No description available.

DNA end-resection and nuclear actin-based movements orchestrate clustering of double-strandbreaks (DSBs) into homology-directed repair (HDR) domains. Using genomic approaches, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. Chromosome conformation capture techniques (Hi-C) reveal multi-scale alterations in genome organization following damage, including changes in chromatin insulation and compartmentalization. Nuclear actin polymerization promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements as visualized by high-throughput translocation assays (HTGTS). Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The advent of the synthesis or manufacturing of controlled structures on submicron scales as well as experimental developments enabling the investigation of physics in speci c biological systems at extremely small length scales underlines the need for dealing with the statistical physics of small systems which are geometrically con ned. A typical example of a system for which physical questions can be answered by means of theoretical modelling is the virus, where polymer genetic material is encapsulated in a protein shell. In this project the role of con nement on polymer chains will be investigated. We investigate how the translocation of polymer from one region to another through a small opening depends on various electrolytic, polymer concentration and wall interaction conditions. This is an extension of the simple, purely entropic, picture in that the interaction terms enter the picture. We employ a variational scheme in deriving our results. / AFRIKAANSE OPSOMMING: Sowel die moontlikheid van beheerbare sintese of vervaardiging van strukture op sub-mikrometer lengteskale asook die koms van eksperimentele metodes vir die ondersoek van biologiese stelsels op baie klein lengteskale onderstreep hoe nodig dit is om die statiestiese sika van klein stelsels met geometriese beperkings te verstaan. 'n Tipiese voorbeeld waar teoretiese metodes vir siese vrae aangewend word is 'n virus, waar die polimeriese genetiese materiaal in 'n proteïen skil beweeg. In die huidge projek word die rol van 'n spesi eke geometriese beperking op polimeerkettings ondersoek. Ons ondersoek hoe die oorplasing van 'n polimeer deur 'n klein opening van een gebied na die ander deur verskillende elektrolietiese, polimeer-konsentrasie en wandinteraksie eienskappe afhang. Dit is 'n uitbreiding van die eenvoudige, volledig entropiese beeld vir oorplasing deurdat wisselwerkings ingesluit word. 'n Variasiebeginsel word aangewend om die resultate af te lei.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2009. / Aims and Background: According to the World Health Organization type 2 diabetes will constitute a major global burden of disease within the next few decades. In agreement, reports show that rapid urbanization and lifestyle changes in South Africa are major factors responsible for these projections. Therefore, any perturbations that alter the regulatory steps that control myocardial glucose uptake by the cardiac-enrich glucose transporter, GLUT4, will lead in the development of diabetic cardiomyopathy and cardiac hypertrophy. Although considerable efforts are been put into unraveling molecular mechanisms underlying this process, less is known regarding the spatio-temporal regulation of GLUT4. In light of this, our specific aim was to establish in vitro fluorescence microscopy- and flow cytometry-based models for visualization and assessment of myocardial GLUT4 translocation using H9c2 cardiac-derived myoblasts. After successful establishment of our in vitro-based model for myocardial GLUT4 translocation, our second aim was to determine the role of the hexosamine biosynthetic pathway (HBP) in this process. Here, we employed HBP modulators to alter flux and subsequently evaluate its effect on myocardial GLUT4 translocation. To further strengthen our hypothesis, we also investigated the role of the HBP in hearts of an in vivo type 