Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type strain for the genus Ampelovirus, family Closteroviridae. There has been only one report that claimed the complete nucleotide sequence of GLRaV-3 (isolate NY-1, AF037268). Here we report the complete sequence of the South African GLRaV-3, isolate GP18 (EU259806) and show a significantly extended 5’ end. We used RLM-RACE to determine the 5’ end of GP18 and found the 5’ UTR to be 737 nt compared to 158 nt in the NY-1 sequence. This extended UTR was found in all other South African isolates of GLRaV-3 that were tested. In two collaborative studies the existence of the extended 5’ UTR was confirmed and further investigated. In the first study (Coetzee et al., 2010), metagenomic data generated by next generation sequencing (Illumina Genome Analyzer II) was analysed for GLRaV-3 specific sequences. Sequences similar to the GP18 isolate confirmed the sequence of the extended 5’ UTR. In the second study (Jooste et al., 2010), three genetic variants were identified and their respective 5’ UTRs studied. Great diversity was observed between the 5’ UTRs of the different genetic variants, however within a variant the 5’ UTR was found to be highly conserved. Grapevine leafroll-associated virus 3 is a positive sense, single stranded RNA virus that has been shown, like other closteroviruses, to produce subgenomic (sg) RNAs during replication. These sgRNAs are deployed for the expression of the ORFs on the 3’ half of the genome. In this study a dsRNA blot confirmed the presence of three, 3’ coterminal sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in GLRaV-3-infected plant material when using a probe directed at the coat protein gene. The specific 5’ terminal nucleotides for these sgRNAs as well as four additional sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were determined by RLM-RACE for GLRaV-3 isolate GP18. The construction of a GLRaV-3 mini-replicon, analogous to RNA1 of Lettuce infectious yellows virus, for the evaluation of putative sg-promoters is also described. / AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ‘n lid van die Closteroviridae familie en die hooflid vir die genus Ampelovirus. Tot dusver was daar net een studie wat die volledige nukleïensuurvolgorde van GLRaV-3 gerapporteer het (isolaat NY-1, AF037268). In hierdie studie rapporteer ons die volledige volgorde van ‘n Suid-Afrikaanse GLRaV-3, isolaat nl. GP18 (EU259806) wat noemenswaardig langer is aan die 5’ kant. RLM-RACE is gebruik om die 5’ eindpunt van GP18 te bepaal en daar is gevind dat die 5’ ongetransleerde streek (UTR) 737 nt lank is in vergelyking met die 158 nt van die NY-1 volgorde. Die verlengde 5’ UTR is gevind in alle Suid-Afrikaanse monsters wat getoets is. Die verlengde 5’ UTR is bevestig en verder bestudeer tydens twee samewerkingsprojekte. In die eerste studie (Coetzee et al., 2010), is metagenomiese data gegenereer deur volgende-generasie volgordebepaling (Illumina Genome Analyzer II) en geanaliseer vir GLRaV-3 spesifieke volgordes. Volgordes soortgelyk aan die GP18 isolaat het die verlengde 5’ UTR volgorde bevestig. In die tweede studie (Jooste et al., 2010), is drie genetiese variante van GLRaV-3 geidentifiseer en hulle onderskeie 5’ UTR volgordes bepaal en bestudeer. Daar is groot diversiteit tussen die 5’ UTRs van die verskillende genetiese variante gevind, maar tussen isolate van dieselfde variant is die volgordes gekonserveerd. Grapevine leafroll-associated virus 3 is ‘n positiewe-sin, enkelstring RNA virus wat al voorheen bewys is om, soos ander closterovirusse, subgenomiese (sg) RNAs te produseer tydens replisering. Hierdie sgRNAs word ingespan vir die uitdrukking van die ORFs op die 3’ helfte van die virusgenoom. In hierdie studie is ‘n dsRNA klad gebruik om die voorkoms van 3’ ko-terminale sgRNAs [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] te bevestig in GLRaV-3 geinfekteerde plantmateriaal deur gebruik te maak van ‘n peiler teen die kapsiedproteïengeen. Die spesifieke 5’ terminale nukleotiedes vir hierdie sgRNAs sowel as vier additionele sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] is bepaal deur gebruik te maak van RLM-RACE op die GLRaV-3 isolaat GP18. Die konstruksie van ‘n GLRaV-3 mini-repliserings konstruk, analoog aan die RNA1 van Lettuce infectious yellows virus, vir die evaluasie van moontlike sg-promotors word ook beskryf.

