Le système RUSH, de l’anglais « Retention using selective hook » est un système développé récemment qui permet d'analyser et de quantifier en temps réel le transport d'une grande diversité de protéines. Le système RUSH permet, grâce à un excès de molécules de Streptavidine (Str.) dirigées dans différents compartiments cellulaires (appelées les hameçons), de retenir des protéines appelées les rapporteurs, comportant un biocapteur fluorescent tel que la GFP (« Green fluorescent protein ») fusionné avec un peptide SBP (« Streptavidin-binding peptide »). L’addition de biotine dans le milieu perturbe l’interaction entre SBP et la Streptavidine, libérant ainsi les rapporteurs de leur hameçon. Basé sur le système RUSH, nous avons établi une méthode de criblage pour identifier des agents pharmacologiques dotés de la capacité à induire la libération d’HMGB1 (« High Mobility Group Box 1 »). La translocation d’HMGB1 depuis le noyau vers le cytoplasme, ainsi que sa sécrétion ou libération passive dans l'espace extracellulaire à travers les membranes plasmiques perméabilisées, représente un signal de danger essentiel à l’activation du système immunitaire. Dans ce système RUSH modifié, une protéine de fusion du Str-NLS3 a été utilisée comme un hameçon nucléaire pour retenir la protéine chimère constituée d'HMGB1, SBP et GFP (HMGB1-SBP-GFP). Lorsque de la biotine est ajoutée en combinaison à des chimiothérapies inductrices de la mort cellulaire immunogène (ICD) telles que les anthracyclines, elle se lie de manière compétitive à Str-NLS3 et permet la libération et la translocation nucléo-cytoplasmique des rapporteurs HMGB1-SBP-GFP. Nous avons utilisé ce système pour des criblages à haut débit visant à identifier des agents induisant le relargage d’HMGB1. Les agents identifiés appartiennent à trois catégories différentes : les inducteurs connus de l’ICD, les inhibiteurs des microtubules et les modificateurs épigénétiques. Leur effet a été confirmé par des méthodes multiples de mesure de la quantité protéique d’HMGB1 nucléaire, cytoplasmique et extracellulaire dans des cellules humaines et murines in vitro ainsi que dans le plasma de souris. Nos données révèlent également que ces agents induisent la libération d’HMGB1 par des mécanismes distincts : arrêt du cycle cellulaire, acétylation des histones ou effets « on-target » par l'inhibition d’ADN méthyltransférase. Il serait alors intéressant d'étudier si les effets décrits ici peuvent contribuer aux effets immunostimulateurs des médicaments utilisés pour le traitement de cancers ou de maladies parasitaires.Le système RUSH permettant la synchronisation et la quantification de la sécrétion des protéines du réticulum endoplasmique (RE) vers l'appareil de Golgi, il permet de cribler un grand nombre de composés afin d’identifier des inhibiteurs des sécrétions candidates. Nous avons conçu et construit une lignée cellulaire humaine exprimant les chimères SBP-GFP sécrétables ainsi que les hameçons Str-KDEL ciblant l’ER ; la biotine permet donc la libération du rapporteur par les voies de sécrétion classiques. Nous avons identifié et validé plusieurs médicaments qui sont capables d’inhiber la sécrétion de protéines : les anti-angineux, les antidépresseurs, les anti-helminthiques, anti-psychotiques, anti-protozoaires, et agents immunosuppresseurs. Ces composés varient dans leur capacité à inhiber la synthèse des protéines et de compromettre la morphologie du RE ou l'intégrité du Golgi. Les données ont ensuite été soumises à une analyse bio-informatique et cette procédure a permis l'identification de quatre groupes en fonction de leur mode d'action. Cette partie démontre la faisabilité et l'utilité d'un nouvel essai de criblage phénotypique basé sur le système RUSH. Nous avons conçu des systèmes de HSC (« High Content Screening ») basés sur le système RUSH, qui ont permis l'identification d'agents pharmacologiques induisant la libération d’HMGB1, ainsi que des inhibiteurs de la sécrétion protéique. / The retention using selective hooks (RUSH) system allows withholding load cargoes with fluorescent biosensor such as green fluorescent proteins (GFP) fused to a streptavidin-binding peptide (SBP) by an excess of streptavidin (Str) molecules that are addressed to different subcellular localizations. Addition of biotin competitively disrupts this interaction, liberating the reporter from its hook. Based on the RUSH system, we developed a screening assay to identify pharmacological agents endowed with HMGB1 (high mobility group box 1) releasing capacities. The translocation of HMGB1 from the nucleus to