Infrared multiple photon dissociation (IRMPD) spectroscopy has been coupled with electronic structure calculations in order to elucidate the structures of several small biological molecules including: uracil, 5-fluorouracil, 5-fluorocytosine, ferulic acid, and a number of their related analogs. IRMPD is a powerful technique, that when combined with electronic structure calculations can provide convincing evidence for the structural characterization of ions in the gas phase. Isomers of uracil and 5-fluorouracil (5-FU) have been characterized by calculations performed at the MP2(full)/aug-cc-pVTZ level of theory; however, infrared multiple photon dissociation spectroscopy experiments proved to be unsuccessful for these species. Geometry optimization and frequency calculations have isolated the dominant isomer(s) for neutral and deprotonated uracil and 5-fluorouracil, along with several cluster interactions involving water, methanol, ammonia, and methylamine. For both uracil and 5-FU, a single relevant neutral isomer was determined, with each isomer existing in the diketo, as opposed to the enol form. Following the deprotonation of this neutral isomer, both uracil and 5-FU were permitted to form anionic cluster ions with water, methanol, ammonia, or methylamine, and based on the relative Gibbs free energies (298 K) of the calculated isomers, relevant cluster interactions were determined. For each cluster, several sites of intramolecular interaction were found to exist; however, interaction at the site of deprotonation was the most favourable in every instance. Ionic hydrogen bond interactions have been found in several clusters formed by 5-fluorocytosine (5-FC). The chloride and trimethylammonium cluster ions, in addition to the cationic and anionic dimers have been characterized by infrared multiple photon dissociation (IRMPD) spectroscopy and electronic structure calculations performed at the B2PLYP/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. IRMPD spectra in combination with calculated spectra and relative energetics have indicated, quite conclusively, that a single isomer for each 5-FC cluster that is likely being observed experimentally except in the case of the anionic dimer, in which a combination of isomers is probable. For the 5-FC-trimethylammonium cluster specifically, the calculated spectrum of the lowest energy isomer matches the experimental spectrum remarkably well. Interestingly, the cationic dimer of 5-FC was found to have a single energetically relevant isomer (Cationic-IV) in which a unique tridentate ionic hydrogen bond interaction is formed. The three sites of intramolecular ionic hydrogen bonds in this isomer interact very efficiently, leading to a significantly large calculated enthalpy of binding of 180 kJ/mol. The magnitude of the calculated binding energy for this species, in combination with the strong correlation between the simulated and IRMPD spectra, indicates that the tridentate-bound dimer is observed predominantly in experiment. Comparison of the calculated relative Gibbs free energies (298 K) for this species with several of the other isomers considered also supports the likelihood of the dominant protonated dimer existing as Cationic-IV. Protonated ferulic acid has been characterized using infrared multiple photon dissociation spectroscopy and electronic structure calculations at the B3LYP/6-311+G(d,p) level of theory. Neutral ferulic acid has been determined to undergo protonation on the carbonyl oxygen of the acid group, forming an ion of m/z 195. Due to its extensively conjugated structure, protonated ferulic acid (m/z 195) is observed to yield three stable fragment ions in IRMPD experiments. It is proposed that two parallel fragmentation pathways of protonated ferulic acid are being observed. First, proton transfer occurs from the carbonyl oxygen to the hydroxyl oxygen within the acid group, resulting in the loss of water and subsequently carbon monoxide, forming ions of m/z 177 and 149, respectively. The second proposed fragmentation pathway undergoes proton transfer from the phenolic group to the methoxy group resulting in loss of methanol and rearrangement to a five-membered ring of m/z 163. IRMPD spectra have been obtained for the ions m/z 195 and m/z 177, and anharmonic calculations have been performed on these species at the B3LYP/6-311+G(d,p) level of theory. The calculated anharmonic spectra for these ions match the experimental spectrum exceptionally well and strongly support the proposed fragmentation mechanisms.