This thesis applies the ab initio techniques of computational chemistry to studies of molecular clusters containing covalent (strong) or van der Waals (weak) bonds formed in chemistry and biochemistry in the temperature range 10-300 K. Van derWaals complexes with an enthalpy of formation from reactants of less than 25 kJ mol-1 and covalent clusters are described in this thesis. The first group of van der Waals complexes involved the molecule carbon monoxide that possesses a small permanent dipole that could lead to dipole - induced dipole interaction and dipole - dipole interaction with another reactant in addition to dispersion. The substrates investigated were methanimine and cyanogen where endergonic unstable molecules were formed, and the clustering of carbon monoxideon a porphin surface leading to the formation of carbon - carbon fragments. TheFaraday effect was invoked to suggest that this was the original method by which thechirality of the D-sugars was selected. Coordination of imino-compounds on thesame surface involving induction and electrostatic interactions could lead to the preferential formation of L-aziridones, hydrolysable to L-amino-acids.The preferred formation of D-ribose, and the more stable D-2-deoxyribose, andnucleotides polymerisable to deoxyribonucleic acids was described. The second group of van der Waals complexes involved the polymerisation of acetylene molecules, to di- and tri-acetylene complexes where the exchange interaction involved the quadrupole moment of the acetylene radical reacting with acetylene or diacetylene. The reaction of carbon monoxide was extended to include its interaction with diacetylene. The entire potential energy surface for the interaction with diacetylene was investigated. The reaction was shown to be endergonic to produce a reactive species, here postulated to rearrange with a reasonable activation energy toform an aldehyde. The energetics of the formation of diacetylene, triacetylene andhigher polymers was briefly investigated. The reactivity of the acetylene polymeraldehydes with other substrates was briefly investigated. This work has apparently laid a firm basis both, qualitative and quantitative, tounderstand some of the weakest interactions in nature involving the simplest ofreactions that have been important in atmospheric chemistry.

The glycophosphatidylinositol(GPI)-linked membrane protein ART2 is an antigenic determinant for T lymphocytes that regulate the expression of diabetes in the BB/W rat model. Though little is understood of the physiologic role of ART2 on T lymphocytes, ART2 is a member of the mono-ADP-ribosyl transferase subgroup ofthe ADP-ribosyl transferase (ART) protein family. The ART protein family, which traditionally has been divided into mono-ADP-ribosyl transferases (mono-ARTs), poly(ADP)-ribose polymerases (PARPs), and ADP-ribosyl cyclases, influences various aspects of cellular physiology including: apoptosis, DNA damage repair, chromatin remodeling, telomere replication, cellular transport, immune regulation, neuronal function, and bacterial virulence. A structural alignment of ART2.2 with chicken PARP indicated the potential for ART2.2 to catalyze ADP-ribose polymers in an activity thought to be specific to the PARP subgroup and important for their regulation of nuclear processes. Kinetic studies determined that the auto-ADP-ribosyl transferase activity of ART2.2 is multitmeric and heterogeneous in nature. Hydroxylamine-cleaved ADP-ribose moieties from the ART2.2 multimers ran as polymers on a modified sequencing gel, and digestion of the polymers with snake-venom phosphodiesterase produced AMP and the poly(ADP)ribose-specific product, PR-AMP, which was resolved by analytical HPLC and structurally confirmed by ESI-MS. The ratio of AMP to PR-AMP was higher than that of PARP raising the possibility that the ART2.2 polymers had a different branching structure than those of PARP. This alternative branching was confirmed by the presence of ribose phosphate polymers in the snake venom phophodiesterase treated samples. The site of the auto-poly(ADP)-ribose modification was determined to be R185, a residue previously proposed to influence the level of auto-ADP ribosylation of ART2.2 by mutational analysis. These data provide the first demonstration of a hybrid between mono-ARTs and PARPs and are the earliest indication that PARP-like enzymes can exist outside the nucleus and on the cell surface.

