186 research outputs found
Cellular, molecular and functional characterisation of YAC transgenic mouse models of Friedreich Ataxia
Full text linkCopyright © 2014 Anjomani Virmouni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.This article has been made available through the Brunel Open Access Publishing Fund.Background - Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene. We have previously established and performed preliminary characterisation of several human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing GAA repeat expansions, Y47R (9 GAA repeats), YG8R (90 and 190 GAA repeats) and YG22R (190 GAA repeats).
Methodology/Principal Findings - We now report extended cellular, molecular and functional characterisation of these FXN YAC transgenic mouse models. FXN transgene copy number analysis of the FRDA mice demonstrated that the YG22R and Y47R lines each have a single copy of the FXN transgene while the YG8R line has two copies. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We identified significant functional deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8R and YG22R FRDA mice compared to Y47R and wild-type control mice. We also confirmed increased somatic GAA repeat instability in the cerebellum and brain of YG22R and YG8R mice, together with significantly reduced levels of FXN mRNA and protein in the brain and liver of YG8R and YG22R compared to Y47R.
Conclusions/Significance - Together these studies provide a detailed characterisation of our GAA repeat expansion-based YAC transgenic FRDA mouse models that will help investigations of FRDA disease mechanisms and therapy.European Union, Ataxia UK and FARA
Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia
Full text linkCopyright @ 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.Muscular Dystrophy Association (USA), the National Health and Medical Research Council (Australia), the Friedreich’s Ataxia Research Alliance (USA), the Brockhoff Foundation (Australia), the Friedreich Ataxia Research Association (Australasia), Seek A Miracle (USA) and the Victorian Government’s Operational Infrastructure Support Program
Friedreich ataxia patient tissues exhibit increased 5-hydroxymethylcytosine modification and decreased CTCF binding at the FXN locus
Full text link© 2013 Al-Mahdawi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,distribution, and reproduction in any medium, provided the original author and source are credited.This article has been made available through the Brunel Open Access Publishing Fund.Friedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, which induces epigenetic changes and FXN gene silencing. Bisulfite sequencing studies have identified 5-methylcytosine (5 mC) DNA methylation as one of the epigenetic changes that may be involved in this process. However, analysis of samples by bisulfite sequencing is a time-consuming procedure. In addition, it has recently been shown that 5-hydroxymethylcytosine (5 hmC) is also present in mammalian DNA, and bisulfite sequencing cannot distinguish between 5 hmC and 5 mC.The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 242193/EFACTS (CS), the Wellcome Trust [089757] (SA) and Ataxia UK (RMP) to MAP
Nilai-nilai Moral yang Terdapat dalam Cerita Rakyat Ine Pare dan Pemanfaatan Pendidikan Karakter untuk Siswa Kelas VII SMP Negeri II Ende
No full textABSTRAKGaa, Alfonsus. 2014. Nilai-nilai Moral yang Terdapat dalam Cerita Rakyat Ine Pare dan Pemanfaatan Pendidikan Karakter untuk Siswa Kelas VII SMP Negeri II EndeTesis, Magister Pendidikan Bahasa Indonesia, Program Pascasarjana Universitas Negeri Malang (UM). Pembimbing: (I) Prof. Dr. Abdus Syukur Ghazali, M.Pd, (II) Prof. Dr. Heri Suwignyo, M.Pd. Kata Kunci: nilai Moral dan pemanfaatan pendidikan karakter siswa.Cerita rakyat Ine Pare dibangun dengan melibatkan unsur-unsur kebudayaan kehidupan masyarakat etnis Ende Lio yang mempunyai adat istiadat dan tradisi tertentu. Cerita rakyat Ine Pare, apa pun bentuknya tidak lepas dari masyarakat pendukungnya. Nilai-nilai budaya yang terdapat dalam cerita rakyat Ine Pare terdiri dari, nilai moral religius, nilai moral individu, nilai moral sosial, dan pemanfaatannya untuk pendidikan karakter siswa. Tujuan umum penelitian ini, yakni, mendeskripsikan nilai moral religius, nilai moral individu, nilai moral sosial, dan pemanfaatannya untuk pendidikan karakter