2 diabetes mouse model. Hypothesis: We hypothesize that increased flux through the HBP impairs myocardial GLUT4 translocation by greater O-linked glycosylation of the insulin signaling pathway, ultimately leading to myocardial insulin resistance. Methods: Rat cardiac-derived H9c2 myoblasts were cultured until ~ 80-90 % confluent for 3 days and thereafter subcultured in Lab-Tek chamber slides (~ 15, 000 cells per well) for 24 hours. Cells were then serum starved for 3 hours by insulin administration of 100 nM for 0, 5 and 30 minutes, respectively. We employed a method to quantify the relative proportion of GLUT4 at the sarcolemma using immunofluorescence microscopy- and flow cytometry-based models for visualization and assessment of myocardial GLUT4 translocation. Using these methods we investigated the role HBP have during GLUT4 translocation. The HBP were then activated through the following: a) high glucose and glutamine concentrations; b) low glucose and glucosamine stimulation; and c) over-expression of the HBP rate- limiting enzyme, i.e. GFAT. Subsequently, cardiac-derived myoblasts were fixed and probed for ~ 24 hours with antibodies specific for intracellular- and membrane-bound GLUT4, anti-myc GLUT4 (9E10) and O-GlcNAc. To assess GLUT4 translocation and O-GlcNAcylation we employed the following secondary antibodies: FITC Green for intracellular-bound GLUT4; and b) Texas Red for membrane-bound GLUT4 (immunofluorescence microscopy) and Phycoerythrin for flow cytometry-based model. Cells were thereafter viewed by multi-dimension imaging using an inverted system microscope (Olympus IX81) and a BD FACS Aria cell sorter for flow cytometric analysis. We also assessed HBP in an in vivo context by probing heart tissue - from insulin resistant db/db mice - with a GFAT monoclonal antibody. Results: The db/db mouse represents an ideal model to confirm our hypothesis in an in vivo context. In agreement, our preliminary results show increased GFAT expression versus heterozygous db/+ controls. Our in vitro model show myocardial GLUT4 translocation at 5 minute peak response when H9c2 cardiac-derived myoblasts were stimulated with 100 nM insulin, and GLUT4 vesicles return to normal after longer insulin stimulatory times (10, 15 and 30 minutes. Myocardial Glut4 v translocation was impaired when cells were stimulated with 100 nM wortmannin. Our transfection based model (immunofluorescence microscopy- and flow cytometry-based models) confirms 5 minute peak response under real time conditions. High glucose concentration (25 mM glucose), glucosamine concentrations (2.5 mM, 5 mM, and 10 mM) and over-expression of GFAT led to an impairment of myocardial GLUT4 translocation. Employment of an HBP activator (50 μM PUGNAc) also caused impairment of myocardial GLUT4 translocation. Myocardial GLUT4 translocation was restored when cells were treated with an HBP inhibitor (40 μM DON). High glucose concentrations (25 mM glucose), glucosamine concentrations (2.5 mM, 5 mM, and 10 mM) and over-expression of GFAT resulted in an increase in O-GlcNAcylation. HBP activation (50 μM PUGNAc) showed an increase in O-GlcNAcylation, while administration of 40 μM DON reversed this effect. Discussion and conclusion: We successfully established an in vitro experimental system to assay myocardial GLUT4 translocation. Our data show that dysregulated flux through the HBP impairs myocardial GLUT4 translocation. It is likely that the HBP becomes dysregulated during the pre-diabetic/early diabetic state and that O-GlcNAcylation of members of the insulin signaling pathway occurs during this stage. This will lead to myocardial insulin resistance, and in the long term, will contribute to the onset of the diabetic cardiomyopathy. Investigations to find unique inhibitors of this maladaptive pathway should therefore result in the development of novel therapeutic agents that will lead to a reduction in the growing global burden of disease for type 2 diabetes and associated cardiovascular diseases.

