kisspeptin were mimicked by the agonist RFRP-3. RF9 itself activated ARN neurons and suppressed only the inhibitory actions of kisspeptin. BIBP-3226 suppressed kisspeptin actions in 50% of neurons. Whole-cell recordings in GPR54KO mice demonstrated that both kisspeptin and RFRP-3 acted directly on the same ARN neurons and activated the same ion channels. Together, these studies demonstrate that kisspeptin can act partly through neuropeptide FF receptors to modulate neuronal activity independent of GPR54 in the mouse brain.
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Keorochana, Narumon, Budsarat Suleesathira et Sritatath Vongkulsiri. « Pigmentary retinopathy and nodular granuloma associated with acute retinal necrosis from varicella zoster virus and human herpes virus type 6 : Case report ». Medicine 102, no 26 (30 juin 2023) : e33958. http://dx.doi.org/10.1097/md.0000000000033958.
Texte intégralRésumé :
Rationale: Acute retinal necrosis (ARN) caused by human herpes virus type 6 (HHV-6) is uncommon. We described a case of consecutive bilateral ARN, which was found to be a coinfection of varicella zoster virus (VZV) and HHV-6 in a 50-year-old woman, not well responded with systemic acyclovir. We showed the atypical findings with corresponding fundus and optical coherence tomography imaging. Patient concerns: She presented with anterior segment inflammation with peripheral retinitis and vasculitis in the left eye with disease progression despite of initial antiviral treatment, end up with retinal detachment. The right eye, subsequently, developed focal retinitis. Diagnosis: ARN was diagnosed by clinical fundus picture, confirmed by polymerase chain reaction (PCR). Interventions: Initially, she was treated with intravenous acyclovir and intravitreal ganciclovir for left eye. Retinal necrosis progressed, followed by retinal detachment. Pars plana vitrectomy with silicone oil was performed. The right eye, subsequently, developed focal retinitis. Medication was switched to intravenous ganciclovir and then oral valganciclovir. Outcomes: Retinitis was resolved, generalized hyperpigmentation appeared as a salt-and-pepper appearance in the right eye. The left eye presented preretinal deposits on silicone-retina interphase along retinal vessels. Spectral-domain optical coherence tomography (SD-OCT) showed multiple hyperreflective nodules on retinal surface. Lessons: ARN from coinfection of VZV and HHV-6 is rare. Preretinal granulomas and generalized hyperpigmentation could be one of the HHV-6 features. HHV-6 should be in the differential diagnosis for ARN. It responds well to systemic ganciclovir.
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Yeo, Shel-Hwa, et Allan E. Herbison. « Estrogen-Negative Feedback and Estrous Cyclicity Are Critically Dependent Upon Estrogen Receptor-α Expression in the Arcuate Nucleus of Adult Female Mice ». Endocrinology 155, no 8 (1 août 2014) : 2986–95. http://dx.doi.org/10.1210/en.2014-1128.
Texte intégralRésumé :
The location and characteristics of cells within the brain that suppress GnRH neuron activity to contribute to the estrogen-negative feedback mechanism are poorly understood. Using adeno-associated virus (AAV)-mediated Cre-LoxP recombination in estrogen receptor-α (ERα) floxed mice (ERαflox/flox), we aimed to examine the role of ERα-expressing neurons located in the arcuate nucleus (ARN) in the estrogen-negative feedback mechanism. Bilateral injection of AAV-Cre into the ARN of ERαflox/flox mice (n = 14) resulted in the time-dependent ablation of up to 99% of ERα-immunoreactive cell numbers throughout the rostrocaudal length of the ARN. These mice were all acyclic by 5 weeks after AAV-Cre injections with most mice in constant estrous. Control wild-type mice injected with AAV-Cre (n = 13) were normal. Body weight was not altered in ERαflox/flox mice. After ovariectomy, a significant increment in LH secretion was observed in all genotypes, although its magnitude was reduced in ERαflox/flox mice. Acute and chronic estrogen-negative feedback were assessed by administering 17β-estradiol to mice as a bolus (LH measured 3 h later) or SILASTIC brand capsule implant (LH measured 5 d later). This demonstrated that chronic estrogen feedback was absent in ERαflox/flox mice, whereas the acute feedback was normal. These results reveal a critical role for ERα-expressing cells within the ARN in both estrous cyclicity and the chronic estrogen negative feedback mechanism in female mice. This suggests that ARN cells provide a key indirect, transsynpatic route through which estradiol suppresses the activity of GnRH neurons.
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Pan, Junhua, Vikram N. Vakharia et Yizhi Jane Tao. « The structure of a birnavirus polymerase reveals a distinct active site topology ». Proceedings of the National Academy of Sciences 104, no 18 (24 avril 2007) : 7385–90. http://dx.doi.org/10.1073/pnas.0611599104.
Texte intégralRésumé :
Single-subunit polymerases are universally encoded in both cellular organisms and viruses. Their three-dimensional structures have the shape of a right-hand with the active site located in the palm region, which has a topology similar to that of the RNA recognition motif (RRM) found in many RNA-binding proteins. Considering that polymerases have well conserved structures, it was surprising that the RNA-dependent RNA polymerases from birnaviruses, a group of dsRNA viruses, have their catalytic motifs arranged in a permuted order in sequence. Here we report the 2.5 Å structure of a birnavirus VP1 in which the polymerase palm subdomain adopts a new active site topology that has not been previously observed in other polymerases. In addition, the polymerase motif C of VP1 has the sequence of -ADN-, a highly unusual feature for RNA-dependent polymerases. Through site-directed mutagenesis, we have shown that changing the VP1 motif C from -ADN- to -GDD- results in a mutant with an increased RNA synthesis activity. Our results indicate that the active site topology of VP1 may represent a newly developed branch in polymerase evolution, and that birnaviruses may have acquired the -ADN- mutation to control their growth rate.
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Sato, Tomohito, Wataru Yamamoto, Atsushi Tanaka, Haruna Shimazaki, Sunao Sugita, Toshikatsu Kaburaki et Masaru Takeuchi. « Viral Loads in Ocular Fluids of Acute Retinal Necrosis Eyes Infected by Varicella-Zoster Virus Treated with Intravenous Acyclovir Treatment ». Journal of Clinical Medicine 9, no 4 (22 avril 2020) : 1204. http://dx.doi.org/10.3390/jcm9041204.
Texte intégralRésumé :
Acute retinal necrosis (ARN) is a rare viral endophthalmitis, and human herpesvirus is the principal pathogen. Early diagnosis and treatment are critical to avoid visual impairment by ARN, and pars plana vitrectomy (PPV) is required in advanced cases. In this study, we evaluated the transition of viral load in ocular fluids of ARN eyes with varicella-zoster virus (VZV) after intravenous acyclovir treatment. Fourteen eyes of 13 patients were analyzed retrospectively. All patients received intravenous acyclovir treatment, and eventually, all eyes underwent PPV. A polymerase chain reaction (PCR) test showed a 100% detection rate in all aqueous humor samples collected before the treatment (Pre-AH), as well as aqueous humor (Post-AH) and vitreous fluid samples (VF), collected during PPV conducted after the treatment. Within eight days or less of acyclovir treatment, viral loads both in AH and VF did not decrease significantly. Furthermore, the viral load of Pre-AH had a strong correlation with that of VH. These data suggest that in ARN eyes with VZV infection, the AH sample for the PCR test was reliable to confirm the pathogen. We propose that short-term treatment of intravenous acyclovir may be insufficient for reducing intraocular viral load, and the Pre-AH sample could be a predictor of viral activity in the eyes after acyclovir treatment.
Thèses sur le sujet "ARN Dependent ARN Polymerase"
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Devert, Anthony. « Etude des ARN Polymérases ARN-dépendantes impliquées dans le RNA silencing ». Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX22086.
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Ce travail de thèse porte sur l’étude des ARN polymérases ARN dépendant impliquées dans le RNA silencing chez Arabidopsis thaliana. Durant ma thèse, la recherche d'interacteurs des RDR, parmi des protéines impliquées dans le RNA silencing, a permis la détection d'interaction entre RDR6 et SDE3, RDR6 et SGS3, mais aussi entre SDE3 et SGS3 en Co-IP et BiFC. Une co-localisation de ces protéines a été observée lorsqu'elles sont produites transitoirement dans des cellules épidermales de N. benthamiana.Un crible d’une banque d’ADNc d’A. thaliana par double hybride de levure, a permis d’isoler des interacteurs potentiels de RDR6. Deux interacteurs potentiels, AtUAP56-1 et U2B’’, sont impliqués dans l’épissage des précurseurs des ARNm. Un effet sur le RNA silencing dans des mutants de l’épissage en 3’ des ARNm était connu et nous avons confirmé l’interaction entre RDR6 et AtUAP56-1 par BiFC. L’étude de lignées mutantes pour AtUAP56-1 a donc été initiée.Une étude biochimique de RDR6 et de RDR2 a été réalisée. Des formes recombinantes de RDR2 et RDR6 ont été produites de façon transitoire dans des feuilles de N. benthamiana, et une étude comparative de RDR2 et RDR6 a été réalisée. Les deux RDR sont actives sur des matrices ARN et ADN, et montrent in vitro une activité amorce-indépendante. De plus, nous avons détecté pour la première fois une activité amorce-dépendante de RDR6 et RDR2. Ces résultats apportent de nouvelles données biochimiques qui sont en accord avec les études menées in vivo et enrichissent les modèles actuels du RNA silencingThe aim of this work was to study RNA-dependent RNA polymerases involved in RNA silencing in Arabidopsis thaliana. During my thesis, the search for RDR interactors among proteins involved in RNA silencing allowed the detection of interactions between RDR6 and SDE3, RDR6 and SGS3, and also between SDE3 and SGS3 using Co-IP and BiFC. In addition, the co-localisation of these proteins was observed when produced transiently in epidermal cells of N. Benthamiana.A screen of an A. thaliana cDNA library by yeast two hybrid allowed us to identify some putative new RDR6 interactors. Two putative RDR6 interactors, AtUAP56 and U2B’’, are known to be involved in pre-miRNA splicing. Furthermore, a link between pre-mRNA 3’ splicing and RNA silencing was previously reported. We also confirmed the interaction between AtUAP56-1 and RDR6 by BiFC. An investigation of A. thaliana of AtUAP56-1 mutants has been initiated.Recombinant RDRs were produced transiently in N. Benthamiana, and a biochemical comparative study of RDR2 and RDR6 performed. We found that RDR2, like RDR6, has a de novo polymerase activity on DNA and RNA templates, and for both RDRs we also showed, for the first time, a primer-dependant synthesis of dsRNA from RNA template. These findings provide important new insights into our understanding of the molecular mechanisms of RNA silencing amplification in Arabidopsis
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Durieux-Trouilleton, Quentin. « Analyse structurale et fonctionnelle de la réplication et de la transcription des Bunyavirus ». Electronic Thesis or Diss., Université Grenoble Alpes, 2024. http://www.theses.fr/2024GRALV028.