siswa. Penelitian ini merupakan penelitian kualitatif dengan pendekatan etnografi.Data penelitian ini berupa data lisan, yaitu, cerita rakyat Ine Pare yang mengandung nilai-nilai moral dan pemanfaatannya untuk pendidikan karakter bagi siswa. Sumber data penelitian ini adalah tua-tua adat atau sesepu suku Lio. Teknik atau cara mengumpulkan, yakni, (1) peneliti melakukan kegiatan wawancara atau merekam, (2) peneliti mengidentifikasi dan mengkode data sesuai dengan aspek yang dikajian, dan (3) penelitian mengklasifikasi data yang telah diperoleh pada kegiatan kedua di atas yang meliputi (1) nilai moral religius, (2) nilai moral individu, (3) nilai moral sosial dan pemanfaatan pendidikan karakter siswa.Berdasarkan analisis data ditemukan bahwa nilai budaya masyarakat etnis Ende Lio dalam kajian ini yang meliputi nilai moral religius, nilai moral individu, dan nilai moral sosial dalam cerita rakyat Ine Pare yang berkaitan dengan pendidikan karakter, nilai religius terdiri atas: (1) percaya kepada Tuhan merupakan suatu sikap yang mempunyai keyakinan atau kepercayaan adanya Tuhan, (2) bersyukur merupakan suatu sikap yang menggucapkan berterima kasih pada Tuhan, dan (3) percaya kepada takdir merupakan suatu sikap dalam kehidupan mempunyai keyakinan bahwa masih adanya ketetapan Tuhan.Nilai moral individu positif, meliputi: (1) kejujuran merupakan sikap dan perilaku yang mencerminkan kesatuan antara pengetahuan, perkataan dan perbuatan (mengetahui yang benar, mengatakan yang benar dan melakukan yang benar), sehingga menjadikan orang yang bersangkutan sebagai pribadi yang dapat dipercaya, (2) kesabaran merupakan suatu sikap tetap dan kuat dalam menghadapi berbagai cobaan, (3) berjiwa besar merupakan suatu sikap mengakui kehebatan, mengakui kesalahan dan kehebatan orang lain, (4) kerja keras merupakan suatu sikap harus berusaha untuk memperbaiki kehidupan dalam hidupnya, (5) mandiri merupakan sikap dan perilaku yang tidak tergantung pada orang lain dalam menyelesaikan berbagai tugas maupun persoalan, dan nilai moral individu negatif meliputi: (1) tidak jujur merupakan suatu sikap dan tingkah laku perbuatannya tidak benar, dan (3) sirik menggambarkan suatu sikap iri hati dan dengki kepada orang lain.Nilai moral sosial tersebut, meliputi: (1) musyawarah merupakan saling memaafkan, saling menasehati antara satu sama lain dalam hidupnya. (2) rela berkorban merupakan suatu sikap yang selalu memberikan bantuan kepada orang lain, dan (3) kebijaksanaan merupakan suatu sikap yang selalu menggunakan akal budinya. Bagi kalangan pendidik, representasi nilai budaya masyarakat etnis Ende Lio ini bisa digunakan sebagai salah satu alternatif bahan pembelajaran bahasa, sastra yang berkaitan dengan pendidikan karakter, budaya, dan etika di antaranya dari sisi pengunaan bahasa dan retorika dalam karya sastra, nilai budaya daerah, dan budi pekerti luhur. Adapun Pemahaman siswa terhadap implementasi yang akan diajarkan oleh guru di sekiolah antara lain: nilai moral religius, nilai moral individu, dan nilai moral sosial yang terdapat pada cerita rakyat Ine Pare. Hasil penelitian ini bisa digunakan sebagai titik tolak untuk penelitian lebih lanjut, misalnya masalah nilai moral budaya yang lain pada masyarakat etnis Ende Lio yang belum terjangkau dalam penelitian ini dan pemakaian pendekatan yang lain atau pendekatan yang berbeda
Stalled DNA Replication Forks at the Endogenous GAA Repeats Drive Repeat Expansion in Friedreich’s Ataxia Cells
Full text linkSummaryFriedreich’s ataxia (FRDA) is caused by the expansion of GAA repeats located in the Frataxin (FXN) gene. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing to disease progression. The mechanism leading to repeat expansion and decreased FXN transcription remains unclear. Using single-molecule analysis of replicated DNA, we detected that expanded GAA repeats present a substantial obstacle for the replication machinery at the FXN locus in FRDA cells. Furthermore, aberrant origin activation and lack of a proper stress response to rescue the stalled forks in FRDA cells cause an increase in 3′-5′ progressing forks, which could enhance repeat expansion and hinder FXN transcription by head-on collision with RNA polymerases. Treatment of FRDA cells with GAA-specific polyamides rescues DNA replication fork stalling and alleviates expansion of the GAA repeats, implicating DNA triplexes as a replication impediment and suggesting that fork stalling might be a therapeutic target for FRDA