Infrared multiple photon dissociation (IRMPD) spectroscopy has been coupled with electronic structure calculations in order to elucidate the structures of several small biological molecules including: uracil, 5-fluorouracil, 5-fluorocytosine, ferulic acid, and a number of their related analogs. IRMPD is a powerful technique, that when combined with electronic structure calculations can provide convincing evidence for the structural characterization of ions in the gas phase. Isomers of uracil and 5-fluorouracil (5-FU) have been characterized by calculations performed at the MP2(full)/aug-cc-pVTZ level of theory; however, infrared multiple photon dissociation spectroscopy experiments proved to be unsuccessful for these species. Geometry optimization and frequency calculations have isolated the dominant isomer(s) for neutral and deprotonated uracil and 5-fluorouracil, along with several cluster interactions involving water, methanol, ammonia, and methylamine. For both uracil and 5-FU, a single relevant neutral isomer was determined, with each isomer existing in the diketo, as opposed to the enol form. Following the deprotonation of this neutral isomer, both uracil and 5-FU were permitted to form anionic cluster ions with water, methanol, ammonia, or methylamine, and based on the relative Gibbs free energies (298 K) of the calculated isomers, relevant cluster interactions were determined. For each cluster, several sites of intramolecular interaction were found to exist; however, interaction at the site of deprotonation was the most favourable in every instance. Ionic hydrogen bond interactions have been found in several clusters formed by 5-fluorocytosine (5-FC). The chloride and trimethylammonium cluster ions, in addition to the cationic and anionic dimers have been characterized by infrared multiple photon dissociation (IRMPD) spectroscopy and electronic structure calculations performed at the B2PLYP/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. IRMPD spectra in combination with calculated spectra and relative energetics have indicated, quite conclusively, that a single isomer for each 5-FC cluster that is likely being observed experimentally except in the case of the anionic dimer, in which a combination of isomers is probable. For the 5-FC-trimethylammonium cluster specifically, the calculated spectrum of the lowest energy isomer matches the experimental spectrum remarkably well. Interestingly, the cationic dimer of 5-FC was found to have a single energetically relevant isomer (Cationic-IV) in which a unique tridentate ionic hydrogen bond interaction is formed. The three sites of intramolecular ionic hydrogen bonds in this isomer interact very efficiently, leading to a significantly large calculated enthalpy of binding of 180 kJ/mol. The magnitude of the calculated binding energy for this species, in combination with the strong correlation between the simulated and IRMPD spectra, indicates that the tridentate-bound dimer is observed predominantly in experiment. Comparison of the calculated relative Gibbs free energies (298 K) for this species with several of the other isomers considered also supports the likelihood of the dominant protonated dimer existing as Cationic-IV. Protonated ferulic acid has been characterized using infrared multiple photon dissociation spectroscopy and electronic structure calculations at the B3LYP/6-311+G(d,p) level of theory. Neutral ferulic acid has been determined to undergo protonation on the carbonyl oxygen of the acid group, forming an ion of m/z 195. Due to its extensively conjugated structure, protonated ferulic acid (m/z 195) is observed to yield three stable fragment ions in IRMPD experiments. It is proposed that two parallel fragmentation pathways of protonated ferulic acid are being observed. First, proton transfer occurs from the carbonyl oxygen to the hydroxyl oxygen within the acid group, resulting in the loss of water and subsequently carbon monoxide, forming ions of m/z 177 and 149, respectively. The second proposed fragmentation pathway undergoes proton transfer from the phenolic group to the methoxy group resulting in loss of methanol and rearrangement to a five-membered ring of m/z 163. IRMPD spectra have been obtained for the ions m/z 195 and m/z 177, and anharmonic calculations have been performed on these species at the B3LYP/6-311+G(d,p) level of theory. The calculated anharmonic spectra for these ions match the experimental spectrum exceptionally well and strongly support the proposed fragmentation mechanisms.