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L’ordre des Bunyavirales constitue un ordre vaste et diversifié de plus de 500 virus à ARN à simple brin négatif segmenté, contenant plusieurs virus émergents et/ou hautement pathogènes pour l'homme. Au sein de cet ordre, la famille des Hantaviridae contient le virus Hantaan (HTNV) qui entraîne des fièvres hémorragiques avec syndrome rénal, ainsi que le virus de Sin Nombre (SNV) qui cause des syndromes pulmonaires graves. Ces maladies sont reliées à des taux de mortalité importants, respectivement 15 % et 50 %. Une seconde famille, celle des Peribunyaviridae, contient des virus entraînant des cas d’encéphalites chez les enfants. C’est notamment le cas du virus de La Crosse (LACV). Aucun vaccin ni médicament n'est actuellement approuvé par les autorités de santé pour les contrer.L’objectif de cette thèse est d’étudier ces virus et plus précisément leur ARN polymérase dépendante de l’ARN, qui est une large protéine multi-fonctionnelle de 250kDa. Cette protéine, aussi appelée L, joue un rôle clé dans la réplication et la transcription du génome viral. Les expériences réalisées se sont principalement focalisées sur la polymérase du virus Hantaan (HTNV-L). Des expériences complémentaires ont également été réalisées sur SNV-L et LACV-L.J’ai utilisé la cryo-microscopie électronique (cryo-ME) comme méthode principale pour déterminer la structure d’HTNV-L. Les premières structures partielles d’HTNV-L ont révélé que la structure apo de HTNV-L adopte une conformation inactive avec une configuration α-hélicoïdale inédite du motif catalytique E. L'analyse structurale de la protéine en présence de l'extrémité 5' de l'ARN viral (vRNA) a montré que la liaison de ce dernier entraîne une cascade de modifications impliquant notamment la réorganisation complète du motif catalytique E en une configuration en β-feuillet canonique, ce qui conduit à des changements conformationnels drastiques dans le site actif. La liaison de l'extrémité 5' de l'ARN viral à HTNV-L est également nécessaire pour le recrutement de l'extrémité 3' de l'ARN viral vers le site actif de la polymérase pour l'initiation de la réplication. Les tests d'activité couplés aux structures cryo-ME d’HTNV-L et LACV-L révèlent les mécanismes impliqués dans les différentes étapes de la réplication du génome, notamment son initiation qui utilise un mécanisme d’amorçage et réalignement. Les structures bloquées à l'élongation de la réplication révèlent ensuite la formation d'un double brin d’ARN matrice/produit dans la cavité du site actif, couplée à des changements conformationnels d’HTNV-L.Dans un second temps, le traitement des images par cryo-ME d’HTNV-L apo a également dévoilé les structures haute résolution d’HTNV-L en trois oligomères différents : des monomères, des dimères symétriques et des hexamères symétriques composés de trimères de dimères. La formation de multimères implique des mouvements drastiques des protomères, montrant la capacité de changement conformationnel d’HTNV-L. Ces oligomères ont été l’opportunité d’observer et de résoudre la structure de chacun des domaines d’HTNV-L, y compris les plus flexibles, révélant enfin la structure complète de cette enzyme essentielle.Ensemble, ces éléments révèlent (i) la capacité de multimérisation d’HTNV-L dans sa forme apo inactive, qui correspond probablement à un système de stockage stabilisant et protecteur pour la protéine, (ii) l'activation de la polymérase déclenchée par la liaison de l'ARN viral et (iii) les mécanismes moléculaires complexes sous-jacents à la réplication du génome. Globalement, ces résultats améliorent considérablement la compréhension des mécanismes impliqués dans la réplication du génome des Bunyavirus et fournissent une base solide pour le développement futur d'antiviraux contre ce groupe de pathogènes émergentsBunyavirales is a large and diverse order of more than 500 segmented negative-stranded single-stranded RNA viruses, including several emerging and/or highly pathogenic human viruses. Within this order the family Hantaviridae includes the Hantaan virus (HTNV) that causes haemorrhagic fever with renal syndrome, and the Sin Nombre virus (SNV), which can lead to severe pulmonary syndromes. These illnesses are linked to significant mortality rate of 15% and 50% respectively. A second family, Peribunyaviridae, includes viruses that cause encephalitis in children, notably La Crosse virus (LACV). There is currently no vaccine or drug approved by health authorities to combat these viruses.The aim of this thesis is to study these viruses and, in particular, their RNA-dependent RNA polymerase, that is a large multi-functional protein of 250kDa. This protein, also called L protein, plays a key role in the replication and transcription of the viral genome. The experiments that I carried out focused mainly on Hantaan virus polymerase (HTNV-L). Complementary experiments were also performed on SNV-L and LACV-L.I used cryo-electron microscopy (cryo-EM) as the main method to determine the structure of HTNV-L. The first partial structures of HTNV-L revealed that its apo structure adopts an inactive conformation with a novel α-helical configuration of the catalytic motif E. Structural analysis of HTNV-L in the presence of the 5'-end of the viral RNA (vRNA) showed that the binding of the latter triggers a cascade of modifications, that notably involves the complete reorganisation of the catalytic motif E into a canonical β-sheet configuration, leading to drastic conformational changes in the active site. Binding of the viral RNA 5'-end to HTNV-L is also required for the recruitment of the viral RNA 3'-end to the polymerase active site for replication initiation. Activity assays coupled with cryo-EM structures of HTNV-L and LACV-L reveal the mechanisms involved in the different steps of genome replication, in particular its initiation, which uses a prime-and-realign mechanism. Structures stalled during replication elongation then reveal the formation of a template/product duplex in the active site cavity, coupled with conformational changes in HTNV-L.Secondly, cryo-EM imaging of apo HTNV-L also revealed the high-resolution structures of HTNV-L in three different oligomers: monomers, symmetric dimers and symmetric hexamers composed of trimers of dimers. Multimer formation involves large movements of the protomers, demonstrating the ability of HTNV-L to undergo conformational changes. These oligomers provided the opportunity to observe and determine the structure of each domain of HTNV-L, including the most flexible ones, finally revealing the complete structure of this essential viral enzyme.Taken together, these elements reveal (i) the ability of HTNV-L to multimerise in its inactive apo form, which probably corresponds to a stabilising and protective storage system for the protein, (ii) the activation of the polymerase triggered by viral RNA binding, and (iii) the intricate molecular mechanisms underlying genome replication. Collectively, these results significantly improve the understanding of the mechanisms involved in Bunyavirus genome replication and provide a solid basis for the future development of antivirals against this group of emerging pathogens
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HERICOURT, FRANCOIS. « Etude moleculaire de l'arn polymerase arn-dependante du virus de la mosaique jaune du navet (tymv) ». Paris 6, 1999. http://www.theses.fr/1999PA066244.
Texte intégralRésumé :
Ce travail de these concerne l'etude de l'arn polymerase arn-dependante (rdrp) d'un virus a arn simple brin de polarite positive, le tymv. L'analyse de cette proteine essentielle a la replication de ce virus a ete entreprise selon 3 axes de recherche : 1) l'etude de ses proprietes biochimiques grace a sa surexpression dans un systeme d'expression eucaryote utilisant un baculovirus recombinant, 2) l'etude de ses interactions avec les autres proteines virales non structurales par la technique du double-hybride et 3) la recherche d'interactions avec des proteines cellulaires soit a) deja soupconnees de participer au complexe de replication, tel que le facteur d'elongation eef1, soit b) encore inconnues, par le criblage d'une banque d'adn, egalement grace au systeme du double-hybride. Les resultats obtenus montrent que la rdrp du tymv est phosphorylee in vivo dans les cellules d'insecte et que certaines especes minoritaires de cette proteine correspondent a des formes ubiquitinees, suggerant ainsi que cette proteine puisse etre degradee par la voie ubiquitine-dependante du proteasome. Par ailleurs, l'etude des interactions entre la rdrp et les autres proteines virales nous ont permis de definir essentiellement 2 interactions entre ces differentes proteines et de delimiter la region minimale necessaire a l'interaction de la rdrp sur elle-meme au niveau de sa partie n-terminale. Enfin, la recherche d'interaction entre la rdrp du tymv et differentes sous-unites du facteur d'elongation eef1 nous a conduit a cloner une nouvelle sous-unite de ce facteur chez arabidopsis thaliana et a purifier le complexe de replication de ce virus chez cette plante modele. En parallele, nous avons isole lors d'un crible double-hybride avec une banque d'adn genomique de levure, 36 candidats capables d'interagir specifiquement avec la rdrp du tymv. De telles proteines constituent des pistes de recherche prometteuses dont l'etude permettra une meilleure comprehension des mecanismes moleculaires de l'interaction entre virus et cellule hote.
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Subissi, Lorenzo. « Biochemical insights into SARS-CoV replication ». Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM5002.
Texte intégralRésumé :
Mon travail de thèse s'est focalisé sur la machinerie enzymatique impliquée dans la réplication du génome ARN du Syndrome Respiratoire Aigu Sévère-Coronavirus (SRAS-CoV). J'ai montré in vitro que l'activité ARN polymérase ARN-dépendante (RdRp) portée par nsp12 nécessite le complexe nsp7/nsp8, qui agit comme facteur de processivité. Grâce à ce complexe polymérase hautement actif, j'ai pu en suite étudier le mécanisme de "proofreading" (correction d'épreuve) associé aux coronavirus, pour lequel seulement des preuves indirectes avaient été assemblées. En effet, les coronavirus codent pour une activité exonucléase 3'-5' (nsp14-ExoN) qui lorsqu'elle est absente, entraine 14-fois plus d'erreurs de réplication en contexte cellulaire. In vitro, nous avons pu montrer que nsp14-ExoN est capable d'exciser l'ARN double brin ainsi qu'un nucléotide mésapparié en 3' de l'ARN en cours d'élongation. J'ai pu apporter pour la première fois une preuve directe de l'existence d'un système de réparation des erreurs au cours de la synthèse, mené par le complexe nsp7/nsp8/nsp12/nsp14. En effet, le complexe nsp7/nsp8/nsp12 ralentit jusqu'à 30-fois quand il rajoute une base mésappariée. Par sequençage, nous avons pu montrer la réparation de cette base mésappariée en presence de nsp14. Enfin, grâce à ce système in vitro nous avons une base pour comprendre l'inefficacité de la ribavirine sur des patients atteints du SRAS. En effet, la ribavirine, incorporée par le complexe polymérase, serait également excisée par nsp14, annihilant tout potentiel effet mutagenique. En conclusion, ce système va permettre de guider le développement d'antiviraux de type nucleoside analogues contre les coronavirusThis work focused on the enzymatic machinery involved in Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) RNA replication and transcription. Firstly, I established a robust in vitro polymerase assay with the canonical SARS-CoV RNA-dependent RNA polymerase (RdRp) nsp12. I showed that nsp12, in order to engage processive RNA synthesis, needs two viral proteins, i.e. nsp7 and nsp8. This nsp7/nsp8 complex not only activates nsp12-RdRp, but also acts as a processivity factor. Thus, using this processive polymerase complex, I could investigate SARS-CoV proofreading for which only indirect evidences were reported. Indeed, coronaviruses encode for a 3'-5' exonuclease (nsp14-ExoN), putatively involved in a mechanism that proofreads coronavirus RNA during viral replication. We first showed in vitro that nsp14-ExoN, which is stimulated by nsp10, is able to excise specifically dsRNA as well as all primer/templates bearing a 3' mismatch on the primer. Moreover, we could confirm by sequencing that a RNA 3' mismatch was indeed corrected in vitro by the nsp7/nsp8/nsp12/nsp14 complex. We provide for the first time direct evidence that nsp14-ExoN, in coordination with the polymerase complex, is able to proofread RNA. Interestingly, using this in vitro system we found an element that could possibly explain the inefficacy of ribavirin therapeutic treatment on SARS-patients: ribavirin, which is incorporated by the SARS-CoV polymerase complex, would also be excised by nsp14. In conclusion, this system will drive future development of antivirals, particularly of the nucleoside analogue type, against coronaviruses
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Duplàa, Cécile. « Analyse quantitative des produits de polymerase chain reaction utilisant l'incorporation de dUTP biotinylé ». Bordeaux 2, 1993. http://www.theses.fr/1993BOR2P045.