Complete nanopore repeat sequencing of SCA27B (GAA-FGF14 ataxia) in Japanese
No full textBackground Although pure GAA expansion is considered pathogenic in SCA27B, non-GAA repeat motif is mostly mixed into longer repeat sequences. This study aimed to unravel the complete sequencing of FGF14 repeat expansion to elucidate its repeat motifs and pathogenicity. Methods We screened FGF14 repeat expansion in a Japanese cohort of 460 molecularly undiagnosed adult-onset cerebellar ataxia patients and 1022 controls, together with 92 non-Japanese controls, and performed nanopore sequencing of FGF14 repeat expansion. Results In the Japanese population, the GCA motif was predominantly observed as the non-GAA motif, whereas the GGA motif was frequently detected in non-Japanese controls. The 5′-common flanking variant was observed in all Japanese GAA repeat alleles within normal length, demonstrating its meiotic stability against repeat expansion. In both patients and controls, pure GAA repeat was up to 400 units in length, whereas non-pathogenic GAA-GCA repeat was larger, up to 900 units, but they evolved from different haplotypes, as rs534066520, located just upstream of the repeat sequence, completely discriminated them. Both (GAA)≥250 and (GAA)≥200 were enriched in patients, whereas (GAA-GCA)≥200 was similarly observed in patients and controls, suggesting the pathogenic threshold of (GAA)≥200 for cerebellar ataxia. We identified 14 patients with SCA27B (3.0%), but their single-nucleotide polymorphism genotype indicated different founder alleles between Japanese and Caucasians. The low prevalence of SCA27B in Japanese may be due to the lower allele frequency of (GAA)≥250 in the Japanese population than in Caucasians (0.15% vs 0.32%–1.26%). Conclusions FGF14 repeat expansion has unique features of pathogenicity and allelic origin, as revealed by a single ethnic study
Pms2 suppresses large expansions of the (GAA·TTC)n sequence in neuronal tissues
No full textCopyright @ 2012 Bourn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Expanded trinucleotide repeat sequences are the cause of several inherited neurodegenerative diseases. Disease pathogenesis is correlated with several features of somatic instability of these sequences, including further large expansions in postmitotic tissues. The presence of somatic expansions in postmitotic tissues is consistent with DNA repair being a major determinant of somatic instability. Indeed, proteins in the mismatch repair (MMR) pathway are required for instability of the expanded (CAG·CTG)(n) sequence, likely via recognition of intrastrand hairpins by MutSβ. It is not clear if or how MMR would affect instability of disease-causing expanded trinucleotide repeat sequences that adopt secondary structures other than hairpins, such as the triplex/R-loop forming (GAA·TTC)(n) sequence that causes Friedreich ataxia. We analyzed somatic instability in transgenic mice that carry an expanded (GAA·TTC)(n) sequence in the context of the human FXN locus and lack the individual MMR proteins Msh2, Msh6 or Pms2. The absence of Msh2 or Msh6 resulted in a dramatic reduction in somatic mutations, indicating that mammalian MMR promotes instability of the (GAA·TTC)(n) sequence via MutSα. The absence of Pms2 resulted in increased accumulation of large expansions in the nervous system (cerebellum, cerebrum, and dorsal root ganglia) but not in non-neuronal tissues (heart and kidney), without affecting the prevalence of contractions. Pms2 suppressed large expansions specifically in tissues showing MutSα-dependent somatic instability, suggesting that they may act on the same lesion or structure associated with the expanded (GAA·TTC)(n) sequence. We conclude that Pms2 specifically suppresses large expansions of a pathogenic trinucleotide repeat sequence in neuronal tissues, possibly acting independently of the canonical MMR pathway.IDB was supported by a postdoctoral fellowship from the National Ataxia Foundation. RMP was supported by Ataxia UK. SA was supported by The Wellcome Trust. This research was made possible by grants from the National Institutes of Health (NIH/NINDS) and the Muscular Dystrophy Association to S.I.B
A novel GAA-repeat-expansion-based mouse model of Friedreich's ataxia.