the cytoplasm and its secretion or passive release through the permeabilized plasma membrane constitutes a major cellular danger signal. Extracellular HMGB1 can interact with specific pattern recognition receptors to stimulate pro-inflammatory and immunostimulatory pathways. In this modified RUSH system, a Str-NLS3 fusion protein was used as a nuclear hook to seize SBP fused with HMGB1 and GFP. When combined with biotin, which competitively binds to Stre-NLS3 to free the HMGB1-SBP-GFP, immunogenic cell death (ICD) inducers such as anthracyclines were able to cause the nucleo-cytoplasmic translocation of HMGB1-SBP-GFP. We used this system for high-content screenings (HCS) to identify HMGB1 releasing agents. Hits fell into three functional categories: known ICD inducers, microtubule inhibitors, and epigenetic modifiers. Their effective action was confirmed by multiple methods monitoring nuclear, cytoplasmic and extracellular HMGB1 pools, both in cultured human or murine cells, as well as in mouse plasma. These agents induced HMGB1 release through a whole set of distinct mechanisms, cell cycle arrest, histone acetylation, or on-target effect. It will be interesting to learn whether such effects may contribute to the immunostimulatory effects of drugs that are used to treat malignant disease or worm infection. For HCS of identification of pharmacological inhibitors of conventional protein secretion, we constructed a human cell line co-expressing soluble secretory-SBP-GFP (ss-SBP-GFP) and Str-KDEL hook within the endoplasmic reticulum (ER) lumen, and biotin addition releases the reporter, ss-SBP-GFP via the conventional Golgi-dependent protein secretion pathway into the culture supernatant. We identified and validated a series of molecularly unrelated drugs including antianginal, antidepressant, anthelmintic, antipsychotic, antiprotozoal and immunosuppressive agents that inhibit protein secretion. These compounds vary in their capacity to suppress protein synthesis and to compromise ER morphology and Golgi integrity, as well as in the degree of reversibility of such effects. These data was then subjected to bioinformatics analysis including correlation analyses, non-supervised hierarchical clustering, and principal component analysis and led to the identification of 4 clusters of agents. We demonstrate the feasibility and utility of a novel RUSH-based phenotypic screening assay. In summary, we built HCS systems based on the improved RUSH sysytem for identification of agents that induce HMGB1 release or inhibit conventional protein secretion.

Master of Science / Department of Plant Pathology / Bernd Friebe / Bikram S. Gill / Common, bread, or hexaploid wheat, Triticum aestivum L. (2n=6x=42, AABBDD), has several relatives in the Triticum/Aegilops complex of the Poaceae family in the Triticeae tribe, which are valuable sources for broadening genetic diversity and may provide genes for disease and pest resistance and general wheat improvement. Other wild relatives of wheat also may be exploited for wheat improvement, such as Haynaldia villosa (L.) Schur. (2n=2x=14, VV). It is a diploid species with resistance to powdery mildew, wheat curl mite colonization, cereal eyespot disease, rust diseases, and wheat spindle streak mosaic virus. H. villosa may harbor many other as yet unidentified traits for wheat improvement. The polyploid nature of bread wheat allows tolerance to genomic changes, because homoeologous chromosomes from other genomes compensate for missing wheat chromosomes. In this experiment, we crossed the disomic alien addition line DA4V (2n=6x=44) with a pair of H. villosa chromosomes added to the wheat chromosome complement with wheat monosomic for chromosome 4D (2n=41) to produce 4D/4V double monosomic plants. According to centric breakage-fusion mechanisms, univalents tend to break at their centromeres at meiotic metaphase I producing telocentric chromosomes with unstable or “sticky” ends that can fuse with the sticky ends of other newly formed telocentric chromosomes. This fusion results in Robertsonian whole-arm translocations that may be compensating if a short arm of one chromosome fuses with a long arm of another. Double monosomic plants were screened cytogenetically and further visualized by genomic in situ hybridization (GISH). Five transfers were identified, including T4DS.4VL and T4VS.4DL translocations, and a T4VS-W.W transfer of unknown wheat origin. These results were confirmed by GISH. The T4DS.4VL and T4VS.4DL translocations are genetically compensating and should be exploited in wheat improvement.