DNA repair proteins in mycobacteria and their physiological importance Mycobacterium tuberculosis, the causative organism of tuberculosis, resides in the host macrophages where it is subjected to a plethora of stresses like reactive oxygen species (ROS) and reactive nitrogen intermediate(RNI) which are generated as a part of the host’s primary immune response. These stresses can damage the cellular components of the pathogen including DNA and its precursors. Two common damages to DNA and its precursors caused by ROS and RNI are oxidation of guanine to 8-oxo-guanine and deamination of cytosine to uracil. Mycobacteria, which are known to have high G+C content, must be more susceptible to such damages, and are thus equipped with the mechanisms to counteract these damages. One such mechanism is to hydrolyse the 8-oxo-dGTP into 8-oxo-dGMP to avoid its incorporation in the DNA during its synthesis. This job is done by a protein called MutT.In mycobacteria four homologs of MutT, namely MutT1, MutT2, MutT3 and MutT4 have been annotated. The second mechanism deals with the repair of uracil residues present in DNA which are generated by deamination of cytosines or incorporation of dUTP during DNA synthesis. This is taken care of by a protein called uracil DNA glycosylase (UDG) which excises uracil by cleaving the N-C1’ glycosidic bond between the uracil and the deoxyribose sugar in a DNA repair pathway called the base excision repair (BER). In this study, the biochemical properties and physiological role of mycobacterial MutT2 and, MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein, have been investigated. I.Biochemical characterization of MutT2 from mycobacteria and its antimutator role. Nucleotide pool, the substrate for DNA synthesis is one of the targets of ROS which is generated in the macrophage upon Mycobacterium tuberculosis infection. Thus, the pathogen is at increased risk of accumulating oxidised guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP. By hydrolysing the damaged guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role inallowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we have purified recombinantM. tuberculosisMutT2 (MtuMutT2) andM. smegmatisMutT2 (MsmMutT2) proteins as representative of slow and fast growing mycobacteria, for the purpose of biochemical characterization. UnlikeEscherichia coliMutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (KmandVmax) revealed thatwhileMtuMutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP,MsmMutT2 hydrolyzes them almost equally well. Also,MsmMutT2 is about 14 times more efficient thanMtuMutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP.Consistent with these observations,MsmMutT2 but notMtuMutT2 rescuesE. colifor MutT deficiency by decreasing both themutation frequency and A to C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins. II.Understanding the biochemical properties of MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein Uracil DNA glycosylases (UDGs) are base excision repair enzymes which excise uracil from DNA by cleaving the N-glycosidic bond. UDGs are classified into 6 different families based on their two functional motifs, i. e.,motif A and motif B. In mycobacteria, there are two uracil DNA glycosylases, Ung and UdgB which belong to Family 1 and Family 5, respectively. In this study, based on the presence of the two functional motifs, we have discovered yet another uracil DNA glycosylase in M. smegmatis, which we have called MsmUdgX.The motif A and motif B of this protein indicate that it does not belong to any of the UDG families already classified but has highest similarity with Family 4 UDGs. Homologs of this protein are also present in several other organisms like M. avium, Streptomyces ceolicolor, Rhodococcus etc., but absent in M. tuberculosis, archaea and eukaryotes. Activity assays of this protein show that unlike other UDGs, MsmUdgX does not excise uracil, but forms a tight complex with uracil containing single stranded (ss) and double stranded (ds) DNAs, as observed by a shifted band in 8M urea-PAGE as well as SDS-PAGE. It also does not recognize other modified nucleotides that we investigated, in DNA. The protein binds to uracil-DNA in a wide range of pH and the minimum substrate required for its binding is pNUNN. Like Family 4 UDG, the protein has Fe-S cluster but it is not as thermostable as the Family 4 UDGs. Addition of different metal ions does not affect its binding property, and even the presence of M. smegmatis cell free extract does not diminish its binding activity. Since this protein binds specifically to uracil in DNA, an application of the protein for detection of uracil in the genomic DNA is proposed. III. Elucidation of the role of KRRIH loop in MsmUdgX by mutational analysis MsmUdgX is a novel uracil DNA glycosylase superfamily protein which has the highest homology to Family 4 UDGs. However, alignment of MsmUdgX amino acid sequence with that of Family 4 UDGs shows that there is an extra stretch of amino acids which is unique to this group of proteins. This stretch, defined by AGGKRRIH is absent in all