Full text linkCopyright © 2015 The Author(s). Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced levels of frataxin protein. We have previously reported the generation of human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing 90-190 GAA repeats, but the presence of multiple GAA repeats within these mice is considered suboptimal. We now describe the cellular, molecular and behavioural characterisation of a newly developed YAC transgenic FRDA mouse model, designated YG8sR, which we have shown by DNA sequencing to contain a single pure GAA repeat expansion. The founder YG8sR mouse contained 120 GAA repeats but, due to intergenerational expansion, we have now established a colony of YG8sR mice that contain ~200 GAA repeats. We show that YG8sR mice have a single copy of the FXN transgene, which is integrated at a single site as confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We have identified significant behavioural deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8sR FRDA mice compared with control Y47R and wild-type (WT) mice. We have also detected increased somatic GAA repeat instability in the brain and cerebellum of YG8sR mice, together with significantly reduced expression of FXN, FAST-1 and frataxin, and reduced aconitase activity, compared with Y47R mice. Furthermore, we have confirmed the presence of pathological vacuoles within neurons of the dorsal root ganglia (DRG) of YG8sR mice. These novel GAA-repeat-expansion-based YAC transgenic FRDA mice, which exhibit progressive FRDA-like pathology, represent an excellent model for the investigation of FRDA disease mechanisms and therapy.The European Union Seventh Framework Programme [FP7/2007–2013] under grant agreement number 242193/EFACTS (C.S. and M.S.), together with funding from Friedreich’s Ataxia Research Alliance (FARA), Ataxia UK and GoFAR (S.A.V. and V.E.) to M.A.P
Electrostatic Analysis of Gate All Around (GAA) Nanowire over FinFET
Full text linkabstract: CMOS Technology has been scaled down to 7 nm with FinFET replacing planar MOSFET devices. Due to short channel effects, the FinFET structure was developed to provide better electrostatic control on subthreshold leakage and saturation current over planar MOSFETs while having the desired current drive. The FinFET structure has an undoped or fully depleted fin, which supports immunity from random dopant fluctuations (RDF – a phenomenon which causes a reduction in the threshold voltage and is prominent at sub 50 nm tech nodes due to lesser dopant atoms) and thus causes threshold voltage (Vth) roll-off by reducing the Vth. However, as the advanced CMOS technologies are shrinking down to a 5 nm technology node, subthreshold leakage and drain-induced-barrier-lowering (DIBL) are driving the introduction of new metal-oxide-semiconductor field-effect transistor (MOSFET) structures to improve performance. GAA field effect transistors are shown to be the potential candidates for these advanced nodes. In nanowire devices, due to the presence of the gate on all sides of the channel, DIBL should be lower compared to the FinFETs.
A 3-D technology computer aided design (TCAD) device simulation is done to compare the performance of FinFET and GAA nanowire structures with vertically stacked horizontal nanowires. Subthreshold slope, DIBL & saturation current are measured and compared between these devices. The FinFET’s device performance has been matched with the ASAP7 compact model with the impact of tensile and compressive strain on NMOS & PMOS respectively. Metal work function is adjusted for the desired current drive. The nanowires have shown better electrostatic performance over FinFETs with excellent improvement in DIBL and subthreshold slope. This proves that horizontal nanowires can be the potential candidate for 5 nm technology node. A GAA nanowire structure for 5 nm tech node is characterized with a gate length of 15 nm. The structure is scaled down from 7 nm node to 5 nm by using a scaling factor of 0.7.Dissertation/ThesisMasters Thesis Electrical Engineering 201
Performance Investigation of SRAM Cells Based on Gate-all-around (GAA) Si Nanowire Transistor for Ultra-low Voltage Applications
No full textIn this paper, the performance metrics (i.e., read and write margins, operation speed, power consumption) of 6T SRAM cell based on gate-all-around (GAA) Si nanowire transistor (SNWT) at 16nm technology node are investigated, as well as the impacts of device variations on GAA SNWT SRAM cells. The results indicate that GAA SNWT SRAM cells have larger read static noise margin, less power-delay product and better tolerance to process variations than planar bulk SRAM cells. And through cell ratio optimization, the GAA SNWT SRAM cells can satisfy the six-sigma (6 sigma) yield at V-DD=0.3V.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000319824700230&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Engineering, Electrical & ElectronicPhysics, AppliedEICPCI-S(ISTP)
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