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.

Dissertation (PhD) -- University of Stellenbosch, 2004. / ENGLISH ABSTRACT: In most plants the efficiency of sucrose production and the systemic distribution thereof are the major determinants of growth, development and yield. The factors governing sugar partitioning co-ordinate its distribution in response to intrinsic and environmental signals. These factors include sugar transporters and invertases as well as metabolites, including sucrose and glucose, which function as signalling molecules to modulate gene expression. The genetic transformation of plants and the subsequent development of transgenic lines with disturbed sugar metabolism have made an unprecedented impact on the study of sugar translocation and -partitioning. For instance, the transformation of plants with a yeast-derived invertase targeted to different subcellular compartments has led to the elucidation of several key aspects of sugar metabolism, including phloem loading mechanisms, the regulation of photosynthesis by sugars, the importance of sugar-metabolism compartmentation with regards to sucrose biosynthesis, storage and distribution, as well as the role of cell-wall invertase in phloem unloading and sink strength. In this study, a similar strategy of transgenic plant analysis was employed to expand our insight into the regulation of sugar partitioning. The yeast-invertase Suc2 gene, from Saccharomyces cere visiae , was overexpressed in either the cytosol, vacuole or apoplast of transgenic tobacco plants. These transgenic lines displayed varying increases in invertase activity, altered sugar levels and consequently disturbed sink-source interactions and sugar partitioning. Transgenic lines overproducing the yeast-derived invertase in either the vacuole (Vac-Inv) or apoplast (Apo-Inv) were utilised to analyse the effect of the altered sugar levels in sink and source organs on the expression of sugar transporters, as well as the endogenous cell wall invertase and inhibitors in these plants. Transcript levels of the sucrose transporter NtSUT1 and hexose transporter NtMST1 encoding genes increased significantly in the source leaves and roots of Vac-Inv lines, whereas increased NtMst1 transcript levels were also detected in the roots of Apo-Inv lines. The increased mRNA levels could be correlated to the altered invertase activities and sugar levels in these tissues. It is concluded that NtSUT1 and NtMST1 are differentially regulated by sucrose and/or hexose content on a transcriptional level. Furthermore, the regulatory effect of the altered sugar levels on transporter expression depended on the subcellular compartment in which the yeast invertase was expressed. It would seem that the subcellular compartmentation of sugar metabolism is also fundamental to the regulation of sugar partitioning. The transcription levels of the endogenous cell wall invertase (CWt) and cell wall invertase inhibitor (Cwi-Inh) genes were examined in the various tissues of Apo-Inv and Vac-Inv lines at both the vegetative and flowering growth stages. In comparison with the control lines, the various tissues of the Apo-Inv and Vac-Inv lines displayed altered Cwi and Cwi-Inh expression levels, depending on the sink-source status and growth stage. However, no obvious correlation between the Cwi and Cwi-Inh expression levels and soluble sugar content of these tissues was found. It is suggested that the post-transcriptional and post-translation control of these proteins by sugars might play an important role in their regulation. Analysis of the Cwi:Cwi-lnh mRNA ratio and growth observations of the various tissues of control as well as Apo-Inv and Vac-Inv lines indicated that this transcription ratio could be an accurate indicator of the sink strength of sink organs. In addition, the influence of sink-source interactions on sugar partitioning was investigated. Reciprocal grafting between Apo-Inv and control lines resulted in scions with an altered sucrose metabolism in either the sink or source organs. These scions were subjected to biomass distribution, soluble sugar quantification and