Porous carbon nanofibers containing silica-coated iron oxide nanoparticles by carbonisation of electrospun amphiphilic copolymer nanocomposites

Van Niekerk, Marthinus Gerhardus Mynhardt (2019-03)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

Introduction Hirschsprung’s disease (HSCR) and African degenerative leiomyopathy (ADL) are rare gastrointestinal disorders affecting neonates and young children. HSCR is characterised by the absence of intrinsic ganglion cells in the distal segment of the intestine; its aetiology has been linked to cellular and molecular mechanisms associated with the enteric nervous system (ENS) development, ADL on the other hand is a distinctive form of visceral myopathy (VSCM) of uncertain aetiology affecting enteric smooth muscles (ESM) of the distal intestine. Gut motility is a result of highly coordinated contractions by muscle layers, neural network and pacemaker intestinal cells of Cajal whose development are controlled by genetic factors. The aetiology of HSCR has been associated with 15 genes linked to ENS development meanwhile ADL has been linked to environmental factors. Actin gamma 2 (ACTG2) is a gene that encodes the ACTG2 protein which is involved in ESM development. Studying the ACTG2 in HSCR patients may ascertain whether individuals affected by HSCR also display muscular dysfunction thereby providing a possible factor in the recurrence of dysmotility post-surgical resection. Additionally, ACTG2 has been identified as the genetic factor in VSCM pathology; therefore, the study may provide novel information regarding the genetic factors of ADL. Aim This project aims to study the genes associated with the development of enteric nervous system (RET, NRG1, SOX10, EDNRB) and smooth muscle cells (ACTG2) that contribute to HSCR and ADL in the South African neonate population. Methods Seventeen whole blood samples were collected from HSCR participants after informed consent; of which only 14 samples were included for genotyping and 9 samples were selected for RNA analysis based on the quality of extracted DNA and RNA respectively. Five whole blood samples were also collected from ADL patients after informed consent. RNA samples from the HSCR cohort were reverse transcribed and quantitative polymerase chain reaction was performed. DNA samples from HSCR and ADL samples were screened for variants in the ACTG2 exons through bidirectional Sanger sequencing. Novel variants were analysed in silico to ascertain their pathogenicity. Results and Discussion In both HSCR and ADL cohorts the variant K119E/R was observed in 64% (9/14) and 60% (3/5) of the study population respectively; K119E/R is likely to function as a disease modifier as it was also observed in the control samples six out nine individuals. Variants S345L and W357G in exon 10 with probable significant effect in the pathogenesis of ESM were identified in the HSCR cohort only. The ADL cohort had polymorphic intronic variants predicted to shift the exonic splice sites namely g>c -IVS12 exon 3 and c>t -IVS3 exon 5. Differential expression of ENS genes EDNRB, RET, SOX10 and NRG1 associated with ENS development in the HSCR cohort was not achieved due to experimental factors. Conclusion ACTG2 encodes an enteric smooth muscle γ-2 actin which plays a pivotal role in the contractile proteins of ESM, thus the data suggests that a muscular component may exist in HSCR aetiology that should be investigated further in vitro and provides further insights into genetic factors that may contribute to ADL pathogenesis.

Inlieding Hirschsprung se siekte (HSCR) en Afrika-degeneratiewe leiomyopatie (ADL) is skaars gastrointestinale afwykings wat neonate en jong kinders raak. HSCR word gekenmerk deur die afwesigheid van intrinsieke ganglion selle in die distale segment van die dunderm; sy etiologie is gekoppel aan sellulêre en molekulêre meganismes wat verband hou met die ontwikkeling van die enteriese senuweestelsel (ENS). ADL, aan die ander kant, is 'n kenmerkende vorm van viscerale myopatie (VSCM) van onseker etiologie wat enteriese gladdespiere (ESM) van die distale dunderm beïnvloed . Gutmotiliteit is 'n gevolg van hoogs gekoördineerde kontraksies deur spierlae, neurale netwerk en pacemaker-intestinale selle van Cajal wie se ontwikkeling deur genetiese faktore beheer word. Die etiologie van HSCR is geassosieer met 15 gene wat verband hou met ENS-ontwikkeling, terwyl ADL gekoppel is aan omgewingsfaktore. Actin gamma 2 (ACTG2) is 'n geen wat kodeer vir die ACTG2 proteïen wat betrokke is by ESM ontwikkeling. Die studie van die ACTG2 in HSCR pasiënte kan vasstel of individue wat deur HSCR geraak word ook spierafwykings toon en sodoende 'n moontlike faktor in die herhaling van dysmotiliteit post-chirurgiese reseksie bied. Daarbenewens is ACTG2 geïdentifiseer as die genetiese faktor in VSCM patologie; daarom kan die studie nuwe inligting verskaf oor die genetiese faktore van ADL. Doel Hierdie projek poog om die gene wat verband hou met die ontwikkeling van die enteriese senuweestelsel (RET, NRG1, SOX10, EDNRB) en gladdespierselle (ACTG2) te bestudeer wat bydra tot HSCR en ADL in die Suid-Afrikaanse neonaatbevolking. Metodes Sewentien volbloedmonsters is na die ingeligte toestemming van die HSCR-deelnemers afgehaal; waarvan slegs 14 monsters vir genotipering ingesluit is en 9 monsters is gekies vir RNA-analise gebaseer op die gehalte van onttrek DNA en RNA onderskeidelik. Vyf volledige bloedmonsters is ook by ADL-pasiënte ingesamel na ingeligte toestemming. RNA monsters van die HSCR kohort was omgekeerde transkribeerde en kwantitatiewe polimerase kettingreaksie uitgevoer. DNA monsters van HSCR en ADL monsters is gesif vir variante in die ACTG2 exons deur tweerigting Sanger volgorde. Nuwe variante is in siliko geanaliseer om hul patogeniteit te bepaal. Resultate en bespreking In beide HSCR- en ADL-kohorte is die variant K119E / R waargeneem in 64% (9/14) en 60% (3/5) van die studiepopulasie; K119E / R sal waarskynlik as 'n siekteveranderings funksie funksioneer, aangesien dit ook in die kontrolemonsters ses uit nege individue waargeneem word. Variante S345L en W357G in exon 10 met waarskynlike beduidende effek in die patogenese van ESM is slegs in die HSCR kohort geïdentifiseer. Die ADL-kohort het polimorfiese introniese variante voorspel om die eksoniese splytareas te verskuif, naamlik g> c -IVS12 exon 3 en c> t -IVS3 exon 5. Differensiële uitdrukking van ENS gene EDNRB, RET, SOX10 en NRG1 wat verband hou met ENS ontwikkeling in die HSCR kohort is nie bereik as gevolg van eksperimentele faktore nie. Afsluiting ACTG2 enkodeer 'n enteriese gladdespier γ-2 actien wat 'n sleutelrol speel in die kontraktiele proteïene van ESM. Die data dui daarop dat 'n spierkomponent bestaan in HSCR etiologie wat verder in vitro ondersoek behoort te word en verdere insigte in genetiese faktore wat kan bydra tot ADL patogenese.

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