Pathogenic bacteria such as M.tuberculosis have evolved several mechanisms to aid their intracellular survival and subvert host defenses. One of the contributing factors is thought to be the production and secretion of large amount of cAMP, Mycobacterial genomes encode a large number of adenylyl cyclases distinct in their structure and regulatory mechanisms. The roles of these enzymes in the physiology and pathogenesis of virulent mycobacteria are only now being elucidated. The roles of phosphodiesterases (PDEs), which serve to lower cAMP levels through degradation are, however, relatively unexplored. The Rv0805 gene was previously shown to code for an active phosphodiesterase from Mycobacterium tuberculosis. Bioinformatics analysis revealed that orthologs of Rv0805 were found even in eukaryotes. Biochemical and structural characterization of Rv0805 revealed that it was a class III cAMP phosphodiesterase. Comparative genomics identified a close ortholog of Rv0805 in M. leprae (ML2210). The genome of M. leprae Encodes only 1,604 predicted proteins and possesses the highest number of pseudogenes, 1,116. The retention of a functional PDE, the ortholog of Rv0805, in the minimal genome of M. leprae is indicative of its importance in cellular physiology. Biochemical characterization of proteins from M. leprae and use of heterologous hosts will help understand this human pathogen better, since there are no tools currently available to genetically manipulate this bacterium. Sequence analysis of ML2210 revealed the presence of conserved motifs and residues known to be critical for catalysis and unique to class III phosphodiesterases. ML2210 shares 83% sequence identity with Rv0805 and 24% sequence identity with the phosphodiesterase from E. coli (cpdA). In vitro biochemical characterization of ML2210 using non-nucleotide colorigenic and cyclic nucleotide substrates revealed that it was an enzymatically active phosphodiesterase. Kinetic parameters of ML2210 with respect ot colorigenic substrates revealed that its catalytic properties were similar to that of Rv0805. However, with respect to hydrolysis of 3’, 5’-cAMP, ML2210 was catalytically more efficient than Rv0805, suggesting that in spite of being orthologs, these enzymes have evolved distinct specificities at their active site. A parallel of monoclonal antibodies raised to Rv0805 was also used understand the differences in the biochemical properties of Rv0805 and ML2210 better. It was observed that only one monoclonal antibody was able to recognize ML2210 by ELISA and not by Western blot analysis. These results revealed that conformational differences between ML2210 and Rv0805 exist. Over-expression of ML2210 in M. smegmatis resulted in a modest decrease in intracellular cAMP levels. Despite the absence of a predicted transmembrane region or a membrane-targeting signal, ML2210 localized to the cell envelop fraction upon over expression in M. smegmatis. Moreover, like Rv0805, over-expression of ML2210 also resulted in perturbation of the cell wall of M. smegmatis, arguing for additional cellular roles of this protein. Orthologs of Rv0805 or ML2210 are found only in slow growing mycobacteria suggesting that other cyclic nucleotide phosphodiesterases could regulate cAMP levels in fast growing mycobacteria like M. smegmatis. Since BLAST results did not retrieve an ortholog of Rv0805 or ML2210, COG1409 (COG database) containing Rv0805 was examined for the presence of other mycobacterial phosphodiesterases. Bioinformatics analysis identified Rv2795c as another PDE from M. tuberculosis. Sequence analysis of Rv2795c revealed the presence of all the motifs conserved in the class III PDEs but Rv2795c shared only 22% sequence identity with Rv0805 and 19% sequence identity with CpdA. Importantly, an ortholog of Rv2795c was identified in M. leprae. Interestingly. Rv2795c and its orthologs branched away from Rv0805, making it phylogenetically distinct and hence warranting further characterization. Recombinant, purified MSMEG_2647 (the Rv2795c ortholog from M. smegmatis) was able to hydrolyze cyclic nucleotides and other phosphodiester substrates in vitro. The Km for colorigenic substrates was higher when compared to the Km of ML2210 or Rv0805 for these substrates. However, the kinetic parameters of MSMEG_2647 for cyclic nucleotides were comparable to those of ML2210 or Rv0805. MSMEG_2647 was a metal dependent enzyme and among the panel of metals tested, Mn2+ supported the highest in vitro catalytic activity of MSMEG_2647. Zn2+ inhibited the catalytic activity of MSMEG_2647. In order to gain insight into the catalysis of MSMEG_2647, the end products of cAMP hydrolysis by MSMEG_2647 were analysed using reverse phase HPLC. The assay revealed that the end products of cyclic nucleotide