C4C]- radiolabelling experiments. The latter revealed an unaltered state of sugar partitioning from the above-ground tissues of the Apo/GUS scions and a significant shift in sugar partitioning towards the roots of the GUS/Apo scions in comparison to the control GUS/GUS scions. Phenotypic changes, opposite to those observed in Apo-Inv lines expressing the heterologous invertase in both sink and source organs, could initially be observed in the GUS/Apo and Apo/GUS scions. However, no significant differences in phenotype or biomass distribution could be observed between the mature GUS/Apo, Apo/GUS and GUS/GUS scions seven weeks postgrafting. This inconsistency between phenotype and sugar partitioning might be explained by an increase in the respiration rate of the tissues as supported by the soluble sugar content. These results highlight the complexity and adaptability of sucrose metabolism and sugar partitioning. In addition, it confirms that sugar partitioning can be modulated by sink-source interactions and emphasise the importance of invertases in the regulation of sugar partitioning through its ability to alter sink strength. This study forms part of the rapidly expanding initiative to unravel the control mechanisms of sugar partitioning. The results obtained in this study confirmed again that the introduction and expression of a single heterologous gene in transgenic plants could provide significant insight into the regulation of this process. It was shown here that the expression of sugar transporters is closely regulated by sugar levels and therefore fulfils a vital function in sugar sensing and consequently the regulation of sugar partitioning. The data presented in this study also demonstrated the intricate and flexible nature of the relationship that exists between sugar metabolism, partitioning and growth phenomena. / AFRIKAANSE OPSOMMING: Die doeltreffendheid van sukroseproduksie, tesame met die sistemiese verspreiding daarvan, is die vernaamste faktore wat die groei, ontwikkeling en opbrengsvermoë van die meeste plante bepaal. Die faktore wat suikerverdeling beheer, funksioneer om suikerverspreiding te koordineer in reaksie op beide inherente- en omgewingsseine. Hierdie faktore sluit suikertransporters en invertases in, asook metaboliete soos sukrose en glukose wat funksioneer as seinmolekule in die modulering van geenuitdrukking. Die genetiese transformasie van plante en die gevolglike daarstelling van transgeniese lyne met veranderde suikermetabolismes het 'n beduidende inwerking op die bestudering van suikervervoer en -verdeling gehad. Byvoorbeeld, die transformasie van plante met 'n gis-invertase geteiken na verskillende sub-sellulêre kompartemente, het tot die toeligting van verskeie aspekte van suikermetabolisme gelei, insluitende dié van floëemladingsmeganismes, die regulering van fotosintese deur suikers, die belang van kompartementalisering ten opsigte van sukrosebiosintese, -opberging en -verspreiding, en die rol van selwand-invertases in floëemontlaaiing en swelgpuntkrag. In hierdie studie is van soortgelyke transgeniese plantontledings gebruik gemaak om 'n dieper insig tot die regulering van suikerverdeling te verkry. Die gis-invertase Suc2 geen, afkomstig van Saccharomyces cerevisiae, is ooruitgedruk in óf die sitosol, vakuool óf apoplastiese ruimte van transgeniese tabakplante. Hierdie transgeniese lyne het wisselende toenames in invertase-aktiwiteite en veranderde suikervlakke getoon, asook gevolglike versteurde bron-swelgpunt interaksies en suikerverdeling. Transgeniese lyne met ooruitdrukking van die gis-invertase in óf die vakuool (Vac-Inv) óf die apoplast (Apo-Inv) is gebruik om die gevolg van die veranderde suikervlakke in bron- en swelgpuntorgane op die uitdrukking van suikertransporters, asook die endogene selwand-invertase en invertase-inhibitor in hierdie plante te bepaal. Transkripsievlakke van die sukrosetransporter NtSut1 en die heksosetransporter, NtMst1, het beduidend toegeneem in die bron-blare en wortels van die Vac-Inv lyne; 'n toename in NtMst1 transkripsievlakke is ook in die wortels van Apo-Inv lyne bevestig. Die toenames in boodskapper