Chirality is the property of objects, including molecules, which are not superimposable on their materialized mirror image. Chiral molecules are omnipresent in living organisms and the constituents of biological macromolecules (proteins and nucleic acids) are chiral. Amino-acids (constituting proteins), ribose and 2-deoxy-ribose (the only chiral constituent of RNA and DNA nucleotides respectively) are furthermore generally present in living organisms only under one of their enantiomeric forms. This is referred to as the homochirality of the living world. The origin of this homochirality is still unexplained, even if many partial scenarios have been proposed in the literature. All scenarios involve the creation of a small enantiomeric excess for certain molecules, amplification of this excess and chirality transfer to other chiral molecules. The origin of homochirality on Earth is closely related to the origin of life, and is currently supposed to have preceded life. As no-one will ever be able to directly observe the phenomena which lead to homochirality, and life, on our planet, the only scientific approach to try and help explain how this occurred is to build scenarios, and test them taking into account all available information on the physical and chemical conditions on the primitive Earth (Earth before life appeared). In our work, we investigated three scenarios related to the origin of homochirality on Earth. One of these scenarios also relates to a very precise step of the origin of life: the selection of beta-d-ribofuranose as component of RNA nucleotides.<p><p>Enantiomeric excesses (up to 15 %) of alpha-methylated alpha-amino-acids have been detected in meteorites which fell on Earth during the 20th century. No enantiomeric excess is detected for the corresponding alpha-hydroxy-acids in the same meteoritic samples and small (2% at most) or no enantiomeric excesses have been measured for non-methylated alpha-amino-acids. In the first part of our work, we investigated if photolysis by circularly polarized light (CPL) in space could be at the origin of the presence (or absence) of an enantiomeric excess for these compounds. Experiments to reproduce UV-CPL photolysis are difficult to undertake: they require high-energy circularly polarized photons, hence the use of a synchrotron. In our work, we used quantum mechanical calculations to obtain the electronic circular dichroïsm (ECD) spectra of two -methylated -amino-acids, their corresponding alpha-hydroxy-acids and one non-methylated alpha-amino-acid. Differences are observed between these spectra, and we propose a scenario to explain the experimental measurements reported here above: the enantioselective photolysis, in the gas phase at low temperatures (20K at most), of the alpha-amino-acids by UV-CPL with lambda>210 nm. Under these conditions no photolysis of the alpha-hydroxy-acids would occur. This scenario concerns the first step in the origin of homochirality on Earth: the creation of a small enantiomeric excess for some chiral molecules.<p><p>The second scenario that we investigated relates to the enantiomeric amplification step of the origin of homochirality on Earth, for which the role of the alpha-amino-acid serine has been suggested in the literature. Serine clusters have been observed in the gas phase by mass spectrometry. Among these clusters the octamer has been shown to be a magic number cluster and to have a preference for homochirality. An enantiomeric amplification via cycles of formation and dissociation of the octamer has been suggested. No complete scenario has however been proposed in the literature to explain how this could have occurred on the primitive Earth, but any scenario would most probably include an aqueous phase. We aimed at determining if the homochiral preference of serine octamers also exists in solution and therefore we first investigated if serine octamers exist in solution. For this study, we used nuclear magnetic resonance and infrared spectroscopies, which are well-adapted to the study of molecular assemblies in solution. We were able to demonstrate that most probably serine clusters are not present in solution, and if they are it could only be in extremely low concentration. The scenario suggested in the literature is discussed in the light of our results and of literature data on serine clusters.