hydrolysis by MSMEG_2647 were different when compared to the end products of hydrolysis of the same substrates by Rv0805 or ML2210. This suggests differences in the architecture of the active site residues of the mycobacterial MPEs. A mutational anlaysis of the active site residues in MSMEG_2647 was carried out to identify residues involved in substrate recognition and metal coordination. Although Rv0805 and MSMEG_2647 shared only a 22% sequence identity, MSMEG_2647 displayed strict conservation in the core MPE motifs. Mutation of the active residues N97 and H98 in Rv0805 had led to an abrogation of its catalytic activity. However, corresponding mutations of N76A and H77A in MSMEG_2647, did not lead to a loss in its catalytic activity. A third mutation known to be important for the catalytic activity of Rv0805 (D19) was incorporated. The corresponding residue at D19 position was mutated to an alanine. The catalytic activity of MSMEG_2647D19AN76AH77A mutant was abrogated, suggesting that while the core MPE motifs are conserved between mycobacterial PDEs, differences in the ensemble of the active site residues contributing to their catalytic activity exist. Thus, at least two biochemically diverse PDE clades are found in mycobacterial species. In order to decipher the function of MSMEG_2647, its expression was monitored during the growth of M. Smegmatis. The promoter of MSMEG_2647 displayed maximum activity during the logarithmic phase of M. smegmatis growth after which its activity declined as M. smegmatis entered the stationary phase. However in contrast to this, the transcript corresponding to msmeg_2647 mRNA was found at both logarithmic and stationary phases. The MSMEG_2647 protein was also detected at both logarithmic and stationary phases of M. smegmatis. These results suggest that additional factors may contribute to the stability of msmeg_2647 mRNA and protein levels. Localization studies of MSMEG_2647 revealed that MSMEG_2647 was present in the cytosol as well as in the cell envelope fractions. Interestingly, over-expression of MSMEG_2647 did not result in a significant increase in PDE activity in various subcellular fractions, suggesting tight regulation on the in vivo activity in various subcellular fractions, suggesting tight regulation on the in vivo activity of MSMEG_2647. In addition, over-expression of MSMEG_2647 in M. smegmatis led to only a modest decrease in cAMP levels in M. smegmatis. These results suggested additional roles of MSMEG_2647 in the biology of mycobacteria. Overexpression of MSMEG_2647 peturbed the integrity of cell wall as assessed by the use of lipophillic indicators of cell growth, crystal violet and malachite green, and a cell wall targeting antibiotic, isoniazid. Analyzing the gene neighborhood of MSMEG_2647 provided an insight into its putative function. It was observed that the stop codon of msmeg_2647 overlapped with the start codon of msmeg_2648 and stop codon of msmeg-2648 overlapped with the start codon of msmeg_2649. RT PCR was carried out at logarhtimic and stationary phases of M. smegmatis growth, which revealed that a polycistronic mRNA was being transcribed. These results confirmed that msmeg_2647, msmeg_2648 and msmeg_2649 were a part of an operon. Interestingly, these three genes as a gene cluster were confined to only those actinobacteria that produced mycolic acids. An operon often encodes products that form multiprotein complexes and operate in a common pathway. Since there were a part of an operon, a GST pull-down approach was employed to test if MSMEG_2647, MSMEG_2648 and MSMEG_2649 could physically interact. It was observed that MSMEG_2647 interacted with MSMEG_2648 and MSMEG_2649. MSMEG_2648 in turn interacted with MSMEG_2649. A role for MSMEG_2647 as a scaffold recruiting MSMEG_2648 and MSMEG_2649 is therefore proposed. In turn, a complex formation with these proteins may regulate the activity of MSMEG_2647. Attempts to generate a knock out of msmeg_2647 in M. smegmatis by homologous recombination were not successful suggesting either the gene was essential or a polar effect on msmeg_2648(an essential gene for the viability of M. smegmatis) may not allow msmeg_2647 to be deleted from the genome of M. smegmatis. In summary, this study has identified and characterized two new phosphodiesterases from mycobacteria, one from the pathogenic mycobacterium, M. leprae and the other, a PDE from M. smegmatis that is conserved in all species of mycobacteria. Several, key biochemical differences were observed using biochemical and biological approaches. It appears that the cellular roles of mycobacterial phsophodiesterases may extend beyond cAMP hydrolysis, with these proteins not only regulating cell wall properties but also acting as scaffolding proteins in the cell.