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Ferrigno, Olivier. « Les elements transposables sine b2 fournissent un promoteur arn polymerase ii mobile ». Nice, 1999. http://www.theses.fr/1999NICE5357.
Texte intégralRésumé :
Les sine (short interspaced elements) sont des composants tres abondants des genomes de mammiferes qui se propagent par retrotransposition. Parmi les sine, la famille des elements b2 constitue approximativement 0,7% de l'adn genomique total de rongeur. Les b2, comme la plupart des sine ont evolue a partir d'un arn de transfert, mais possedent aussi une region unique, d'origine inconnue, de 70 pb a leur extremite 3. Nous avons localise un de ces membres dans un intron du gene lama3 qui code pour les isoformes de la chaine 3 de la laminine-5 murine. Nous avons identifie un nouveau transcrit 3 dont le site d'initiation est situe dans cette sequence b2 au niveau de la region unique et est derive du brin antisens au transcrit ressemblant aux arnt synthetises par la pol iii. Nos etudes ont montre tout d'abord que la nouvelle transcription in vivo par la pol ii du gene lama3 est conduite par un petit fragment de 70 pb appartenant exclusivement a la sequence b2 et est dirigee par un element initiateur et une boite tata en amont. Cette sequence initiatrice est reconnue par le facteur de transcription usf et l'expression ectopique de ce facteur peut stimuler fortement la transcription par la pol ii de l'element b2 in vivo. Ces resultats mettent en evidence pour la premiere fois la presence d'un promoteur pol ii dans un element sine. Nous avons ensuite elargi nos etudes en montrant que la grande majorite des elements b2 possedent un promoteur pol ii, et que ce promoteur possede des similitudes de sequences avec plusieurs promoteurs de genes codant pour des proteines. Ces donnees indiquent que les elements sine b2 ont le potentiel de distribuer un promoteur pol ii fonctionnel et regulable a travers le genome. Ceci a pu, au cours de l'evolution, resulter en la creation de nouveaux arnm qui fournissent le materiel de base pour la selection naturelle.
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Swale, Christopher. « RNA binding and assembly of human influenza A virus polymerases ». Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV053/document.
Texte intégralRésumé :
Le virus de la grippe A est un virus à ARN négatif appartenant à la famille des Orthomyxoviriadea dont la réplication se produit dans le noyau des cellules infectées. L'organisation du génome est segmentée en huit segments d'ARNv de polarité négative, codant pour un minimum de 16 protéines virales différentes. Ces ARN viraux (ARNv) sont en complexe avec de nombreuses copies de nucléoprotéines et liés par leurs extrémités 5' et 3' au complexe hétérotrimérique de l'ARN-polymérase ARN-dépendante composé des sous unités PA, PB1 et PB2. Cet assemblage macromoléculaire (ARNv / polymérase / NP) nommée Ribonucléoprotéine (RNP) constitue une entité génomique indépendante. Dans le contexte de la RNP, l'ARN-polymérase assure à la fois la transcription et la réplication du génome ARNv. En assurant ces deux fonctions, l'ARN-polymérase joue un rôle majeur dans la réplication virale et constitue une cible antivirale privilégiée. Les travaux de recherche présentés dans cette thèse se concentrent sur les éléments structuraux participants à l'assemblage de l'ARN polymérase et son interaction avec les avec les ARNv. Pour atteindre ces objectifs, notre laboratoire, en collaboration avec d'autres groupes, a mis en place un système d'expression en polyprotéines permettant d'exprimer la polymérase. Plus encore, cette méthode a aussi permis de reconstituer des complexes entre l'ARN-polymérase et des partenaires cellulaires, notamment RanBP5 qui appartient à la famille des importines-βInfluenza A virus is a negative-strand RNA virus belonging to the Orthomyxoviriadea family whose replication occurs in the nucleus of infected cells. The genome organisation of influenza virus is segmented in eight vRNA segments of negative polarity coding for at least 16 different viral proteins. Each vRNA is bound to multiple copies of nucleoprotein (NP) and to the heterotrimeric RNA-dependent RNA-polymerase complex (PA, PB1 and PB2) through its 5' and 3' extremities. This macromolecular assembly (vRNA/polymerase/NP) forms the ribonucleoprotein (RNP) particle, which acts as a separate genomic entity within the virion. The RNP complex is at the core of viral replication and in the context of RNPs, the polymerase performs both transcription and replication of the vRNA genome. As such, the polymerase constitutes a major antiviral drug target. The research work presented within this thesis focuses on the underlying determinants of the RNA polymerase assembly process and its interaction with its vRNA genome. To fulfill these goals, our lab, in collaboration with other groups, has set up a novel polyprotein expression system to express the polymerase but also to reconstitute polymerase and cellular partner complexes, notably RanBP5, which belongs to the importin-β family
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Azevedo, Jacinthe. « Caractérisation d'une nouvelle famille de protéines susceptibles d'interagir avec une ARN polymerase plastidiale ». Université Joseph Fourier (Grenoble), 2005. https://tel.archives-ouvertes.fr/tel-00011722.
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Les ARN polymérases de type phagique, codées dans le noyau (NEP; Nuclear Encoded RNA Polymerase), assurent une partie de la transcrIption du génome plastidial des végétaux supérieurs. Ces travaux mettent en évidence l'existence d'une nouvelle famille de protéines d'A. Thaliana susceptibles d"interagir avec les NEP des plantes dicotylédones: les protéines NIP (NEP Interacting Protein). Les protéines NIP sont retrouvées uniquement dans le végétaux supérieurs et leur synthèse est dépendante de la lumière. Elles présentent un domaine d'interaction protéine-protéine RING fmger en C terminal. Nous avons montré pour la première fois par inununodétection que ces protéines sont intégrées aux membranes thylacoïdiennes et qu'elles retiennent probablement les NEP plastidiales à la surface de la membrane. Cette interaction avec les membranes pourrait apporter une nouvelle vision du fonctionnement des NEP dans le chloroplastes des plantes dicotylédonesThe phage-like RNA polymerases, encoded in the nucleus (NEP; Nuclear Encoded RNA Polymerase) ensure partial!y plastidial genome transcription in higher plants. This work underlined the existence of a new protein family potentially able to interact with NEP in dicotyledon plants: NIP proteins (NEP Interacting Protein). NIP proteins are only present in higher plants and their synthesis is light dependant. Their C-terminal region presents a RINC finger protein-protein interacting domain. Using immundetection, we show the first time that NIP proteins are integrated into thylakoid membranes, keeping probably NEP close to the membrane on the stroma side. This association to membranes offers new insights into NEP activity in chloroplasts of dicotyledon plants
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Gu, Bo. « Co-transcriptional processing of pre-mRNA : Effects of RNA polymerase II carboxyl-terminal domain modification ». Paris 6, 2012. http://www.theses.fr/2012PA066202.
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La transcription par l’ARN polymerase II (RNAPII) est un processus complexe qui inclue initiation, échappement du promoteur, elongation et terminaison. Chaque étape est régulée par un grand nombre d’éléments en cis et de facteurs en trans. La maturation des transcrits est aussi une succession d’étapes incluant la formation d’une coiffe en 5’, épissage puis clivage suivi de polyadénylation en 3’. Il est couramment admis que la transcription par la RNAPII et la maturation des transcrits est largement couplée et que les différents processus s’influencent mutuellement. En recherchant des ARNs non-codants pouvant réguler l’activité de la RNAPII chez les mammifères, nous avons trouvé que le snARN U1 était constitutivement associé à la RNAPII qu’elle soit en train de transcrire ou non. En outre, une analyse par microscopie de fluorescence a montré que cette interaction était indépendante de l’épissage qui est l’une des principales fonctions de U1 snRNA. Je me suis particulièrement intéressé aux modifications du domaine CTD de la RNAPII qui est une caractéristique de cette polymerase eukaryote. J’ai trouvé que la phosphorylation de la serine 2 était importante pour assurer l’épissage co-transcriptionnel et une terminaison en 3’ correcte. Je montre aussi que la stimulation de la terminaison par l’épissage est médiée par le facteur U2AF65. Enfin, j’analyse l’importance de la phosphorylation de la serine 2 du CTD sur l’épissage alternatif et le recrutement des facteurs TAF15, PSF et P54Transcription of RNA polymerase II (RNAPII) is a highly complex procedure, including initiation, promoter escape, elongation and termination. Each step of transcription is regulated by a variety of cis-elements and trans-factors. Maturation of RNAPII transcripts is also a complicated course consisting of transcript capping, splicing and 3’ end processing. It is widely accepted that RNAPII transcription and its RNA processing are interactional and different processes of RNAPII transcript maturation influence each other. In the attempt of finding the non-coding RNAs that can regulate the activity of RNAPII in mammals, we found that U1 snRNA is associated with RNAPII no matter if RNAPII is transcribing or not. Moreover, using fluorescence microscopy, we showed that the interaction between U1 snRNA and RNAPII is independent on splicing, which is the main function of U1 snRNA. During the investigation of the effect of RNAPII on transcript maturation, I focused on post-translational phosphorylation of the carboxyl-terminal domain of RNAPII, which is the unique domain of RNAPII in all RNAPI, II and III. I found that the phosphorylation of CTD serine 2 residues is required for constitutive splicing as well as 3’ end processing. I also provided the evidence to show the effect of splicing on 3’ end processing via the splicing factor U2AF65. Furthermore, I showed the effect of CTD serine 2 residue phosphorylation on alternative splicing and the recruitment of TAF15, PSF and P54 to the transcription sites. Finally, I summarized the unknown points in my study and proposed the perspective
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Gajda, Anna Ewa. « Regulation of a gene transcription by RNA polymerase III in Saccharomyces cerevisiae : the role of evolutionarily conserved domains of the Maf1 protein, RNA polymerase III repressor ». Paris 11, 2010. http://www.theses.fr/2010PA112224.