<p><p>As last hypothesis, we investigated a possible scenario for the selection of beta-d-ribofuranose as component of RNA nucleotides. The currently known prebiotic synthesis pathways to ribose also lead to the formation of many other carbohydrates, and ribose is only a minor product of these syntheses. Our hypothesis is that beta-d-ribofuranose could have been selected through favorable interactions with alpha-amino-acids already present on the primitive Earth under one enantiomeric form. Indeed, it is plausible that a peptidic world emerged before the presence of RNA and that homochiral alpha-amino-acids were present on Earth when RNA was synthesized. Under this hypothesis, we investigated the role that alpha-l-amino-acids could have played in the selection of alpha-d-ribofuranose as component of RNA nucleotides. This work is related to the last step of the origin of homochirality: chirality transfer. Our scenario was investigated via nuclear magnetic resonance studies of the interaction between alpha-amino-acids and carbohydrates. We were able to show that, in the systems that we studied, when an interaction occurs it is very weak (affinity constant less than 1M−1) and non enantioselective. Our results most probably discard the role that alpha-amino-acids alone could have played in the selection of beta-d-ribofuranose as component of RNA nucleotides, but does not discard the role that peptides could have played in this selection. / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

Raman optical activity (ROA) is a powerful probe of the structure and behaviour of biomolecules in aqueous solution for a number of important problems in molecular biology. Although ROA is a very sensitive technique for studying biological samples, it is a very weak effect and the conditions of high concentration and long data collection time required limit its application for a wide range of biological samples. These limitations could possibly be overcome using the principle of surface enhanced Raman scattering (SERS). The combination of ROA with SERS in the form of surface enhanced ROA (SEROA) could be a solution for widening the application of ROA. In the last few years, the generation of reliable SEROA spectra of biomolecules has been problematic due to non-homogenous colloidal systems forming and low signal-to-noise ratios which complicated detection of the true SEROA signal from the analyte. L- and D-enantiomers give full or partially mirror image chiroptical spectra, this property of enantiomers can be employed to prove the chiroptical activity of the SEROA technique. In this thesis we employed a hydrophilic polycarbopol polymer as stabilising media which has led to the first report of mirror image SEROA bands for enantiomeric structures. This new technique of incorporating the hydrogel polymer as a means to stabilise the colloidal system has proven to be reliable in obtaining high quality SEROA spectra of D- and L-enantiomers of ribose and tryptophan. In an extension of the hydrogel-stabilised SEROA work, we also demonstrate that single nanoparticle plasmonic substrate such as silver silica nanotags can enhance the weak ROA effect. These dye tagged silica coated silver nanoparticles have enabled a chiral response to be transmitted from a chiral analyte to the plasmon resonance of an achiral metallic nanostructure. The measurement of mirror image SERROA bands for the two enantiomers of each of ribose and tryptophan was confirmed for this system. The generation of SEROA for both systems was achieved and confirmed SEROA as a new sensitive tool for analysis of biomolecular structure. In a related project, Raman and ROA spectra were measured for adenosine and seven of its derivative ribonucleotides. Both of these spectroscopic techniques are shown to be sensitive to the site and degree of phosphorylation, with a considerable number of marker bands being identified for these ribonucleotides. Moreover, the SERS studies of these ribonucleotides were also performed. The obtained SERS spectra were shown similar features that confirm these analytes interact with the surface in a similar manner, hence limiting the structural sensitivity of this method towards phosphate position. Short dipeptides such as diketopiperazine (DKP) have been investigated during the last decades as both natural and synthetic DKPs have a wide variety of biological activities. Raman and ROA spectra of linear and cyclic dialanine and diserine were measured to charecterize their solution structures. Density functional theory (DFT) calculations were carried out by a collaborator to assist in making vibrational band assignments. Considerable differences were observed between the ROA bands for the cyclic and linear forms of both dialanine and diserine that reflect large differences in the vibrational modes of the polypeptide backbone upon cyclicization. In this study, the ROA spectra of cyclic dialanine and diserine have been reported for the first time which demonstrated that ROA spectroscopy when utilised in combination with computational modelling clearly provides a potential tool for characterization of cyclic peptides.