Tuberculosis continues to be one of the major causes of morbidity and mortality worldwide. Several mycobacterial species such as M. tuberculosis and M. africanum are responsible for causing this disease in humans. Reports of high cAMP levels in mycobacterial species (as compared to other bacteria such as E. coli) suggested that this second messenger may play an important role in the biology of mycobacteria. Further, it was reported that infection with mycobacteria led to an increase in the cAMP levels within the host macrophage. More recent studies have shown that this cAMP increase may be due to bacterially derived cAMP, hinting at a role for cAMP in mycobacterial pathogenesis. Given this background, the study of cAMP in mycobacteria proves to be an interesting field of research. Signalling through cAMP involves an interaction of this cyclic nucleotide with a cAMP-binding protein. These proteins typically contain a cyclic nucleotide-binding domain (CNB domain) linked to another (effector) domain. The CNB domain is thought to allosterically control the activity of the effector domain, thus mediating cellular responses to altered cAMP levels. For example, in the case of eukaryotic protein kinase A (PKA), binding of cAMP to the CNB domain results in relieving the inhibitory effects of the regulatory subunit on the catalytic subunit. The catalytic subunit then phosphorylates its target substrates, eliciting a variety of cellular responses. This work involves the characterisation of novel cAMP-binding proteins from mycobacteria, in an attempt to better understand cAMP signalling mechanisms in these organisms. The genome of M .tuberculosis H37Rv is predicted to code for ten CNB domain-containing proteins. One of these genes is Rv0998 (KATmt). KATmt was found to contain a GCN5 related N-acetyltransferase (GNAT) domain linked to a CNB domain. KATmt finds orthologues throughout the genus Mycobacterium, thereby suggesting its role in the basic physiology of these organisms. In addition, such a domain fusion is unique to mycobacteria and hence promises to deliver insights into the biology of this medically important genus. Presented here are the biochemical and functional characterisation of KATmt and its orthologue from M. smegmatis, MSMEG_5458 (KATms). Recombinant KATms bound cAMP with high affinity, validating the functionality of its CNB domain. Mutational and analogue-binding studies showed that the biochemical properties of the CNB domain were similar to mammalian protein kinase A and G-like CNB domains. The substrate for the GNAT acetyltransferase domain was identified to be a universal stress protein from M. smegmatis (MSMEG_4207). MSMEG_4207 was acetylated at a single lysine residue (Lys 104) by KATms in vitro. Further, cAMP binding to KATms increased the initial rate of acetylation of MSMEG_4207 by 2.5-fold, suggesting allosteric control of acetyltransferase activity by the CNB domain. To ascertain that KATms acetylated MEMEG_4207 in vivo, an in-frame deletion of the KATms gene was generated in M. smegmatis (ΔKATms). MSMEG_4207 was immunoprecipitated from wild-type M. smegmatis and the ΔKATms strains, followed by mass spectrometric analysis. Acetylated MSMEG_4207 was only present in the wild-type strain, confirming that KATms and MSMEG_4207 is an in vivo enzyme-substrate pair. Key biochemical differences were observed between KATms and KATmt. KATmt had an affinity for cAMP in the micromolar range, close to three log orders lower than that of KATms. In addition, KATmt showed strictly cAMP-dependent acetylation of MSMEG_4207. This demonstrates that orthologous proteins often evolve under varied selective pressures, resulting in divergent properties. Using a combination of bioluminescence resonance energy transfer (BRET) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS), the conformational changes that occur upon cAMP binding to the CNB domain of KATms were monitored. A BRET-based conformation sensor was constructed for KATms by inserting KATms between GFP2 (green fluorescent protein) and Rluc (Renilla luciferase). An increase in BRET upon cAMP binding to the sensor was observed. HDXMS analysis revealed that besides the CNB domain, the only other region that showed conformational changes in KATms upon cAMP-binding was the linker region. To confirm that the linker region was important in propagating the effects of cAMP-binding to the acetyltransferase domain, an additional construct for BRET analysis encompassing the CNB domain and the linker region was generated. The magnitude of the increase in BRET was similar to the full length BRET-based sensor, validating the crucial role of the linker region in propagating cAMP-mediated conformational changes. A ‘PXXP’ motif found in the linker region, showed maximum exchange in HDXMS analysis. Mutation of both these proline residues to alanine in KATms, as well as KATmt, resulted in decoupling of cAMP-binding and allosteric potentiation of acetyltransferase activity. In contrast to the intricate parallel allosteric relays observed in other CNB domain-containing proteins, the CNB domain in KATms functions as a simpler cyclic nucleotide binding-induced switch involving stabilization of the CNB and linker domain alone. Therefore, KATms is an example of a primordial CNB domain where conformational changes are a consequence of binding-induced ordering alone. Using a computational approach, putative substrate proteins of KATmt from M. tuberculosis were identified. The substrate specificity of lysine acetyltransferases is determined loosely by a consensus sequence around the lysine residue which is acetylated. Using this property of protein acetyltransferases, the genome of M. tuberculosis H37Rv was mined for proteins harboring lysine residues in a similar sequence context as seen in MSMEG_4207. In vitro biochemical analysis of some of the predicted substrates helped confirm a subset of enzymes belonging to the fatty acyl CoA synthetase (FadD) class as substrates of KATmt. The acetylation of FadDs by KATmt was cAMP-dependent. In each of the four proteins tested, acetylation was found to occur at a single conserved lysine residue. To confirm that FadDs were acetylated by KATmt in vivo, BCG_1055, the orthologue of KATmt in M. bovis BCG, was deleted using the specialised transduction method. FadD13, one of the FadDs acetylated by KATmt in vitro, was immunoprecipitated from wild-type M. bovis and the ΔBCG_1055 strains using a FadD13-specific polyclonal antibody. Acetylated FadD13 was almost completely absent in ΔBCG_1055 but substantial amounts of acetylated FadD13 were present in the wild-type strain, indicating that FadD13 was indeed an in vivo substrate of KATmt. The functional consequences of acetylation of FadDs were analysed using an in vitro fatty acyl CoA synthetase assay. The activities of FadD2 and FadD13 were inhibited on acetylation with KATmt, while acetylation of FadD5 resulted in the formation of a novel product. Therefore, modification of the highly conserved lysine residue in these enzymes by acetylation led to loss or alteration of their enzymatic activity, suggesting that acetylation may be used as a regulatory mechanism to modulate the activities of some of the FadDs by KATmt in a cAMP-dependent manner. Given the extensive role of FadDs in cell wall biosynthesis and lipid degradation in mycobacteria, it seems possible that post-translational control by KATmt in a cAMP-dependent manner constitutes a novel mechanism utilised by these bacteria to regulate these pathways. This direct regulation of protein lysine acetylation by cAMP appears to be unique to mycobacteria, as orthologues of KATmt are not found outside this genus. In addition, the biochemical differences between KATmt and its orthologue from M. smegmatis KATms, indicate species specific variation, on a common theme. This study is the first report of protein lysine acetylation in mycobacteria. In addition to the identification of several proteins subject to this post-translational modification, the effect of acetylation on the enzymatic activities of some of them has been elucidated.