RNA kon gekorreleer word met die veranderde invertase-aktiwiteite en suikervlakke in hierdie weefsels. Die gevolgtrekking word gemaak dat NtSUT1 en NtMST1 differensieël gereguleer word op transkripsionele vlak deur die sukrose en/of heksose inhoud van weefsels. Meer nog, die regulerende effek van die veranderde suikervlakke op transporteruitdrukking het afgehang van die subsellulêre kompartement waarin die gis-invertase uitgedruk is. Dit wil dus voorkom dat die subsellulêre kompartementalisering van suikermetabolisme fundamenteel tot die deurgee en waarneming van suikerseine is, met In gevolglike eweneens belangrike rol in die regulering van suikerverdeling. Die transkripsievlakke van beide die endogene selwand-invertase (CWI) en die selwand-invertase-inhibitor (CWI-Inh) enkoderende gene is in verskeie weefsels van die Apo-Inv en Vac-Inv lyne, tydens beide die vegetatiewe- en blomstadia, bestudeer. Die onderskeie weefsels van die Apo-Inv en Vac-Inv lyne het, in vergelyking met die kontrole lyne, veranderde Cwi en Cwi-inh transkripsievlakke getoon wat bepaal is deur bron-swelgpunt status en groeistadium. Geen duidelike korrelasie kon tussen beide Cwi en Cwi-inh uitdrukkingsvlakke en oplosbare suiker inhoud gevind word nie. Daar word voorgestel dat post-transkripsionele en posttranslasionele beheer deur suikers 'n belangrike rol in die regulering van hierdie proteïne speel. Bestudering van die Cwi:Cwi-lnh mRNA verhouding, asook groei verskynsels van die onderskeie weefsels van kontrole en Apo-Inv en Vac-Inv lyne, dui daarop dat hierdie transkripsievlak-verhouding moontlik 'n akkurate aanwyser van die swelgpuntkrag van 'n swelgpuntorgaan kan wees. Voorts is die invloed van bron-swelgpuntorgaan interaksies op suikerverdeling ondersoek. Omgekeerde enting tussen Apo-Inv en kontrole lyne het entlote met gemodifiseerde suikermetabolisme in óf hul bron- óf hul swelgpuntorgane tot gevolg gehad. Hierdie entlote is aan biomassaverspreidings-, oplosbare suiker kwantifisering en C4C]-radiomerking eksperimente onderwerp. Hierdie resultate het gewys dat, in vergelyking met die kontrole (GUS/GUS) ente, daar geen verandering in die status van suikerverdeling vanaf die bogrondse plantdele in die Apo/GUS ente is nie, maar wel 'n beduidende verskuiwing in suikerverdeling na die wortels van die GUS/Apo ente. Fenotipiese veranderinge, wat teenoorgesteld van dié teenwoordig in die Apo- Inv lyne waar die heteroloë invertase in beide bron en swelgpuntorgane uitgedruk word, is aanvanklik in die GUS/Apo en Apo/GUS ente waargeneem. Geen verskille in fenotipe of biomassa-verspreiding kon egter sewe weke na die entings prosedures tussen die GUS/Apo, Apo/GUS and GUS/GUS ente gevind word nie. Dit mag verduidelik word deur 'n moontlike toename in respirasietempo in die betrokke weefsels; die oplosbare suikervlakke wat in die verskillende ente aangeteken is ondersteun dié moontlikheid. Hierdie resultate as geheelonderstreep die kompleksiteit en aanpasbaarheid van suikermetabolisme en -verdeling. Verder bevestig dit dat suikerverdeling beïnvloed kan word deur bron-swelgpunt interaksies, asook die belang van invertases in die regulering van suikerverdeling gegewe die vermoë om swelgpuntkrag te verander. Hierdie studie vorm deel van 'n vinnig groeiende inisiatief om die beheermeganismes van suikerverdeling te ontrafel. Die resultate verkry in hierdie studie bekragtig die belang van rekombinante DNA tegnologie in die bestudering van fundamentele plantprosesse. Die invoeging en uitdrukking van 'n geteikende gisinvertase in transgeniese plante het gelei tot veranderde suikervlakke en bronswelgpunt interaksies in hierdie lyne met die gevolglike ontginning van waardevolle inligting ten opsigte van die regulering van suikerverdeling in reaksie tot interne seine. Daar is aangetoon dat suikertransporters onlosmaakbaar gekoppel is aan die deurgee en waarneming van suikerseine, spesifiek op die vlak van transkripsionele regulering, en dus ook die regulering van suikerverdeling. Voorts wys die resultate op die komplekse en aanpasbare aard van die verhouding wat bestaan tussen suikermetabolisme, -verdeling en groeiverskynsels.