Texte intégralRésumé :
Dans l'environnement, la levure doit faire face à des conditions variées qui nécessitent une adaptation rapide du métabolisme cellulaire. Une des premières réponses est l'inhibition de la transcription par l'ARN polymérase III (Pol III). La protéine Maf1, le seul régulateur de la machinerie de la Pol III chez Saccharomyces cerevisiae (Sc), est conservée au cours de l'évolution. Les protéines Maf1 des Eucaryotes contiennent deux domaines A et BC phylogénétiquement conservés. Ce travail de thèse a cherché à identifier le rôle de ces domaines dans la fonction de la protéine ScMaf1. J'ai construit une banque de mutants de Maf1, identifié les changements dans leurs séquences ainsi que leurs phénotypes. En utilisant la technique du double-hybride, j'ai montré que les domaines A et BC interagissent physiquement et que l'extrémité N-terminale de 34 acides aminés du domaine A est le fragment minimal nécessaire à cette interaction. Grâce à un crible génétique, j'ai mis en évidence que les mutations du domaine BC (D250E et V260D-N344I) permettent de restaurer l'activité de Maf1 mutée dans le domaine A (K35E). Cette restauration est observable pour le phénotype, la répression efficace de la transcription par la Pol III, le niveau de phosphorylation et la localisation cellulaire de Mafl. La technique du double-hybride m'a permis aussi de montrer que la mutation K35E inactive partiellement l'interaction entre les domaines de Maf1 qui est restaurée par les mutations suppresseurs D250E et V260D-N344I. Les résultats permettent de conclure que : « la répression de la transcription par la Pol III requiert l'interaction physique des domaines de Maf1»Yeast cell encounters numerous environ mental situations that require a rapid and efficient adaptation of cellular melabolism to changing Iife conditions. One of the first responses is the inhibition of RNA polymerase III (Pol llI) transcription. The Maf1 protein, the unique negative regulator of the Pol III apparatus in Saccharomyces cerevisiae (Sc), is conserved through evolution. The family of eukaryotic Maf1 share highly conserved amino acid sequence with two easily recognizable regions called A and BC domains. The work performed during this PhD thesis concerns the role of these evolutionary conserved domains in the activity of ScMaf1. I have constructed a Iibrary of Maf1, identified and localized the mutations of corresponding Maf1 proteins and studied the phenotype. Using yeast two-hybrid system, I have found the A and BC domains interact physically and defined the minimal 34 aa fragment of the A domain involved in this interaction. Using genetic screen for internal suppressor mutations, I have identified that mutations localized in BC domain (D250E, V260D-N344I) recovered the activity of Maf1 mutated in A domain (K35E), as deduced from no defected growth, efficient Pol III repression, phosphorylation and cellular localization of identified suppressors. The identified K35E mutation disrupted physical interaction between Maf1 domains unless the presence of additional D250E or V260D-N344I suppressor mutations occurred. The Take Home message from the results obtained during my PhD thesis is that: "Full repression of Pol III requires the physical interaction between Maf1 domains"
Livres sur le sujet "ARN Dependent ARN Polymerase"
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(Editor), Sankar Adhya, et Susan Garges (Editor), dir. RNA Polymerase and Associated Factors, Part C, Volume 370 (Methods in Enzymology). Academic Press, 2003.
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(Editor), M. J. McPherson, B. D. Hames (Editor) et G. R. Taylor (Editor), dir. PCR 2 : A Practical Approach (Practical Approach Series). Oxford University Press, USA, 1995.
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(Editor), M. J. McPherson, B. D. Hames (Editor) et G. R. Taylor (Editor), dir. PCR 2 : A Practical Approach (Practical Approach Series). Oxford University Press, USA, 1995.
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White, Thomas J., Michael A. Innis, David H. Gelfand et John J. Sninsky. PCR Protocols : A Guide to Methods and Applications. Elsevier Science & Technology Books, 2012.
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PCR Protocols : A Guide to Methods and Applications. Academic Press, 1989.
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PCR protocols : A guide to methods and applications. San Diego : Academic Press, 1990.
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PCR Protocols : A Guide to Methods and Applications. Academic Press, 1989.
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PCR Protocols : A Guide to Methods and Applications. Academic Press, 1989.
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(Editor), Michael A. Innis, David H. Gelfand (Editor), John J. Sninsky (Editor) et Thomas J. White (Editor), dir. PCR Protocols : A Guide to Methods and Applications. Academic Press, 1989.
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Vaheri, Antti, James N. Mills, Christina F. Spiropoulou et Brian Hjelle. Hantaviruses. Oxford University Press, 2011. http://dx.doi.org/10.1093/med/9780198570028.003.0035.
Texte intégralRésumé :
Hantaviruses (genus Hantavirus, family Bunyaviridae) are rodent- and insectivore-borne zoonotic viruses. Several hantaviruses are human pathogens, some with 10-35% mortality, and cause two diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Hantaviruses are enveloped and have a three-segmented, single-stranded, negative-sense RNA genome. The L gene encodes an RNA-dependent RNA polymerase, the M gene encodes two glycoproteins (Gn and Gc), and the S gene encodes a nucleocapsid protein. In addition, the S genes of some hantaviruses have an NSs open reading frame that can act as an interferon antagonist. Similarities between phylogenies have suggested ancient codivergence of the viruses and their hosts to many authors, but increasing evidence for frequent, recent host switching and local adaptation has led to questioning of this model. Infected rodents establish persistent infections with little or no effect on the host. Humans are infected from aerosols of rodent excreta, direct contact of broken skin or mucous membranes with infectious virus, or rodent bite. One hantavirus, Andes virus, is unique in that it is known to be transmitted from person-to-person. HFRS and HCPS, although primarily affecting kidneys and lungs, respectively, share a number of clinical features, such as capillary leakage, TNF-, and thrombocytopenia; notably, hemorrhages and alterations in renal function also occur in HCPS and cardiac and pulmonary involvement are not rare in HFRS. Of the four structural proteins, both in humoral and cellular immunity, the nucleocapsid protein appears to be the principal immunogen. Cytotoxic T-lymphocyte responses are seen in both HFRS and HCPS and may be important for both protective immunity and pathogenesis. Diagnosis is mainly based on detection of IgM antibodies although viral RNA (vRNA) may be readily, although not invariably, detected in blood, urine and saliva. For sero/genotyping neutralization tests/RNA sequencing are required. Formalin-inactivated vaccines have been widely used in China and Korea but not outside Asia. Hantaviruses are prime examples of emerging and re-emerging infections and, given the limited number of rodents and insectivores thus far studied, it is likely that many new hantaviruses will be detected in the near future.
Chapitres de livres sur le sujet "ARN Dependent ARN Polymerase"
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Merkl, Philipp E., Christopher Schächner, Michael Pilsl, Katrin Schwank, Catharina Schmid, Gernot Längst, Philipp Milkereit, Joachim Griesenbeck et Herbert Tschochner. « Specialization of RNA Polymerase I in Comparison to Other Nuclear RNA Polymerases of Saccharomyces cerevisiae ». Dans Ribosome Biogenesis, 63–70. New York, NY : Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2501-9_4.
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AbstractIn archaea and bacteria the major classes of RNAs are synthesized by one DNA-dependent RNA polymerase (RNAP). In contrast, most eukaryotes have three highly specialized RNAPs to transcribe the nuclear genome. RNAP I synthesizes almost exclusively ribosomal (r)RNA, RNAP II synthesizes mRNA as well as many noncoding RNAs involved in RNA processing or RNA silencing pathways and RNAP III synthesizes mainly tRNA and 5S rRNA. This review discusses functional differences of the three nuclear core RNAPs in the yeast S. cerevisiae with a particular focus on RNAP I transcription of nucleolar ribosomal (r)DNA chromatin.
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Ravelonandro, Michel, et Pascal Briard. « Biogenesis and functional RNAi in fruit trees. » Dans RNAi for plant improvement and protection, 40–46. Wallingford : CABI, 2021. http://dx.doi.org/10.1079/9781789248890.0005.
Texte intégralRésumé :
Abstract In plants, genome expression is linked to the transcribed mRNAs that are synthesized by RNA polymerase. Following its move to the cytoplasm, the generated mRNA is briefly translated to the encoded protein. If transcription and translation are dependent on the family of RNA polymerase, these two phenomena could be interfered with through the process designated as gene regulation. Thus, large molecules of RNA (single-stranded or double-stranded) consequently sliced into small molecules produce nascent small interfering RNA ranging from 21 to 27 nucleotides. This chapter revisits the biogenesis of these two types of RNAi, miRNA and siRNA, and notably their involvement in plant gene regulation. Following their sequential transcription and their specific involvement, we will consider the sources and roles of RNA interference in plants and we will look at their detection in fruit crops. We discuss their applications and the risk assessment studies in fruit crops.
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Ravelonandro, Michel, et Pascal Briard. « Biogenesis and functional RNAi in fruit trees. » Dans RNAi for plant improvement and protection, 40–46. Wallingford : CABI, 2021. http://dx.doi.org/10.1079/9781789248890.0040.
Texte intégralRésumé :
Abstract In plants, genome expression is linked to the transcribed mRNAs that are synthesized by RNA polymerase. Following its move to the cytoplasm, the generated mRNA is briefly translated to the encoded protein. If transcription and translation are dependent on the family of RNA polymerase, these two phenomena could be interfered with through the process designated as gene regulation. Thus, large molecules of RNA (single-stranded or double-stranded) consequently sliced into small molecules produce nascent small interfering RNA ranging from 21 to 27 nucleotides. This chapter revisits the biogenesis of these two types of RNAi, miRNA and siRNA, and notably their involvement in plant gene regulation. Following their sequential transcription and their specific involvement, we will consider the sources and roles of RNA interference in plants and we will look at their detection in fruit crops. We discuss their applications and the risk assessment studies in fruit crops.
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Pilsl, Michael, et Christoph Engel. « Structural Studies of Eukaryotic RNA Polymerase I Using Cryo-Electron Microscopy ». Dans Ribosome Biogenesis, 71–80. New York, NY : Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2501-9_5.
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AbstractTechnical advances have pushed the resolution limit of single-particle cryo-electron microscopy (cryo-EM) throughout the past decade and made the technique accessible to a wide range of samples. Among them, multisubunit DNA-dependent RNA polymerases (Pols) are a prominent example. This review aims at briefly summarizing the architecture and structural adaptations of Pol I, highlighting the importance of cryo-electron microscopy in determining the structures of transcription complexes.
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Flohr, Alexander, Catharina Rohde, Savitha Devarajamohalla Narayana et Andrea Osburg. « Evaluation of Strength and Modulus of Elasticity of Polymer-Modified Cement Concrete (PCC) Under Thermal Impact Within a Defined Service Temperature Range ». Dans Springer Proceedings in Materials, 513–21. Cham : Springer Nature Switzerland, 2024. http://dx.doi.org/10.1007/978-3-031-72955-3_52.