Family 4 UDGs and the region KRRIH of the strtch is quite conserved amongst all UdgX proteins. Homology modelling of MsmUdgX, using a Family 4 UDG (TthUdgA) shows that this extra stretch of amino acids forms an outloop near the enzyme active site. Another unique difference between MsmUdgX and Family 4 UDGs is in the motif A where MsmUdgX has GEQPG and the Family 4 UDGs haveGE(A/G)PG. Our work on MsmUdgX has shown that, unlike other UDGs, this protein does not excise uracils, but forms a tight complex with the uracil containing DNA. This unique tight uracil binding property as well as KRRIH amino acid stretch has not been observed for any uracil DNA glycosylase superfamily proteins. So, to gain insight into the role of KRRIH and glutamine (Q) of motif A in MsmUdgX family of proteins, site directed mutagenesis was done in this region and we observed that mutation of His109 of the KRRIH loop to serine (S) leads to a gain of uracil excision activity, whereas changing the R107 to S, ‘RRIH’ to ‘SSAS’ or deleting the loop altogether leads to loss of its complex formation activity. Further, mutation of H109 to other amino acids like G, Q and A also shows uracil excision activity. Mutation of the glutamine in the motif A to alanine so that it is exactly similar to that of Family 4 UDGs, does not affect its uracil binding activity. This observation indicates that the KRRIH loop has an important role in the tight binding and/or uracil excision activity of MsmUdgX. Crystal structure of MsmUdgX in complex with uracil-DNA oligo and MsmUdgX H109S mutants are being studied.IV. Physiological importance of MsmUdgX in M. smegmatis MsmUdgX is a uracil DNA glycosylase superfamily protein which binds tightly to uracil (in DNA) without excising it. To elucidate its role in M. smegmatis, knockout of udgX was generated. Growth comparison of the wild type and the ΔudgX strains does not show any growth differences under the conditions tested. However, overexpression of MsmUdgX in recA deficient strains of E. coli as well as M. smegmatis leads to their retarded growth. Retarded grown is also observed in strains deficient in other DNA repair proteins that work in conjunction with RecA. These observations indicate that repair/release of MsmUdgX-uracil DNA complex might be a RecA dependent process.

La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.

The ability of RNA bases to self-assemble into larger structures is an important research area relevant to the origins of life. In the RNA helix the bases are arranged on a sugar-phosphate carcass but it has been suggested that the initial ordering could form on a flat surface. This thesis is an attempt to establish experimentally whether the complementary RNA bases, adenine and uracil, have the ability to self-assemble into large ordered structures when adsorbed on metal and mineral surfaces. The Au (111) surface was chosen as a preferred substrate as it is flat, relatively free of defects, chemically inert and reconstructs in a characteristic pattern of corrugation lines, which provide a reference for crystallographic directions. Six of the molecular phases shown were observed for the first time with molecular resolution and the possible two-dimensional arrangements of adenine and uracil molecules for these phases are proposed. The pure adenine and pure uracil structures have chiral unit cells and in the case of pure uracil alternating monochiral domains within the polychiral islands are created. Well-ordered intricate uracil-adenine bimolecular networks were also observed. The self-assembly of both uracil and adenine appears to be weakly influenced by the surface crystallography. The (100) surface of the mineral pyrite (FeS₂) was chosen as the alternative substrate as it is the most common face that occurs naturally in pyrite crystals. The experiments show the formation of small adenine and uracil crystals at the terrace edges. Neither uracil nor adenine were observed to form a monolayer on the surface of the terraces. The results of the experiments described in this thesis are very interesting in terms of establishing the possible mechanisms for creating regular chiral molecular networks and provide a useful insight into the role of surfaces in the processes of self-assembly of RNA bases.

La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.

Infusões prolongadas de 5-fluorouracil são mais seguras e potencialmente mais efetivas no tratamento do câncer de cólon metastático do que infusões rápidas da mesma medicação. No entanto, infusões prolongadas requerem a disponibilidade de um acesso venoso central, bem como de bombas de infusão dispendiosas. O desenvolvimento de fluoropirimidinas orais permitiu que pacientes fossem expostos ao 5-fluorouracil por longo tempo, com maior conveniência. UFT e leucovorin administrados três vezes ao dia demonstraram previamente uma eficácia equivalente, com menor toxicidade, quando comparados a um regime convencional de infusão rápida de 5- fluorouracil e leucovorin. Este estudo com 98 pacientes foi desenhado e conduzido com objetivo de demonstrar equivalência no tempo de progressão com o