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

The ten extant species in the genus Equus are separated by less than 3.7 million years of evolution. Three lines of investigation were pursued to further characterize equid genome organization. 1.) The Przewalski.s wild horse (E. przewalskii, EPR) has a diploid chromosome number of 2n=66, while the domestic horse (E. caballus, ECA) has 2n=64. A comparative gene map for E. przewalskii was constructed using 46 bacterial artificial chromosome (BAC) probes previously mapped to 38 of 44 E. caballus chromosome arms and ECAX. BAC clones were hybridized to metaphase spreads of E. przewalskii and localized by fluorescent in situ hybridization (FISH). No exceptions to homology between E. przewalskii and E. caballus were identified, except for ECA5, a metacentric chromosome with homology to two acrocentric chromosome pairs, EPR23 and EPR24. 2.) The onager (E. hemionus onager, EHO) has a modal diploid chromosome number 2n=56 and a documented chromosome number polymorphism within its population, resulting in individuals with 2n=55. Construction of a comparative gene map of a 2n=55 onager by FISH using 52 BAC probes previously mapped to 40 of 44 E. caballus chromosome arms and ECAX identified multiple chromosome rearrangements between E. caballus and E. h. onager. 3.) A centric fission (Robertsonian translocation) polymorphism has been documented in 5 of the ten extant equid species, namely, E. h. onager, E. h. kulan, E. kiang, E. africanus somaliensis, and E. quagga burchelli. BAC clones containing equine (E. caballus, ECA) genes SMARCA5 (ECA2q21 homologue to human (HSA) chromosome 4p) and UCHL1 (ECA3q22 homologue to HSA4q) were FISH mapped to metaphase spreads for individuals possessing the chromosome number polymorphism. These probes mapped to a single metacentric chromosome and two unpaired acrocentrics showing that the centric fission polymorphism involves the same homologous chromosome segments in each species and has homology to HSA4. These data suggest the polymorphism is either ancient and conserved within the genus or has occurred recently and independently within each species. Since these species are separated by 1-3 million years of evolution, the persistence of this polymorphism would be remarkable and worthy of further investigations.

La maturation des cellules lymphoïdes T est un processus thymique hautement régulée où se mettent en place de manière ordonnée et successive les réarrangements des loci du TCRδ, γ, β et enfin α. Ceci nécessite des événements de recombinaisons somatiques V(D)J qui font intervenir les protéines RAG1/2, les séquences RSS jouxtant les segments V, D et J et des éléments régulateurs (enhancers) assurant une cis-régulation de ce processus. Le contrôle de la recombinaison V(D)J se fait grâce à divers mécanismes incluant des mécanismes épigénétiques, l'intervention de facteurs de transcription et la conformation/séquence des RSS (la règle 12/23 et la restriction B12/23). Les leucémies aiguës lymphoblastiques T (LAL-T) sont des proliférations malignes de blastes de phénotype thymique immature. Elles présentent des caractéristiques communes avec les progéniteurs T dont elles dérivent. Les anomalies cytogénétiques les plus fréquentes sont des translocations chromosomiques avec les loci TCRα/δ et TCRβ. La dérégulation, par translocation chromosomique, de nombreux oncogènes, comme le gène à homéodomaine TLX, ont été décrits dans les LAL-T. Les mécanismes moléculaires de ces anomalies, et notamment le rôle joué par la machinerie V(D)J ainsi que les mécanismes moléculaires intimes du blocage de différenciation observé restent cependant peu connus. Les leucémies, TLX1 ou TLX3 positive, se caractérisent par un blocage de maturation au stade cortical. Celui-ci se