Thesis (PhD (Agric)) -- Stellenbosch University, 2001. / ENGLISH ABSTRACT: Chromosome arm 7DL of common wheat carries genes for agronomically important traits such as leaf rust, stem rust, Russian wheat aphid and eye spot resistance. Some of these genes occur on introgressed foreign chromatin, which restricts their utility in breeding. The 7DL genetic maps are poorly resolved, which seriously hampers attempts to manipulate the genes and introgressed regions in breeding. This dissertation represents an attempt to improve our knowledge of the relative map positions of three resistance genes that have significant potential for use in local breeding programmes. The leaf rust resistance gene, Lr19, is located on a Thinopyrum ponticum-derived translocation which occupies a large part of the terminal end of 7DL. The translocation also carries genes for less favourable traits such as yellow flour colour. Attempts have been made to reduce the size of the translocation through allosyndetic pairing induction; the primary aims being to remove deleterious genes and to minimise the amount of foreign chromatin associated with Lr19 so it can be recombined with other useful 7DL genes. Twenty-nine 'Indis'-derived Lr 19 deletion mutants were previously produced by gamma irradiation and a physical map was constructed. In this study, the set of mutant lines were further analysed using 144 Sse8387I/Msei and 32 EcoRI/Msel amplified fragment length polymorphism (AFLP) primer combinations. The previous physical map, which was based on five restriction fragment length polymorphism (RFLP) markers and five structural gene loci, was extended and now includes 95 novel AFLP markers (86 Sse8387I/Msei and 9EcoRI!Msel markers), of which seven map close to Lr 19. Most of the deletions could be ordered according to size and the improved map has already been used to characterise shortened recombinant forms of the Lr 19 translocation. An unsuccessful attempt was made to convert one of the seven markers closest to Lr 19 into a sequence-specific marker. However, an AFLP marker located distally from Lr 19 was successfully converted into a sequence-specific marker in collaboration with other researchers. An attempt was also made to map and tag the Russian wheat aphid (RWA) resistance gene, Dn5. A doubled haploid mapping population consisting of 94 lines was created and typed for Dn5, four microsatellite loci and the endopeptidase locus, Ep-Dl. The Dn5 locus mapped 25.4 cM and 28.6 cM distally from Xg.vm111 and Xg.vm437, respectively, but was not linked to Xgwm428, Xgwm3 7 or Ep-Dl. Tagging of Dn5 was attempted by screening twelve homozygous resistant and seven homozygous susceptible F2 lines from a cross between 'Chinese Spring' and 'PI 294994' with 70 Sse8387IIi\1sei AFLP primer combinations. Only two potentially useful polymorphisms (one in coupling and one in repulsion phase) were identified. Conversion of the coupling phase marker to a sequence-specific marker was not successful. The eyespot resistance gene, Pchl , was derived from Triticum ventricosum and is present in the wheat VPM-1. Close association between Pchl and the endopeptidase Ep-Dlb allele has been reported previously. Pchl/Ep-Dl was tagged by screening ten wheat genotypes (each homozygous for the confirmed presence or absence of Pchl and/or Ep-Dl b) with 36 Sse83 87I/ Msei AFLP primer combinations. Three AFLP markers were closely associated with Pchl I Ep-D 1, one of which was targeted for conversion into a sequence-specific marker. The sequence-specific marker contained a microsatellite core motif and was found to be useful for tagging Pchl!Ep-Dl. A genetic distance of 2 cM was calculated between the novel microsatellite marker and Ep-Dl. The microsatellite marker was also polymorphic for the Lr 19 translocation and it was possible to map it between the Wsp-Dl and Sr25 loci. In this dissertation, mapping and/or tagging of three important resistance genes were achieved. Due to the fact that all markers used in these studies were not polymorphic between all of the targeted regions, it was not possible to fully integrate the data obtained for the three regions. / AFRIKAANSE OPSOMMING: Chromosoom arm 7DL van broodkoring dra gene vir agronomies-belangrike kenrnerke soos blaarroes, stamroes, Russiese koringluis en oogvlek weerstand. Sommige van hierdie gene kom voor in blokke spesie-verhaalde chromatien wat hul bruikbaarheid in teling beperk. Die genetiese kaarte van 7DL is swak ontwikkel en dit maak dit baie moeilik om hierdie gene en spesie-verhaalde streke tydens teling te manipuleer. Hierdie proefskrif verteenwoordig 'n paging om kennis van die relatiewe kaart liggings van drie weerstandsgene, met betekenisvolle potensiaal in plaaslike