Dyslipidemia can be a comorbidity of both insulin-resistance and atherosclerosis. Hypertriglyceridemia is common in hyperglycemia and is associated with hypoalphalipoproteinemia (low HDL) and with altered nucleotide or purinergic signaling. We therefore hypothesized that extracellular nucleotides may affect hepatic lipoprotein metabolism. Our studies confirm this view and show that nucleotides regulate cellular proteolytic pathways in liver cells and thereby control lipoprotein secretion and their metabolism by hepatic lipase (HL). Treatment of liver cells with the nucleotide, adenosine diphosphate (ADP), stimulates VLDL-apoB100 and apoE secretion, but blocks HDL-apoA-I and HL secretion. ADP functions like a proteasomal inhibitor to block proteasomal degradation and stimulate apoB100 secretion. Blocking the proteosome is known to activate autophagic pathways. The nucleotide consequently stimulates autophagic degradation in liver cells and increases cellular levels of the autophagic proteins, LC3 and p62. Confocal studies show that ADP increases cellular LC3 levels and promotes co-localization of LC3 and apoA-I in an autophagosomal degradation compartment. ADP acts through the G-protein coupled receptor, P2Y13, to stimulate autophagy and block both HDL and HL secretion. Overexpression of P2Y13 increases cellular LC3 levels and blocks the induction of both HDL and HL secretion, while P2Y13 siRNA reduce LC3 protein levels and cause up to a ten-fold stimulation in HDL and HL secretion. P2Y13 gene expression regulates autophagy through the insulin receptor (IR-β). A reduction in P2Y13 expression increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, while increasing P2Y13 expression inhibits the activation of IR-β and Akt. Experiments with epitope-labeled apoA-I and HL show that activation of purinergic pathways has no effect on the internalization and degradation of extracellular apoA-I and HL, which confirms the view that nucleotides primarily impact intracellular protein transport and degradation. In conclusion, elevated blood glucose levels may promote dyslipidemia by stimulating purinergic signaling through P2Y13 and IR-β and perturbing the intracellular degradation and secretion of both HDL and VLDL.

Empreintes moléculaires pour la libération contrôlée des analogues de nucléosides : la ribavirine pour le traitement du virus Influenza A par voie pulmonaire et l’adénosine-5’-monophosphate.Nous nous sommes concentrés dans un premier lieu au développement de différentes formulations de MIPs enbulk sous forme hydrogels afin de mettre en place des systèmes de libération contrôlée de la ribavirine sous différents stimuli. Ensuite, nous nous sommes basés sur l’approche de « Dummy-template » en utilisant la 2’,3’,5’-tri-O-acétylribavirine afin de diminuer la polarité de la ribavirine et ainsi accéder à differents solvants aprotique pour mieux stabiliser le complexe de pré-polymèrisation. Cette étude a été faite en utilisant de nouveaux monomères synthétisés au sein du laboratoire et en les comparants avec un monomère commercial tel que l’acide méthacrylique. Ces différents MIPs ont montré des différences d’adsorption vis à vis de la ribavirine mais aussi des profils et des cinétiques de relargage différents et dépendant du milieu de libération ou de la température.Dans un deuxième temps, nous avons transposé certaines formulations utilisant la 2’,3’,5’-tri-O-acétyl-ribavirine comme molécule template vers la synthèse de billes imprimées. Les MIPs sphériques obtenus ont permis d’avoir la géométrie et le diamètre recherchés pour être administrés par voie pulmonaire. L’incorporation de différents co-monomères ont permis de modifier l’architecture de ces billes en les rendant thermosensibles ou fluorescentes.Finalement, nous avons, cette fois, synthétisé des polymères imprimés pour la libération contrôlée de l’adénosine-5’-monophosphate. Dans cette étude, nous avons étudié la libération à partir de la forme sphérique obtenue par polymérisation en émulsion de pickering inverse. / This thesis report presents the synthesis of