définit notamment par la présence de réarrangements complets du TCRβ, mais sans réarrangement du TCRα ni expression détectable du récepteur membranaire TCRαβ. Par un système de gène rapporteur, nous avons mis en évidence l'action répressive des protéines à homéodomaines TLX1 et TLX3 sur l'Enhancer Alpha (Eα). Puis l'interaction protéique entre ETS1 et TLX1/3 a été mise en évidence in vivo dans des lignées TLX positives. Enfin, nous montrons le recrutement des protéines TLX1/3, via ETS1, sur l'Eα. De manière intéressante, l'inactivation directe (knock-down par shRNA TLX1) ou indirecte (surexpression d'un TCRαβ exogène) de cette répression a pour conséquence une reprise de la différenciation T suivie d'une mort cellulaire par apoptose. De manière notable, cette apoptose est observé uniquement en condition de culture sur OP9DL1. Les cellules restent ainsi dépendantes du blocage de maturation dont la levée semble incompatible avec leur survie malgré l'accumulation de nombreux évènements oncogéniques. Parallèlement, en analysant les translocations TCRα/δ-TLX1 dans les LAL-T et dans le thymus d'individus sains, nous avons identifié un mécanisme d'auto-sélection clonal par addiction oncogénique. L'étude des translocations TCR-oncogènes sur une série de 280 LAL-T, nous a permis de confirmer la survenue des translocations TCRα/δ-oncogènes dans 19% de cas et TCRβ-oncogènes dans 14% des cas de LAL-T. Ce travail descriptif a permis l'identification de quatre nouveaux oncogènes impliqués dans les translocations TCR dans les LAL-T (LEF1, GNAQ, NKX2.4, IL2RB). Le clonage moléculaire des points de cassure a permis de mettre en évidence une absence d'intervention de la machinerie RAG au niveau de la cassure double brin de l'oncogène, soit une translocation de type 2. De façon intéressante, nous montrons un découplage entre le moment de survenue de la translocation (stade DN) et l'activation de l'oncogène (cortical, pré-αβ). Enfin nous avons mise en évidence un mécanisme de dérégulation oncogénique sensiblement différent en fonction du locus du TCR (α/δ ou β) impliqué dans la translocation. Cette étude descriptive, a permis de pointer l'implication fréquente des segments Dδ2 et Dδ3 dans les translocations chromosomiques et nous a amené à analyser les étapes les plus précoces de la thymopoïèse humaine. Nous avons pu mettre en évidence un ordonnement strict des réarrangements précoces de ce locus : Dδ2-Dδ3 précédant toujours les réarrangements impliquant les segments Jδ1. Le premier réarrangement Dδ2-Dδ3 à s'effectuer est détectable au stade de maturation Early Thymic Precursor ETP (CD34+ /CD1a-/CD7+/dim). De façon importante, nous montrons que le facteur de transcription RUNX1 est directement impliqué dans ce remaniement par sa fixation sur le Dδ2-23RSS et son interaction avec RAG1. Ainsi l'inactivation de RUNX1 dans des CD34+ de sang cordon, cultivés en condition de différenciation T a pour conséquence une absence totale de réarrangement Dδ2-Dδ3. L'ensemble de ces données met bien en évidence le rôle majeur de RUNX1 dans l'ordonnement précoce des réarrangements du TCRδ et pointe pour la première fois que ce locus, comme celui du TCRβ, est contrôlé par la restriction B12/23 chez l'Homme contrairement à ce qui est décrit chez la souris. Au final l'ensemble de ces expériences montre que l'oncogenèse et l'ontogénie T sont étroitement liées et qu'ainsi le blocage du processus de maturation physiologique T est un élément central du processus oncogénique dont les cellules leucémiques restent dépendantes pour leur survie. Ces résultats soulignent le potentiel de l'étude des LAL-T pour une meilleure compréhension de l'ontogénie T et ouvrent des perspectives originales de thérapie ciblée " différenciante ".

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.