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AbstractPolymer-modified cement mortars (PCM) and concretes (PCC) are mainly used in concrete repair and restoration exhibiting improved durability, suitable chemical resistance, and beneficial adhesion strength compared to unmodified cementitious materials. Due to these favorable properties, the material is increasingly implemented in construction. Commonly, the modifiers applied to cementitious binders consist of thermoplastic polymers, which feature a change in the deformation behavior under the influence of different temperatures. Despite the distinct temperature-dependent properties of the polymers, the load-dependent deformation behavior of PCM and PCC was barely investigated within a service temperature range. To make statements about the effect of polymers on the load bearing and elastic deformation behavior of PCM and PCC, the engineering properties of the material have to be experimentally assessed under thermal conditioning. Accordingly, the compressive and flexural strength as well as dynamic and static modulus of elasticity of seven different PCM mixtures were characterized while the specimens were exposed to service temperatures of −20 ℃, 20 ℃, and 60 ℃. After the specimens were thermally conditioned in a climate chamber, the samples were transferred to the equally conditioned test machine and tested in the proposed temperature scope. The experimental results reveal influential changes in all tested mechanical attributes for the modified system within the applied service temperature range compared to an unmodified reference. This knowledge is essential to further investigate the temperature impact on the material and develop appropriate prediction models for the application of polymer-modified cementitious materials in construction and the integration in design guidelines.
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Wang, Teresa S. F., krista L. Conger, William C. Copeland et Martha P. Arroyo. « Eukaryotic DNA polymerases ». Dans Eukaryotic DNA Replication, 67–92. Oxford University PressOxford, 1999. http://dx.doi.org/10.1093/oso/9780199636815.003.0003.
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Abstract DNA polymerases are the fundamental enzymes that synthesize cellular DNA to produce two identical daughter chromosomes. Cellular DNA polymerases in conjunction with DNA repair enzymes play critical roles for the transmission and maintenance of error-free genetic information from one generation to the next. In the last decade, genetic studies in yeast, biochemical studies of in vitro replication of the simain virus 40 (SV40) origin-containing DNA, and isolation of genes and cDNAs of DNA polymerases from budding yeast, fission yeasts, and mammalian cells led to the identification of five distinct DNA template-dependent DNA polymerases, named polymerase α, β, γ, δ, and ε(1,2).
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Sclavi, Bianca, Pascal Roux et Henri Bue. « Nucleotide incorporation by DNA dependent RNA and DNA polymerases ». Dans DNA-Protein Interactions, 239–56. Oxford University PressOxford, 2000. http://dx.doi.org/10.1093/oso/9780199636921.003.0018.
Texte intégralRésumé :
Abstract The steady-state analysis of the’ incorporation of nucleotide substrates by nucleic acid polymerases has be’en a subject of intensive research for many years. It has provided interesting re’ Wards in its specific field, for example a heller understanding of the faithfi.11 or the incorrect incorporation of nucleotides into zin elongating template by a specific enzyme. It has also proved to be of great use for a more indirect purpose, namely the understanding of how, in a time-resolved manner, nucleic acid polymerases interact with their cognate templates as the copying process is initiated or proceeds at steady state. This topic the subject of this chapter. Such assays were initially used in a rather crude. analytical manner. For example run-off transcription experiments were inserted into purification protocols to assess the degree of purity of a preparation of RNA polymerase or the extent of activation generated by a given factor. We describe here how more quantitative assays, performed with purified enzymes and templates, provide significant information on the interactions occurring between the two partners. Such assays arc essential prerequisites for the establishment of a plausible kinetic scheme which will in turn be challenged by the specific molecular methods developed in other chapters of this volume.
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Anand, Lallit, Ken Kamrin et Sanjay Govindjee. « Temperature dependence of linear viscoelastic response ». Dans Introduction to Mechanics of Solid Materials, 355–64. Oxford University PressOxford, 2022. http://dx.doi.org/10.1093/oso/9780192866073.003.0021.
Texte intégralRésumé :
Abstract This chapter considers the effects of temperature on the linear viscoelastic response of materials. Representative results from Dynamics Mechanical Analysis (DMA) experiments for measurement of the storage modulus, loss modulus, and tan δ as a function of temperature for amorphous and semi-crystalline polymers are discussed, and the glass transition temperature (Tg) and melt temperatures (Tm, for semi-crystalline polymers) are introduced. The effects of temperature on the stress-relaxation and creep-compliance functions are considered, and the concepts of time-temperature equivalence and shift factors for thermo-rheologically simple materials are introduced.
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Epstein, Irving R., et John A. Pojman. « Polymer Systems ». Dans An Introduction to Nonlinear Chemical Dynamics. Oxford University Press, 1998. http://dx.doi.org/10.1093/oso/9780195096705.003.0017.
Texte intégralRésumé :
In the classic 1967 film “The Graduate” the protagonist, Benjamin (Dustin Hoffman), is attempting to plan his postcollege path. His neighbor provides one word of advice, “Plastics.” This counsel has become part of American culture and is often parodied. But, it is good advice, because not since the transformations from stone to bronze and then to iron have new materials so completely transformed a society. Plastics made from synthetic polymers are ubiquitous, from Tupperware to artificial hearts. About half the world’s chemists work in polymer-related industries. In this chapter, we will survey some of the work that has been done in applying nonlinear dynamics to polymerization processes. These systems differ from those we have considered so far because they do not involve redox reactions. We will consider polymerization reactions in a CSTR that exhibit oscillations through the coupling of temperature-dependent viscosity and viscosity-dependent rate constants. Emulsion polymerization, which produces small polymer particles dispersed in water, can also oscillate in a CSTR. Both types of systems are important industrially, and their stabilities have been studied by engineers with the goal of eliminating their time-dependent behavior. Our favorite oscillating system, the Belousov-Zhabotinsky reaction, can be used to create an isothermal periodic polymerization reaction in either a batch or continuous system. This, however, is not a practical system because of the cost of the reagents. In most industrial processes, nonlinear behavior is seen not as an advantage but as something to be avoided. However, we will look at several reaction-diffusion systems that have desirable properties precisely because of their nonlinear behavior. Replication of RNA is autocatalytic and can occur as a traveling front. Since not all RNA molecules replicate equally well, faster mutants gradually take over. At each mutation, the front propagates faster. Evolution can be directly observed in a test tube. Propagating polymerization fronts of synthetic polymers may be useful for making new materials, and they are interesting because of the rich array of nonlinear phenomena they show, with pulsations, convection, and spinning fronts. Finally, we will consider photopolymerization systems that exhibit spatial pattern formation on the micron scale, which can be used to control the macroscopic properties.
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Patil, Shalini. « VISCO ELASTIC BEHAVIOUR OF POLYMERS ». Dans Futuristic Trends in Chemical Material Sciences & ; Nano Technology Volume 3 Book 1, 151–54. Iterative International Publishers, Selfypage Developers Pvt Ltd, 2024. http://dx.doi.org/10.58532/v3bdcs1ch11.
Texte intégralRésumé :
In Polymeric materials mechanical properties exhibit two ideal special cases hence these are known as visco elastic materials. The property of stress is a function of strain and function of time for visco elastic material. The very common characteristic features of visco elastic materials are a time dependent strain response to a constant stress and a constant strain response to a time dependent stress. When the applied stress is less or zero to the materials, the material recovers slowly with a time. This type of effects also observed in metals at very high temperatures whereas in plastics they show this type of behaviour at room temperature.
Actes de conférences sur le sujet "ARN Dependent ARN Polymerase"
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Ikegwu, Ugochukwu M., Victor M. Zavala et Reid C. Van Lehn. « Screening Green Solvents for Multilayer Plastic Films Separation ». Dans Foundations of Computer-Aided Process Design, 763–70. Hamilton, Canada : PSE Press, 2024. http://dx.doi.org/10.69997/sct.162050.
Texte intégralRésumé :
This paper introduces a computational framework for selecting green solvents to separate multilayer plastic films, particularly those challenging to recycle through mechanical means. The framework prioritizes the selective dissolution of polymers while considering solvent toxicity. Initial screening relies on temperature-solubility dependence, utilizing octanol-water partition coefficients (LogP) to identify non-toxic solvents (LogP = 3). Additionally, guidelines from GlaxoSmithKline (GSK), Registration, Evaluation, Authorization, and Restriction of Chemical Regulation (REACH), and the US Environmental Protection Agency (EPA) are employed to screen for green solvents. Molecular-scale models predict temperature-dependent solubilities and LogP values for polymers and solvents. The framework is applied to identify green solvents for separating a multilayer plastic film composed of polyethylene (PE), ethylene vinyl alcohol (EVOH), and polyethylene terephthalate (PET). The case study demonstrates the framework's effectiveness in identifying environmentally friendly solvents and balancing trade-offs between solvent toxicity and solubility. Furthermore, the framework informs process design by screening for suitable green solvents in selective dissolution processes, potentially leading to the development of more sustainable dissolution processes and the identification of easily recyclable polymer blends in multilayer plastic films.
2
Shuchi, Nuren, Tyler Adams, V. Paige Stinson, Micheal J. McLamb, Dustin Louisos, Glenn D. Boreman, Michael G. Walter et Tino Hofmann. « Complex Dielectric Function of Photochromic Thiazolothiazole Embedded Polymers Determined by Spectroscopic Ellipsometry ». Dans CLEO : Applications and Technology, JTh2A.10. Washington, D.C. : Optica Publishing Group, 2024. http://dx.doi.org/10.1364/cleo_at.2024.jth2a.10.
Texte intégralRésumé :
The complex dielectric function of photochromic thiazolo[5,4-d]thiazole embedded in polymer is reported in the spectral range from 0.8 to 2.5 eV. Strong absorption bands in the measured spectral range are observed depending upon its redox states.
3
Mungu�a-L�pez, Aurora del C., Panzheng Zhou, Ugochukwu M. Ikegwu, Reid C. Van Lehn et Victor M. Zavala. « A Fast Computational Framework for the Design of Solvent-Based Plastic Recycling Processes ». Dans Foundations of Computer-Aided Process Design, 814–19. Hamilton, Canada : PSE Press, 2024. http://dx.doi.org/10.69997/sct.175924.
Texte intégralRésumé :
Multilayer plastic films are widely used in packaging applications because of their unique properties. These materials combine several layers of different polymers to protect food and pharmaceuticals from external factors such as oxygen, water, temperature, and light. Unfortunately, this design complexity also hinders the use of traditional recycling methods, such as mechanical recycling. Solvent-based separation processes are a promising alternative to recover high-quality pure polymers from multilayer film waste. One such process is the Solvent-Targeted Recovery and Precipitation (STRAPTM) process, which uses sequential solvent washes to selectively dissolve and separate the constituent components of multilayer films. The STRAPTM process design (separation sequence, solvents, operating conditions) changes significantly depending on the design of the multilayer film (the number of layers and types of polymers). Quantifying the economic and environmental benefits of alternative process designs is essential to provide insights into sustainable recycling and film (product) design. In this work, we present a fast computational framework that integrates molecular-scale models, process modeling, techno-economic and life-cycle analysis to evaluate STRAPTM designs. The computational framework is general and can be used for complex multilayer films or multicomponent plastic waste streams. We apply the proposed framework to a multilayer film commonly used in industrial food packaging. We identify process design configurations with the lowest economic and environmental impact. Our analysis reveals trends that can help guide process and product design.
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Alisafaei, F., Seyed Hamid Reza Sanei et Chung-Souk Han. « Length Scale Dependent Deformation in Polymers ». Dans ASME 2012 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/imece2012-88060.