L’ostéonécrose (ON) et les fractures (FR) sont des complications qui prennent de plus en plus place dans le traitement pédiatrique de la leucémie aiguë lymphoblastique (LAL). L’ON peut être causée par différents facteurs, dont principalement l’utilisation de glucocorticoïdes. Les glucocorticoïdes sont administrés lors du traitement de la leucémie dans le but d’initier l’apoptose des cellules malignes tout en ayant un effet anti-inflammatoire. Cependant, l’utilisation de ces corticostéroïdes comprend des effets secondaires sérieux, notamment le développement d’ostéonécrose. Des variantes génétiques peuvent mettre certains patients plus à risque que d’autres. Plusieurs gènes ont déjà été signalés comme régulés par les actions glucocorticoïdes (GC). Les variations génétiques présentes dans les régions régulatrices de ces gènes peuvent affecter leur fonctionnement normal et, en fin de compte, de déterminer un risque accru de développer l’ON associé au traitement contre la leucémie. Pour cette raison, plusieurs polymorphismes ont été identifiés et étudiés dans la cohorte QcALL de Ste-Justine, concernant les gènes suivants : ABCB1, ACP1, BCL2L11, NFKB1, PARP1, et SHMT1. Ces gènes jouent majoritairement un rôle dans les mécanismes d’action des glucocorticoïdes, mais quelques-uns ont plutôt un effet direct sur le développement d’ostéonécrose. Nos recherches ont démontré une corrélation entre ces polymorphismes et l’apparition d’ostéonécrose chez les patients de la cohorte QcALL, traités aux glucocorticoïdes. L'incidence cumulative de l'ostéonécrose a été évaluée rétrospectivement chez 305 enfants atteints de la leucémie qui ont subi un traitement à l’hôpital Ste-Justine selon les protocoles DFCI de Boston (87-01, 91-01, 95-01 et 2000-01). Parmi les huit polymorphismes de BCL2L11 étudiés, les 891T> G (rs2241843) et 29201C> T (rs724710) ont été significativement associés à ON (p = 0.01 et p = 0.03, respectivement). L'association du polymorphisme 891T> G a été modulée par le type de corticostéroïde (CS), l’âge, le sexe et le groupe à risque (p ≤ 0,05). Le polymorphisme 29201C> T était particulièrement apparent chez les patients à haut risque (p = 0,003). La même étude était conduite en parallèle sur des patients de la cohorte DFCI de Boston (N = 192), et montrait des résultats significatifs pour les polymorphismes étudiés. En conclusion, les résultats de cette étude permettront de confirmer l’association de ces polymorphismes au développement d’ON chez les patients de LLA traités aux GC. / Osteonecrosis (ON) and fractures (FR) are complications that take place in the treatment of children acute lymphoblastic leukemia (cALL). They can be caused by various factors, mainly using glucocorticoids. The corticosteroids, dexamethasone (DXM) and prednisone (PDN) are administered during the treatment of leukemia to initiate apoptosis of malignant cells; while having an anti-inflammatory effect. However, the use of these corticosteroids has severe side effects, including the development of osteonecrosis. Moreover, some patients develop resistance to treatment, and are at risk of developing side effects. The genetic variants predispose some patients at higher risk than others. Several genes have been previously reported as up- or down regulated by the GCs actions. The genetic variations present in gene coding or regulatory regions can affect their function and ultimately determine an increased risk of developing ON associated to ALL therapy. Therefore, we investigated the association between several single nucleotide polymorphisms (SNPs) in six candidate genes: BCL2L11, NFKB1, PARP1, ABCB1, ACP1, and SHMT1. These genes play a role in the mechanisms of action of glucocorticoids, but some have more of a direct effect on the development of osteonecrosis. Our research has shown a correlation between these polymorphisms and the occurrence of osteonecrosis in patients in the QCALL cohort, treated with glucocorticoids. Cumulative incidence of osteonecrosis was assessed retrospectively in 305 children with ALL who underwent treatment with DFCI protocols (87-01, 91-01, 95-01 and 2000-01) in childhood ALL cohort from Quebec (QcALL). Among the eight tag BCL2L11 polymorphisms studied the 891T>G (rs2241843) and 29201C>T (rs724710) were significantly associated with ON (p = 0.01 and p = 0.03, respectively). Association of 891T>G polymorphism was modulated by type of corticosteroid (CS), age, sex and risk group (p ≤ 0.05 and that of 29201C>T was particularly apparent among high risk (p = 0.003) patients. These polymorphisms have shown significant ON association in several QcALL risk groups, mainly in corticosteroid groups, age < 10 years, and high risk (HR) group. Furthermore, the same study was conducted in parallel with patients in the replication (DFCI) cohort (N = 192), and we showed significant genetic association results for all studied polymorphisms. In conclusion, this study identifies that some ALL children have a high incidence of ON during the treatment that is highly associated with polymorphisms in different genes regulated by corticosteroids and ALL prognostic factors.