uso de UFT e leucovorin administrados duas vezes ao dia, com o uso da mesma combinação administrada três vezes ao dia. Objetivos secundários incluíram análise de toxicidade, resposta objetiva e sobrevida global. O tempo mediano de progressão foi de 3,8 meses, comparado com 3,5 meses observados com o uso da medicação três vezes ao dia e a taxa de resposta foi de 11%, com uma sobrevida mediana de 12,8 meses, sendo comparável aos resultados de 12% e 12,4 meses obtidas com o uso da combinação três vezes ao dia. A incidência de diarréia com graus 3 e 4 foi de 30% no regime de administração duas vezes ao dia, e 21% no de três vezes ao dia. Esses resultados sugerem que o uso de UFT e leucovorin duas vezes ao dia tem eficácia e toxicidade similares àquelas obtidas com o uso da mesma medicação três vezes ao dia. / Prolonged infusions have been shown to be safer and potentially more effective than bolus regimens of 5- fluorouracil as treatment for advanced colorectal cancer. However, infusional 5- fluorouracil requires central venous access and costly infusion pumps. Development of oral fluoropyrimidines has allowed longer exposures to 5-fluorouracil with increased convenience. UFT and leucovorin given thrice daily showed improved safety and no significant difference in survival or response rate compared with bolus 5- fluorouracil and leucovorin. This study with 98 patients was conducted to evaluate whether UFT and leucovorin given twice daily provided comparable time to progression (TTP) to the same combination administered three times a day. Secondary objectives included evaluation of toxicity, overall tumor response rate, and survival. Median time to progression was 3.8 months, compared with 3.5 months observed with the thrice-daily regimen. The twice-daily regimen had a response rate of 11% and median survival of 12.8 months, comparable to the 12% and 12.4 months seen with the thrice-daily regimen. The incidence of grade 3-4 drug-related diarrhea was 30% on the twice-daily and 21% on the thrice-daily schedule. Results suggest that the twice-daily schedule has similar safety and efficacy to the thrice-daily schedule.

La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.

Infusões prolongadas de 5-fluorouracil são mais seguras e potencialmente mais efetivas no tratamento do câncer de cólon metastático do que infusões rápidas da mesma medicação. No entanto, infusões prolongadas requerem a disponibilidade de um acesso venoso central, bem como de bombas de infusão dispendiosas. O desenvolvimento de fluoropirimidinas orais permitiu que pacientes fossem expostos ao 5-fluorouracil por longo tempo, com maior conveniência. UFT e leucovorin administrados três vezes ao dia demonstraram previamente uma eficácia equivalente, com menor toxicidade, quando comparados a um regime convencional de infusão rápida de 5- fluorouracil e leucovorin. Este estudo com 98 pacientes foi desenhado e conduzido com objetivo de demonstrar equivalência no tempo de progressão com o uso de UFT e leucovorin administrados duas vezes ao dia, com o uso da mesma combinação administrada três vezes ao dia. Objetivos secundários incluíram análise de toxicidade, resposta objetiva e sobrevida global. O tempo mediano de progressão foi de 3,8 meses, comparado com 3,5 meses observados com o uso da medicação três vezes ao dia e a taxa de resposta foi de 11%, com uma sobrevida mediana de 12,8 meses, sendo comparável aos resultados de 12% e 12,4 meses obtidas com o uso da combinação três vezes ao dia. A incidência de diarréia com graus 3 e 4 foi de 30% no regime de administração duas vezes ao dia, e 21% no de três vezes ao dia. Esses resultados sugerem que o uso de UFT e leucovorin duas vezes ao dia tem eficácia e toxicidade similares àquelas obtidas com o uso da mesma medicação três vezes ao dia. / Prolonged infusions have been shown to be safer and potentially more effective than bolus regimens of 5- fluorouracil as treatment for advanced colorectal cancer. However, infusional 5- fluorouracil requires central venous access and costly infusion pumps. Development of oral fluoropyrimidines has allowed longer exposures to 5-fluorouracil with increased convenience. UFT and leucovorin given thrice daily showed improved safety and no significant difference in survival or response rate compared with bolus 5- fluorouracil and leucovorin. This study with 98 patients was conducted to evaluate whether UFT and leucovorin given twice daily provided comparable time to progression (TTP) to the same combination administered three times a day. Secondary objectives included evaluation of toxicity, overall tumor response rate, and survival. Median time to progression was 3.8 months, compared with 3.5 months observed with the thrice-daily regimen. The twice-daily regimen had a response rate of 11% and median survival of 12.8 months, comparable to the 12% and 12.4 months seen with the thrice-daily regimen. The incidence of grade 3-4 drug-related diarrhea was 30% on the twice-daily and 21% on the thrice-daily schedule. Results suggest that the twice-daily schedule has similar safety and efficacy to the thrice-daily schedule.