Texte intégralRésumé :
Length scale dependent deformation of polymers has been observed in different experiments including micro-beam bending and indentation tests. Here the length scale dependent deformation of polydimethylsiloxane is examined in indentation testing at length scales from microns down to hundreds of nanometers. Strong indentation size effects have been observed in these experiments which are rationalized with rotation gradients that can be related to Frank elasticity type molecular energies known from liquid crystal polymers. To support this notion additional experiments have been conducted where Berkovich and spherical indenter tips results have been compared with each other.
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Sanei, Seyed Hamid Reza, F. Alisafaei et Chung-Souk Han. « Indenter Tip Dependence in the Determination of Elastic Modulus in Polymers ». Dans ASME 2013 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/imece2013-64831.
Texte intégralRésumé :
The two most common outputs of nanoindentation experiment are hardness and elastic modulus. Length scale dependent deformation in polymers has however been observed in different experiments such as microbeam bending, torsional thin wires and indentation testing which may affect the mechanical testing. Unlike in metals where the size dependency is attributed to necessary geometry dislocations, the origin of length scale dependent deformation in polymers is not well understood. In this study, elastic modulus of polydimethylsiloxane (PDMS) is determined using both Berkovich and spherical tips. Observing different trends for elastic modulus upon the change of indentation depth using these two different tips brings up the question which tip should be used to get the real mechanical properties of PDMS which is discussed here. Surface roughness, surface effects and the imperfection of the Berkovich indenter tip are negligible at the studied length scale.
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Han, Chung-Souk, Andrew J. Wrucke et Partha Majumdar. « Indentation Depth Dependent Hardness in Polydimethylsiloxane ». Dans ASME 2011 International Mechanical Engineering Congress and Exposition. ASMEDC, 2011. http://dx.doi.org/10.1115/imece2011-64335.
Texte intégralRésumé :
Size dependent deformation in polymers has been observed in various experiments including microbeam bending, foams, composites and indentation. For indentation depths from 100 microns down to hundreds of nanometers strong increases in the hardness has been observed where the hardness has been determined with a Berkovich indenter tip on polydimethylsiloxane. These observations are related to other existing experimental data of the literature and possible rationales for these indentation size effects are discussed.
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Kratzok, Max, Anil Saigal et Michael Zimmerman. « Temperature-Dependent Impact Properties of ABS Polymer ». Dans ASME 2021 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2021. http://dx.doi.org/10.1115/imece2021-71382.
Texte intégralRésumé :
Abstract Polymeric materials are composed of chains of molecules known as monomers which are held together by secondary bonds. Amorphous polymers have a glass transition temperature above which their behavior transitions from glassy to viscoelastic, meaning they act like both a viscous liquid and an elastic solid. This concept may seem familiar to anyone who has used Silly Putty®; bouncing a ball of Silly Putty causes the material to behave elastically whereas it will flow into a puddle if left on a table overnight. Time temperature superposition (TTS) describes the dependence of viscoelastic mechanical properties on time and temperature. Repeating the Silly Putty experiment at a different temperature will change how long it takes to reach the same end mechanical property. The Williams-Landel-Ferry (WLF) equation empirically defines the relationship between a temperature shift and a shift in the timescale for a specific material property. It has been widely used for materials undergoing low rates of strain (e.g. creep, stress relaxation), but it applies to any property of viscoelastic behavior. This paper examines modeling the temperature-dependent impact behavior of polymers based on the WLF equation. Although polymers experience viscoelastic behavior for an incredibly short time prior to fracture, the strong temperature dependence and past literature suggest the validity of the WLF equation to higher rates of strain as demonstrated herein for the energy absorption of acrylonitrile-butadiene-styrene (ABS) undergoing high-velocity multiaxial impact tests.
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Hanselle, Felix, Dennis Kleinschmidt et Florian Brüning. « A Cost-effective Determination of Pressure - and Temperature-Dependent Viscosity of Polymers by Linking Conventional Viscosity Data to PVT Data ». Dans The Nordic Rheology Conference. University of Stavanger, 2024. http://dx.doi.org/10.31265/atnrs.775.
Texte intégralRésumé :
The viscosity of polymer melts is dependent on various factors such as shear rate, temperature, pressure and molecular structure. High-pressure capillary rheometery (HPCR) can be used to determine viscosity as a function of shear rate and temperature in the shear rate range relevant for injection molding and extrusion processing. Conventional HPCR measurements cannot determine the pressure dependence of viscosity so that it is typically neglected. Particularly at high pressures and low shear rates, the viscosity is therefore underestimated. However, it is possible to determine the pressure dependency using a counter pressure chamber or actively controlled counter pressure viscometer. Nevertheless, these devices are rarely available, and the measuring effort is high compared to conventional measurements. In order to be able to represent the pressure-dependent material behavior and thus improve the accuracy of process simulations in a cost-effective way, the aim of this paper is to use the free volume approach via the coupled equations of state according to Simha and Somcynsky1 to link the temperature and pressure dependence of the melt density to the viscosity. The model was extended according to Utracki and Sedlacek2–4 and applied to true viscosity data at constant shear stresses in the process relevant apparent shear rate range from 1 to 5000 1/s. The necessary viscosity data for the investigated PP and PC at different temperatures in the typical processing range were determined using a conventional HPCR, and a pvT measuring device was used to determine the melt density. The hole fraction as a measure for the free volume is calculated at each shear stress through the coupled equations of state and linked to the true viscosity through error square minimization at the mean pressure in the capillary. This allows for the recalculation of an isobaric viscosity curve at different pressure and temperature levels. For validation of the model viscosities were also measured at various pressure levels using a counter pressure chamber to determine an experimental pressure coefficient. The model results for the investigated materials show a high agreement with the experimentally determined pressure coefficients.
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Estler, R. C., et N. S. Nogar. « Mass Spectral Identification of UV-Laser Photoablation Products from Polymers ». Dans Microphysics of Surfaces, Beams, and Adsorbates. Washington, D.C. : Optica Publishing Group, 1987. http://dx.doi.org/10.1364/msba.1987.wa3.
Texte intégralRésumé :
Recently, much attention has been given to the laser photoablation of polymeric films because of applications to the etching processes involved in the microelectronics industry. For a number of compounds, including PMMA ( polymethylmethacrylate), PET (poly (ethylene terephthalate)), polycarbonate, and polyimide, it has been observed1 that short wavelength (193 nm) laser ablation results in precise and clean etching, while long wavelength (≥248 nm) ablation can result in melting, charring and burning. It has been proposed that the differences cited above in the ablative photodecomposition (APD) are a manifestation of the variation of photochemical quantum yields with wavelength, or possibly with intensity. If the quantum yield(s) for various photolytic processes are in fact wavelength dependent, one would expect that dependence to be manifest as a change in the distribution of volatilized products. We report here the direct observation of such quantum yield variations via mass spectrometric studies of the photoablation products.
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Chen, Pin-Chuan, Hong Wang, Daniel S. Park, Sunggook Park, Dimitris E. Nikitopoulos, Steven A. Soper et Michael C. Murphy. « Protein Adsorption in a Continuous Flow Microchannel Environment ». Dans ASME 2008 International Mechanical Engineering Congress and Exposition. ASMEDC, 2008. http://dx.doi.org/10.1115/imece2008-68094.
Texte intégralRésumé :
Protein adsorption is a critical issue in microfluidic devices especially for those reactions depending on proteins like the polymerase chain reaction (PCR). Understanding protein absorption phenomena in different geometry microchannels and evaluating the efficiency of dynamic coating, which has been using as a method to prevent protein adsorption, are important tasks. Two different sets of microchannels were designed and fabricated on polymers. Bovine serum albumin (BSA) was used as a model protein for quantification of and monitoring the protein loss in different microchannel geometries. Up to 58% of the BSA was lost after flowing a 2030 mm long microchannel. The BSA adsorption rate changed along the microchannel. Smaller microchannels required a longer time to achieve protein saturation point. Dynamic coating was shown to be a time consuming and inefficient method to prevent protein adsorption in a continuous flow environment.
Rapports d'organisations sur le sujet "ARN Dependent ARN Polymerase"
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Gal-On, Amit, Shou-Wei Ding, Victor P. Gaba et Harry S. Paris. role of RNA-dependent RNA polymerase 1 in plant virus defense. United States Department of Agriculture, janvier 2012. http://dx.doi.org/10.32747/2012.7597919.bard.
Texte intégralRésumé :
Objectives: Our BARD proposal on the impact of RNA-dependent RNA polymerase 1 (RDR1) in plant defense against viruses was divided into four original objectives. 1. To examine whether a high level of dsRNA expression can stimulate RDR1 transcription independent of salicylic acid (SA) concentration. 2. To determine whether the high or low level of RDR1 transcript accumulation observed in virus resistant and susceptible cultivars is associated with viral resistance and susceptibility. 3. To define the biogenesis and function of RDR1-dependent endogenous siRNAs. 4. To understand why Cucumber mosaic virus (CMV) can overcome RDR1-dependent resistance. The objectives were slightly changed due to the unique finding that cucumber has four different RDR1 genes. Background to the topic: RDR1 is a key plant defense against viruses. RDR1 is induced by virus infection and produces viral and plant dsRNAs which are processed by DICERs to siRNAs. siRNAs guide specific viral and plant RNA cleavage or serve as primers for secondary amplification of viral-dsRNA by RDR. The proposal is based on our preliminary results that a. the association of siRNA and RDR1 accumulation with multiple virus resistance, and b. that virus infection induced the RDR1-dependent production of a new class of endogenous siRNAs. However, the precise mechanisms underlying RDR1 induction and siRNA biogenesis due to virus infection remain to be discovered in plants. Major conclusions, solutions and achievements: We found that in the cucurbit family (cucumber, melon, squash, watermelon) there are 3-4 RDR1 genes not documented in other plant families. This important finding required a change in the emphasis of our objectives. We characterized 4 RDR1s in cucumber and 3 in melon. We demonstrated that in cucumber RDR1b is apparently a new broad spectrum virus resistance gene, independent of SA. In melon RDR1b is truncated, and therefore is assumed to be the reason that melon is highly susceptible to many viruses. RDR1c is dramatically induced due to DNA and RNA virus infection, and inhibition of RDR1c expression led to increased virus accumulation which suggested its important on gene silencing/defense mechanism. We show that induction of antiviral RNAi in Arabidopsis is associated with production of a genetically distinct class of virus-activated siRNAs (vasiRNAs) by RNA dependent RNA polymerase-1 targeting hundreds of host genes for RNA silencing by Argonaute-2. Production of vasiRNAs is induced by viruses from two different super groups of RNA virus families, targeted for inhibition by CMV, and correlated with virus resistance independently of viral siRNAs. We propose that antiviral RNAi activate broad-spectrum antiviral activity via widespread silencing of host genes directed by vasiRNAs, in addition to specific antiviral defense Implications both scientific and agricultural: The RDR1b (resistance) gene can now be used as a transcription marker for broad virus resistance. The discovery of vasiRNAs expands the repertoire of siRNAs and suggests that the siRNA-processing activity of Dicer proteins may play a more important role in the regulation of plant and animal gene expression than is currently known. We assume that precise screening of the vasiRNA host targets will lead in the near future for identification of plant genes associate with virus diseases and perhaps other pathogens.