new polymeric cargos associated with molecular imprinting technology for the controlled release of nucleoside analogs: ribavirin for the treatment of pulmonary influenza A and adenosine 5'-monophosphate.At first, we focused on the development of different formulations of bulk MIPs in hydrogel form with the aim of setting up controlled release systems for ribavirin under different stimuli. Then, we carried out a "Dummy-template"approach using 2 ', 3', 5'-tri-O-acetyl-ribavirin in order to reduce the polarity of ribavirin so that we could explore different aprotic solvents to better stabilize the pre-polymerization complex. This study was accomplished by the use of new monomers synthesized within the laboratory and by comparing them with a commercial monomer such as methacrylic acid.These different MIPs showed differences in adsorption with respect to ribavirin but also different release profiles and kinetics depending on the release medium or the temperature.Secondly, we transposed the best formulations using 2 ', 3', 5'-tri-O-acetyl-ribavirin as template molecule towards the synthesis of imprinted beads. The spherical MIPs obtained showed desired geometry and diameter to be administeredby the pulmonary route. The incorporation of various co-monomers allowed to modify the architecture of these beads bymaking them thermosensitive or fluorescent.Lastly, this time, we have synthesized imprinted polymers for the controlled release of adenosine-5'-monophosphate. In this part, we studied the release from the spherical shape obtained by inverse Pickering emulsion polymerization.

Le somnambulisme est un trouble du sommeil fréquent qui appartient à la famille des parasomnies NREM. Malgré des décennies de recherche, sa pathophysiologie reste peu comprise. Les études de familles et les études de jumeaux démontrent qu’une forte composante héréditaire est en jeu. Toutefois, très peu d’études moléculaires ont été menées afin d’identifier des gènes impliqués et il n’y a toujours pas de consensus quant au mode de transmission dans les familles. Cet ouvrage contient deux études distinctes qui tenteront de répondre à ces deux problèmes. L’objectif de la première étude était de déterminer si des variants génétiques dans le gène Adénosine désaminase (ADA) étaient enrichis dans la population somnambule en comparaison avec les dormeurs sains. Le gène entier a été séquencé chez 251 patients somnambules provenant de Montréal et de Montpellier ainsi que chez 94 sujets contrôles sans histoire personnelle ni familiale de somnambulisme. Aucun variant génétique n’était enrichi chez les patients somnambules en comparaison avec les dormeurs sains et les bases de données génétiques publiques. Dans la deuxième étude, le premier objectif était de déterminer le mode de transmission du somnambulisme chez 20 familles canadiennes-françaises. Le deuxième objectif était de mesurer le risque récurrent ainsi que le risque relatif pour la fratrie et les enfants des patients index. Dans notre cohorte, le somnambulisme se transmettait principalement selon un mode autosomal dominant à pénétrance réduite. Les risques récurrents pour les apparentés de premier degré étaient : à vie 0,48 à 0,56, durant l’enfance 0,43 à 0,56 et à l’âge adulte 0,14 à 0,35. Les risques relatifs pour les apparentés de premier degré étaient : à vie 6,96 à 8,12, durant l’enfance 1,48 à 4,06 et à l’âge adulte 4,67 à 11,67 supérieurs à la population générale. D’autres études moléculaires comme le séquençage de l’exome et les études de liaison génétique dans les familles seront nécessaires afin d’identifier de nouveaux gènes candidats qui pourront agir à titre de biomarqueurs. Cela permettrait de faciliter le diagnostic et ultimement développer des approches thérapeutiques ciblées. / Sleepwalking is a common sleep disorder and it belongs to the family of NREM parasomnias. Despite decades of research, its pathophysiology remains poorly understood. Family and twin studies show that a strong hereditary component is involved. However, very few molecular studies have been conducted to identify the