tee! programme, te verbreed. Die blaarroes weerstandsgeen, Lr 19, kom voor op 'n Thinopyrum ponticum-verhaalde translokasie wat 'n groot terminale gedeelte van 7DL beslaan. Die translokasie dra ook gene vir minder gewensde kenrnerke soos gee! meelkleur. Pogings is aangewend om die translokasie deur homoeoloe parings-induksie te verkort. Die doe! was om nadelige gene te verplaas en die hoeveelheid vreemde chromatien geassosieer met Lr 19 te minimiseer sodat dit met ander nuttige gene op 7DL gerekombineer kan word. Nege-en-twintig 'Indis'-verhaalde Lr 19 delesie mutante is vroeer met gamma bestraling geproduseer en gebruik om 'n fisiese kaart op te stel. Teenswoordig is die stel mutante verder ontleed met behulp van 144 Sse8387I!Msei en 32 EcoRII Msel amplifikasie-fragment-lengte-polimorfisme (AFLP) inleier kombinasies. Die bestaande fisiese kaart, wat gebaseer was op vyf restriksie-fragment-lengte-polimorfisme (RFLP) merkers en vyf strukturele geen loki, is uitgebrei en sluit nou 95 unieke AFLP merkers (86 Sse8387I/Msel en 9EcoRI/Msel merkers) in, waarvan sewe naby aan Lr19 karteer. Die meeste van die delesies kon op grond van hulle grootte gegroepeer word en die verbeterde fisiese kaart is alreeds gebruik om verkorte rekombinante vorms van die Lr 19 translokasie te karakteriseer. 'n Onsuksesvolle paging is aangewend om een van die sewe merkers naaste aan Lr 19 om te skakel na 'n volgorde-spesifieke merker. 'n AFLP merker wat distaal van Lr 19 karteer is egter wel suksesvol in samewerking met ander navorsers omgeskakel na 'n volgordespesifieke merker. 'n Paging is ook aangewend om die Russiese koringluis (RKL) weerstandsgeen, Dn5, te karteer en merkers gekoppel aan die geen te identifiseer. 'n Verdubbelde-haplo!ede karteringspopulasie van 94 lyne is geskep en getipeer vir Dn5, vier mikrosatelliet loki en die endopeptidase lokus, Ep-D1. Die Dn5 lokus karteer 25.4 cM en 28.6 cM distaal van Xgwml11 en Xgwm437, respektiewelik, maar was me gekoppel met Xgwm428, Xgwm37 of Ep-D1 me. Twaalf homosigoties weerstandbiedende en sewe homosigoties vatbare F2 lyne uit die kruising: 'Chinese Spring' I 'PI 294994' is met 70 Sse8387VMsel AFLP inleier kombinasies getoets in 'n poging om merkers vir Dn5 te identifiseer. Slegs twee moontlik bruikbare polimorfismes (een in koppelings- en een in repulsie fase ), is ge'identifiseer. Omskakeling van die koppelingsfase merker na 'n volgorde-spesifieke merker was onsuksesvol. Die oogvlek weerstandsgeen, Pch1, is uit Triticum ventricosum oorgedra en kom voor in die koringlyn, VPM-1. Noue koppeling van Pch1 en die endopeptidase alleel, Ep-D1 b, is vantevore gerapporteer. Merkers is vir P chl I Ep-D 1 gevind deur tien koring genoti pes ( elkeen homosigoties vir die bevestigde teenwoordigheid of afwesigheid van Pch1 en/of Ep-D1 b) te toets met 36 Sse83871/kfsel AFLP inleier kombinasies. Drie AFLP merkers is gevind wat nou koppel met Pchl!Ep-D1 , waarvan een gekies is vir omskakeling na 'n volgorde-spesifieke merker. Die volgorde-spesifieke merker het 'n mikrosatelliet kernmotief bevat en was nuttig as merker vir Pch1/Ep-D1. 'n Genetiese afstand van 2 cM is tussen die unieke mikrosatelliet merker en Ep-D1 bereken. Die mikrosatelliet merker was ook polimorfies vir die Lr 19 translokasie en dit is tussen die Wsp-D1 en Sr25 loki gekarteer. Kartering en/of identifikasie van merkers vir drie belangrike weerstandsgene was suksesvol in hierdie studie. Omdat al die merkers wat gebruik is, nie polimorf was tussen al die streke van belang nie, was dit nie moontlik om die data vir elk van die drie streke ten volle te integreer nie.

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

Phenotypic effects and transmission rates of the extra chromosome in interspecific trisomics of Cucurbita moschata cv. Butternut (2n C. moschata + 1 C. palmata chromosome) were compared with those of a primary trisomic of C. moschata. Based on gross morphological similarities, 17 interspecific trisomic lines were placed in six phenotypic groups, suggesting that six different C. palmata chromosomes were recovered. Fruit from one of the interspecific trisomics exhibited the hard rind of C. palmata, indicating that this is a dominant trait carried on one chromosome. Some phenotypic effects of the extra chromosome were similar in both the interspecific and primary trisomics, showing a chromosomal effect due to genic imbalance. Transmission of the extra chromosome through the female ranged from 15% to 32% for the C. palmata chromosomes, and was 44% in the primary trisomic. None of the extra chromosomes were transmitted through the male parent.

No description available.

Abstract not available

published_or_final_version / Pathology / Master / Master of Medical Sciences