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Vakharia, Vikram, Shoshana Arad, Yonathan Zohar, Yacob Weinstein, Shamila Yusuff et Arun Ammayappan. Development of Fish Edible Vaccines on the Yeast and Redmicroalgae Platforms. United States Department of Agriculture, février 2013. http://dx.doi.org/10.32747/2013.7699839.bard.
Texte intégralRésumé :
Betanodaviruses are causative agents of viral nervous necrosis (VNN), a devastating disease of cultured marine fish worldwide. Betanodavirus (BTN) genome is composed of two single-stranded, positive-sense RNA molecules. The larger genomic segment, RNA1 (3.1 kb), encodes the RNA-dependent RNA polymerase, while the smaller genomic segment, RNA 2 (1.4kb), encodes the coat protein. This structural protein is the host-protective antigen of VNN which assembles to form virus-like particles (VLPs). BTNs are classified into four genotypes, designated red-spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV), and striped jack nervous necrosis virus (SJNNV), based on phylogenetic analysis of the coat protein sequences. RGNNV type is quite important as it has a broad host-range, infecting warm-water fish species. At present, there is no commercial vaccine available to prevent VNN in fish. The general goal of this research was to develop oral fish vaccines in yeast and red microalgae (Porphyridium sp.) against the RGNNV genotype. To achieve this, we planned to clone and sequence the coat protein gene of RGNNV, express the coat protein gene of RGNNV in yeast and red microalgae and evaluate the immune response in fish fed with recombinantVLPs antigens produced in yeast and algae. The collaboration between the Israeli group and the US group, having wide experience in red microalgae biochemistry, molecular genetics and large-scale cultivation, and the development of viral vaccines and eukaryotic protein expression systems, respectively, was synergistic to produce a vaccine for fish that would be cost-effective and efficacious against the betanodavirus infection.
3
Morrison, Mark, et Joshuah Miron. Molecular-Based Analysis of Cellulose Binding Proteins Involved with Adherence to Cellulose by Ruminococcus albus. United States Department of Agriculture, novembre 2000. http://dx.doi.org/10.32747/2000.7695844.bard.
Texte intégralRésumé :
At the beginning of this project, it was clear that R. albus adhered tightly to cellulose and its efficient degradation of this polysaccharide was dependent on micromolar concentrations of phenylacetic acid (PAA) and phenylpropionic acid (PPA). The objectives for our research were: i) to identify how many different kinds of cellulose binding proteins are produced by Ruminococcus albus; ii) to isolate and clone the genes encoding some of these proteins from the same bacterium; iii) to determine where these various proteins were located and; iv) quantify the relative importance of these proteins in affecting the rate and extent to which the bacterium becomes attached to cellulose. BARD support has facilitated a number of breakthroughs relevant to our fundamental understanding of the adhesion process. First, R. albus possesses multiple mechanisms for adhesion to cellulose. The P.I.'s laboratory has discovered a novel cellulose-binding protein (CbpC) that belongs to the Pil-protein family, and in particular, the type 4 fimbrial proteins. We have also obtained genetic and biochemical evidence demonstrating that, in addition to CbpC-mediated adhesion, R. albus also produces a cellulosome-like complex for adhesion. These breakthroughs resulted from the isolation (in Israel and the US) of spontaneously arising mutants of R. albus strains SY3 and 8, which were completely or partially defective in adhesion to cellulose, respectively. While the SY3 mutant strain was incapable of growth with cellulose as the sole carbon source, the strain 8 mutants showed varying abilities to degrade and grow with cellulose. Biochemical and gene cloning experiments have been used in Israel and the US, respectively, to identify what are believed to be key components of a cellulosome. This combination of cellulose adhesion mechanisms has not been identified previously in any bacterium. Second, differential display, reverse transcription polymerase chain reaction (DD RT-PCR) has been developed for use with R. albus. A major limitation to cellulose research has been the intractability of cellulolytic bacteria to genetic manipulation by techniques such as transposon mutagenesis and gene displacement. The P.I.'s successfully developed DD RT- PCR, which expanded the scope of our research beyond the original objectives of the project, and a subset of the transcripts conditionally expressed in response to PAA and PPA have been identified and characterized. Third, proteins immunochemically related to the CbpC protein of R. albus 8 are present in other R. albus strains and F. intestinalis, Western immunoblots have been used to examine additional strains of R. albus, as well as other cellulolytic bacteria of ruminant origin, for production of proteins immunochemically related to the CbpC protein. The results of these experiments showed that R. albus strains SY3, 7 and B199 all possess a protein of ~25 kDa which cross-reacts with polyclonal anti-CbpC antiserum. Several strains of Butyrivibrio fibrisolvens, Ruminococcus flavefaciens strains C- 94 and FD-1, and Fibrobacter succinogenes S85 produced no proteins that cross-react with the same antiserum. Surprisingly though, F. intestinalis strain DR7 does possess a protein(s) of relatively large molecular mass (~200 kDa) that was strongly cross-reactive with the anti- CbpC antiserum. Scientifically, our studies have helped expand the scope of our fundamental understanding of adhesion mechanisms in cellulose-degrading bacteria, and validated the use of RNA-based techniques to examine physiological responses in bacteria that are nor amenable to genetic manipulations. Because efficient fiber hydrolysis by many anaerobic bacteria requires both tight adhesion to substrate and a stable cellulosome, we believe our findings are also the first step in providing the resources needed to achieve our long-term goal of increasing fiber digestibility in animals.
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Grumet, Rebecca, et Benjamin Raccah. Identification of Potyviral Domains Controlling Systemic Infection, Host Range and Aphid Transmission. United States Department of Agriculture, juillet 2000. http://dx.doi.org/10.32747/2000.7695842.bard.
Texte intégralRésumé :
Potyviruses form one of the largest and most economically important groups of plant viruses. Individual potyviruses and their isolates vary in symptom expression, host range, and ability to overcome host resistance genes. Understanding factors influencing these biological characteristics is of agricultural importance for epidemiology and deployment of resistance strategies. Cucurbit crops are subject to severe losses by several potyviruses including the highly aggressive and variable zucchini yellow mosaic virus (ZYMV). In this project we sought to investigate protein domains in ZYMV that influence systemic infection and host range. Particular emphasis was on coat protein (CP), because of known functions in both cell to cell and long distance movement, and helper component-protease (HC-Pro), which has been implicated to play a role in symptom development and long distance movement. These two genes are also essential for aphid mediated transmission, and domains that influence disease development may also influence transmissibility. The objectives of the approved BARD project were to test roles of specific domains in the CP and HC-Pro by making sequence alterations or switches between different isolates and viruses, and testing for infectivity, host range, and aphid transmissibility. These objectives were largely achieved as described below. Finally, we also initiated new research to identify host factors interacting with potyviral proteins and demonstrated interaction between the ZYMV RNA dependent RNA polymerase and host poly-(A)-binding protein (Wang et al., in press). The focus of the CP studies (MSU) was to investigate the role of the highly variable amino terminus (NT) in host range determination and systemic infection. Hybrid ZYMV infectious clones were produced by substituting the CP-NT of ZYMV with either the CP-NT from watermelon mosaic virus (overlapping, but broader host range) or tobacco etch virus (TEV) (non- overlapping host range) (Grumet et al., 2000; Ullah ct al., in prep). Although both hybrid viruses initially established systemic infection, indicating that even the non-cucurbit adapted TEV CP-NT could facilitate long distance transport in cucurbits, after approximately 4-6, the plants inoculated with the TEV-CPNT hybrid exhibited a distinct recovery of reduced symptoms, virus titer, and virus specific protection against secondary infection. These results suggest that the plant recognizes the presence of the TEV CP-NT, which has not been adapted to infection of cucurbits, and initiates defense responses. The CP-NT also appears to play a role in naturally occurring resistance conferred by the zym locus in the cucumber line 'Dina-1'. Patterns of virus accumulation indicated that expression of resistance is developmentally controlled and is due to a block in virus movement. Switches between the core and NT domains of ZYMV-NAA (does not cause veinal chlorosis on 'Dina-1'), and ZYMV-Ct (causes veinal chlorosis), indicated that the resistance response likely involves interaction with the CP-NT (Ullah and Grumet, submitted). At the Volcani Center the main thrust was to identify domains in the HC-Pro that affect symptom expression or aphid transmissibility. From the data reported in the first and second year report and in the attached publications (Peng et al. 1998; Kadouri et al. 1998; Raccah et al. 2000: it was shown that: 1. The mutation from PTK to PAK resulted in milder symptoms of the virus on squash, 2. Two mutations, PAK and ATK, resulted in total loss of helper activity, 3. It was established for the first time that the PTK domain is involved in binding of the HC-Pro to the potyvirus particle, and 4. Some of these experiments required greater amount of HC-Pro, therefore a simpler and more efficient purification method was developed based on Ni2+ resin.
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Schwartz, Bertha, Vaclav Vetvicka, Ofer Danai et Yitzhak Hadar. Increasing the value of mushrooms as functional foods : induction of alpha and beta glucan content via novel cultivation methods. United States Department of Agriculture, janvier 2015. http://dx.doi.org/10.32747/2015.7600033.bard.