genes involved and there is still no consensus on the mode of transmission in families. This Master’s thesis contains two separate studies which will attempt to address these two problems. The aim of the first study was to determine whether genetic variants in the Adenosine Deaminase (ADA) gene were enriched in the sleepwalking population compared to healthy sleepers. The entire gene was sequenced in 251 sleepwalking patients from Montreal and Montpellier as well as in 94 control subjects with no personal or family history of sleepwalking. No genetic variants were enriched in sleepwalking patients compared to healthy sleepers and public genetic databases. In the second study, the first objective was to determine the mode of transmission of sleepwalking in 20 French-Canadian families. The second objective was to measure the recurrence risk as well as the relative risk for siblings and children of index patients. In our cohort, sleepwalking was transmitted mainly in an autosomal dominant mode with reduced penetrance. The recurrence risks for first-degree relatives were: lifetime 0.48 to 0.56, in childhood 0.43 to 0.56, and in adulthood 0.14 to 0.35. The relative risks for first-degree relatives were: lifetime 6.96 to 8.12, in childhood 1.48 to 4.06 and in adulthood 4.67 to 11.67 higher than the general population. Further molecular studies, such as exome sequencing, and genetic linkage studies in families will be needed in order to identify new candidate genes that can act as biomarkers. This would allow the development of an independent test for the diagnosis and ultimately have implications for targeted therapeutic approaches.

L’ATP est l’agoniste naturel du récepteur P2Y11. Ce nucléotide ne peut cependant pas être utilisé comme agent thérapeutique car, in vivo, il s’hydrolyse rapidement en ADP ou en AMP qui ne possèdent qu’une faible activité pour le récepteur. D’où l’intérêt de disposer d’analogues de synthèse moins sensibles à l’hydrolyse et possédant une affinité égale ou supérieure à celle de l’ATP.<p><p>Le premier objectif que nous nous sommes fixés au cours de notre thèse de doctorat fut de mettre au point un schéma de synthèse permettant d’obtenir des analogues de l’adénosine-5’-triphosphate [1] portant un motif thioalkyle ou thioaryle en position 2 de la base ainsi qu’un groupement dichlorométhylène entre les phosphores b et g & / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Nucleoside 5'-Diphosphate Kinase (NdPK EC 2.7.4.6) is an enzyme (phosphotransferase) with extraordinary characteristics due to its unique ability to transfer phosphor groups to interconvert all nucleoside di- and triphosphates as a part of the DNA synthesis. Due to Biovica International AB's use of signaling and complementing enzymes in their in vitro diagnostic (IVD) test assays for Thymidine Kinase activity, an investigation was proposed to evaluate NdPK, which is a complementing enzyme in the assay. The aim of the study was to evaluate the enzymatic turnover of the enzyme NdPK with a spectrophotometric assay to obtain the specific activity (Units/mg solid protein). To determine the specific activity, enzyme kinetic methodology was applied, including the Michaelis-Menten model. In this study, the method is proposed as a general internal control procedure for the company, as a tool for function control of the different purchased enzymes used in their products in development. Results from the study reflects the different methods used to gain the specific activity for NdPK, where they were compared with the already specified specific activity from the manufacturing company. The results were auspicious, but before the method's authorization as an internal quality procedure, a few amendments are in mind. For instance, determining a method for the graphical readings, validating the method for quality control, and investigating if the method is applicable to other complementing enzymes. In conclusion, the method for determining the specific activity of the enzyme NdPK can be done, by executing the procedure of colorimetric enzyme assay.