Texte intégralRésumé :
During the granting period, we performed the following projects: Firstly, we differentially measured glucan content in several pleurotus mushroom strains. Mushroom polysaccharides are edible polymers that have numerous reported biological functions; the most common effects are attributed to β-glucans. In recent years, it became apparent that the less abundant α-glucans also possess potent effects in various health conditions. In our first study, we explored several Pleurotus species for their total, β and α-glucan content. Pleurotuseryngii was found to have the highest total glucan concentrations and the highest α-glucans proportion. We also found that the stalks (stipe) of the fruit body contained higher glucan content then the caps (pileus). Since mushrooms respond markedly to changes in environmental and growth conditions, we developed cultivation methods aiming to increase the levels of α and β-glucans. Using olive mill solid waste (OMSW) from three-phase olive mills in the cultivation substrate. We were able to enrich the levels mainly of α-glucans. Maximal total glucan concentrations were enhanced up to twice when the growth substrate contained 80% of OMSW compared to no OMSW. Taking together this study demonstrate that Pleurotuseryngii can serve as a potential rich source of glucans for nutritional and medicinal applications and that glucan content in mushroom fruiting bodies can be further enriched by applying OMSW into the cultivation substrate. We then compared the immune-modulating activity of glucans extracted from P. ostreatus and P. eryngii on phagocytosis of peripheral blood neutrophils, and superoxide release from HL-60 cells. The results suggest that the anti-inflammatory properties of these glucans are partially mediated through modulation of neutrophileffector functions (P. eryngiiwas more effective). Additionally, both glucans dose-dependently competed for the anti-Dectin-1 and anti-CR3 antibody binding. We then tested the putative anti-inflammatory effects of the extracted glucans in inflammatory bowel disease (IBD) using the dextran sulfate sodium (DSS)–induced model in mice. The clinical symptoms of IBD were efficiently relieved by the treatment with two different doses of the glucan from both fungi. Glucan fractions, from either P. ostreatus or P. eryngii, markedly prevented TNF-α mediated inflammation in the DSS–induced inflamed intestine. These results suggest that there are variations in glucan preparations from different fungi in their anti-inflammatory ability. In our next study, we tested the effect of glucans on lipopolysaccharide (LPS)-induced production of TNF-α. We demonstrated that glucan extracts are more effective than mill mushroom preparations. Additionally, the effectiveness of stalk-derived glucans were slightly more pronounced than of caps. Cap and stalk glucans from mill or isolated glucan competed dose-dependently with anti-Dectin-and anti-CR-3 antibodies, indicating that they contain β-glucans recognized by these receptors. Using the dextran sulfate sodium (DSS)-inflammatory bowel disease mice model, intestinal inflammatory response to the mill preparations was measured and compared to extracted glucan fractions from caps and stalks. We found that mill and glucan extracts were very effective in downregulatingIFN-γ and MIP-2 levels and that stalk-derived preparations were more effective than from caps. The tested glucans were equally effective in regulating the number of CD14/CD16 monocytes and upregulating the levels of fecal-released IgA to almost normal levels. In conclusion, the most effective glucans in ameliorating some IBD-inflammatory associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii. These spatial distinctions may be helpful in selecting more effective specific anti-inflammatory mushrooms-derived glucans. We additionally tested the effect of glucans on lipopolysaccharide-induced production of TNF-α, which demonstrated stalk-derived glucans were more effective than of caps-derived glucans. Isolated glucans competed with anti-Dectin-1 and anti-CR3 antibodies, indicating that they contain β-glucans recognized by these receptors. In conclusion, the most effective glucans in ameliorating IBD-associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii grown at higher concentrations of OMSW. We conclude that these stress-induced growing conditions may be helpful in selecting more effective glucans derived from edible mushrooms. Based on the findings that we could enhance glucan content in Pleurotuseryngii following cultivation of the mushrooms on a substrate containing different concentrations of olive mill solid waste (OMSW) and that these changes are directly related to the content of OMSW in the growing substrate we tested the extracted glucans in several models. Using dextran sulfate sodium (DSS)–inflammatory bowel disease (IBD) mice model, we measured the colonic inflammatory response to the different glucan preparations. We found that the histology damaging score (HDS) resulting from DSS treatment reach a value of 11.8 ± 2.3 were efficiently downregulated by treatment with the fungal extracted glucans, glucans extracted from stalks cultivated at 20% OMSWdownregulated to a HDS value of 6.4 ± 0.5 and at 80% OMSW showed the strongest effects (5.5 ± 0.6). Similar downregulatory effects were obtained for expression of various intestinal cytokines. All tested glucans were equally effective in regulating the number of CD14/CD16 monocytes from 18.2 ± 2.7 % for DSS to 6.4 ± 2.0 for DSS +glucans extracted from stalks cultivated at 50% OMSW. We finally tested glucans extracted from Pleurotuseryngii grown on a substrate containing increasing concentrations of olive mill solid waste (OMSW) contain greater glucan concentrations as a function of OMSW content. Treatment of rat Intestinal epithelial cells (IEC-6) transiently transfected with Nf-κB fused to luciferase demonstrated that glucans extracted from P. eryngii stalks grown on 80% OMSWdownregulatedTNF-α activation. Glucans from mushrooms grown on 80% OMSW exerted the most significant reducing activity of nitric oxide production in lipopolysaccharide (LPS) treated J774A.1 murine macrophages. The isolated glucans were tested in vivo using the Dextran Sodium Sulfate (DSS) induced colitis in C57Bl/6 mice and found to reduce the histology damaging score resulting from DSS treatment. Expression of various intestinal cytokines were efficiently downregulated by treatment with the fungal extracted glucans. We conclude that the stress-induced growing conditions exerted by OMSW induces production of more effective anti-inflammatory glucans in P. eryngii stalks.
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African Open Science Platform Part 1 : Landscape Study. Academy of Science of South Africa (ASSAf), 2019. http://dx.doi.org/10.17159/assaf.2019/0047.
Texte intégralRésumé :
This report maps the African landscape of Open Science – with a focus on Open Data as a sub-set of Open Science. Data to inform the landscape study were collected through a variety of methods, including surveys, desk research, engagement with a community of practice, networking with stakeholders, participation in conferences, case study presentations, and workshops hosted. Although the majority of African countries (35 of 54) demonstrates commitment to science through its investment in research and development (R&D), academies of science, ministries of science and technology, policies, recognition of research, and participation in the Science Granting Councils Initiative (SGCI), the following countries demonstrate the highest commitment and political willingness to invest in science: Botswana, Ethiopia, Kenya, Senegal, South Africa, Tanzania, and Uganda. In addition to existing policies in Science, Technology and Innovation (STI), the following countries have made progress towards Open Data policies: Botswana, Kenya, Madagascar, Mauritius, South Africa and Uganda. Only two African countries (Kenya and South Africa) at this stage contribute 0.8% of its GDP (Gross Domestic Product) to R&D (Research and Development), which is the closest to the AU’s (African Union’s) suggested 1%. Countries such as Lesotho and Madagascar ranked as 0%, while the R&D expenditure for 24 African countries is unknown. In addition to this, science globally has become fully dependent on stable ICT (Information and Communication Technologies) infrastructure, which includes connectivity/bandwidth, high performance computing facilities and data services. This is especially applicable since countries globally are finding themselves in the midst of the 4th Industrial Revolution (4IR), which is not only “about” data, but which “is” data. According to an article1 by Alan Marcus (2015) (Senior Director, Head of Information Technology and Telecommunications Industries, World Economic Forum), “At its core, data represents a post-industrial opportunity. Its uses have unprecedented complexity, velocity and global reach. As digital communications become ubiquitous, data will rule in a world where nearly everyone and everything is connected in real time. That will require a highly reliable, secure and available infrastructure at its core, and innovation at the edge.” Every industry is affected as part of this revolution – also science. An important component of the digital transformation is “trust” – people must be able to trust that governments and all other industries (including the science sector), adequately handle and protect their data. This requires accountability on a global level, and digital industries must embrace the change and go for a higher standard of protection. “This will reassure consumers and citizens, benefitting the whole digital economy”, says Marcus. A stable and secure information and communication technologies (ICT) infrastructure – currently provided by the National Research and Education Networks (NRENs) – is key to advance collaboration in science. The AfricaConnect2 project (AfricaConnect (2012–2014) and AfricaConnect2 (2016–2018)) through establishing connectivity between National Research and Education Networks (NRENs), is planning to roll out AfricaConnect3 by the end of 2019. The concern however is that selected African governments (with the exception of a few countries such as South Africa, Mozambique, Ethiopia and others) have low awareness of the impact the Internet has today on all societal levels, how much ICT (and the 4th Industrial Revolution) have affected research, and the added value an NREN can bring to higher education and research in addressing the respective needs, which is far more complex than simply providing connectivity. Apart from more commitment and investment in R&D, African governments – to become and remain part of the 4th Industrial Revolution – have no option other than to acknowledge and commit to the role NRENs play in advancing science towards addressing the SDG (Sustainable Development Goals). For successful collaboration and direction, it is fundamental that policies within one country are aligned with one another. Alignment on continental level is crucial for the future Pan-African African Open Science Platform to be successful. Both the HIPSSA ((Harmonization of ICT Policies in Sub-Saharan Africa)3 project and WATRA (the West Africa Telecommunications Regulators Assembly)4, have made progress towards the regulation of the telecom sector, and in particular of bottlenecks which curb the development of competition among ISPs. A study under HIPSSA identified potential bottlenecks in access at an affordable price to the international capacity of submarine cables and suggested means and tools used by regulators to remedy them. Work on the recommended measures and making them operational continues in collaboration with WATRA. In addition to sufficient bandwidth and connectivity, high-performance computing facilities and services in support of data sharing are also required. The South African National Integrated Cyberinfrastructure System5 (NICIS) has made great progress in planning and setting up a cyberinfrastructure ecosystem in support of collaborative science and data sharing. The regional Southern African Development Community6 (SADC) Cyber-infrastructure Framework provides a valuable roadmap towards high-speed Internet, developing human capacity and skills in ICT technologies, high- performance computing and more. The following countries have been identified as having high-performance computing facilities, some as a result of the Square Kilometre Array7 (SKA) partnership: Botswana, Ghana, Kenya, Madagascar, Mozambique, Mauritius, Namibia, South Africa, Tunisia, and Zambia. More and more NRENs – especially the Level 6 NRENs 8 (Algeria, Egypt, Kenya, South Africa, and recently Zambia) – are exploring offering additional services; also in support of data sharing and transfer. The following NRENs already allow for running data-intensive applications and sharing of high-end computing assets, bio-modelling and computation on high-performance/ supercomputers: KENET (Kenya), TENET (South Africa), RENU (Uganda), ZAMREN (Zambia), EUN (Egypt) and ARN (Algeria). Fifteen higher education training institutions from eight African countries (Botswana, Benin, Kenya, Nigeria, Rwanda, South Africa, Sudan, and Tanzania) have been identified as offering formal courses on data science. In addition to formal degrees, a number of international short courses have been developed and free international online courses are also available as an option to build capacity and integrate as part of curricula. The small number of higher education or research intensive institutions offering data science is however insufficient, and there is a desperate need for more training in data science. The CODATA-RDA Schools of Research Data Science aim at addressing the continental need for foundational data skills across all disciplines, along with training conducted by The Carpentries 9 programme (specifically Data Carpentry 10 ). Thus far, CODATA-RDA schools in collaboration with AOSP, integrating content from Data Carpentry, were presented in Rwanda (in 2018), and during17-29 June 2019, in Ethiopia. Awareness regarding Open Science (including Open Data) is evident through the 12 Open Science-related Open Access/Open Data/Open Science declarations and agreements endorsed or signed by African governments; 200 Open Access journals from Africa registered on the Directory of Open Access Journals (DOAJ); 174 Open Access institutional research repositories registered on openDOAR (Directory of Open Access Repositories); 33 Open Access/Open Science policies registered on ROARMAP (Registry of Open Access Repository Mandates and Policies); 24 data repositories registered with the Registry of Data Repositories (re3data.org) (although the pilot project identified 66 research data repositories); and one data repository assigned the CoreTrustSeal. Although this is a start, far more needs to be done to align African data curation and research practices with global standards. Funding to conduct research remains a challenge. African researchers mostly fund their own research, and there are little incentives for them to make their research and accompanying data sets openly accessible. Funding and peer recognition, along with an enabling research environment conducive for research, are regarded as major incentives. The landscape report concludes with a number of concerns towards sharing research data openly, as well as challenges in terms of Open Data policy, ICT infrastructure supportive of data sharing, capacity building, lack of skills, and the need for incentives. Although great progress has been made in terms of Open Science and Open Data practices, more awareness needs to be created and further advocacy efforts are required for buy-in from African governments. A federated African Open Science Platform (AOSP) will not only encourage more collaboration among researchers in addressing the SDGs, but it will also benefit the many stakeholders identified as part of the pilot phase. The time is now, for governments in Africa, to acknowledge the important role of science in general, but specifically Open Science and Open Data, through developing and aligning the relevant policies, investing in an ICT infrastructure conducive for data sharing through committing funding to making NRENs financially sustainable, incentivising open research practices by scientists, and creating opportunities for more scientists and stakeholders across all disciplines to be trained in data management.