Chromatin is organized dynamically to accommodate different biological processes. One of the factors required for proper chromatin organization is a group of complexes called condensins. Most eukaryotes have two conserved condensins (I and II) required for chromosome segregation. C. elegans has a third condensin (IDC) that specializes in dosage compensation, a process that down-regulates X gene dosage in XX hermaphrodites to match the dosage in XO males. How the three condensins are regulated is not well understood. Here, I present the discovery and characterization of a novel condensin regulator, SMCL-1. We identified SMCL-1 through purification of a MAP-tagged condensin subunit. Condensins are comprised of SMC ATPases and regulatory CAP proteins; SMCL-1 interacts most abundantly with condensin SMC subunits and resembles the ATPase domain of SMC proteins. Interestingly, the SMCL-1 protein has residues that differ from SMC consensus and potentially render SMCL-1 incapable of hydrolyzing ATP. Worms harboring smcl-1 deletion are viable and show no detectable phenotype. However, deleting smcl-1 in a condensin hypomorph mildly suppresses condensin I and IDC mutant phenotypes, suggesting that SMCL-1 functions as a negative regulator of condensin I and IDC. Consistent with this, overexpression of SMCL-1 leads to condensin loss-of-function phenotypes such as lethality, segregation defects and disruption of IDC localization on the X chromosomes. Homology searches based on the unique ATPase domain of SMCL-1 reveal that SMCL-1-like proteins are present only in organisms also predicted to have condensin IDC. Taken together, we conclude that SMCL-1 is a negative modulator of condensin functions and we propose a role for SMCL-1 in helping organisms adapt to having a third condensin by maintaining the balance among three condensin complexes.

This study has determined the cellular site of adenosine deaminase (ADA) expression in the mouse during development from Days 5 through 13 (day vaginal plug was found = Day 0) of gestation. Developmental expression of ADA progressed in two overlapping phases defined genetically (maternal vs. embryonal) and according to region (decidual vs. placental). In the first phase, ADA enzyme activity increased almost 200-fold in the antimesometrial region (decidua capsularis + giant trophoblast cells) from Days 6 through 9 of gestation but remained low in the mesometrial region. Immunohistochemical staining revealed a major localization of ADA to the secondary decidua. In the second phase, ADA activity increased several-fold in the placenta (labyrinth + basal zones) from Days 9 through 13 of gestation but remained low in the embryo proper. Immunohistochemical staining revealed a major localization of ADA to secondary giant cells, spongiotrophoblast, and labyrinthine trophoblast. Regression of decidua capsularis and growth of the spongiotrophoblast population accounted for an antimesometrial to placental shift in both ADA enzyme activity and a 40-kDa immunoreactive protein band. To verify a shift from maternal to fetal expression, studies were performed with two strains of mice (ICR, Eday) homozygous for a different ADA isozyme (ADA-A, ADA-B). Blastocysts homozygous for Adab were transferred to the uterus of pseudopregnant female recipients homozygous for Adaa. The isozymic pattern in chimeric embryo-decidual units analyzed at Days 7, 9, 11, and 13 revealed a predominance of maternal-encoded enzyme at Days 7 through 11 of gestation and a shift to fetal-encoded enzyme by Day 13. Thus, maternal expression of ADA in the antimesometrial decidua may play a role during establishment of the embryo in the uterine environment, whereas fetal expression of ADA in the trophoblast might be important to placentation.

<p>This work studies the effect of dopamine (DA) and adenosine triphosphate (ATP) on lipid membranes using a number of complementary experimental methods. These methods include Dynamic Light Scattering to measure electrostatic surface potentials, solid-state Nuclear Magnetic Resonance to measure the degree of lipid acyl chain order, Electron Paramagnetic Resonance to measure changes in membrane viscosity, and X-ray diffuse scattering to measure structural and material parameters of lipid bilayers. It is shown that both DA and ATP have a measurable affinity to the lipid-water interface even in the absence of specialized biological receptors. These results are important for understanding the function of DA and ATP in cellular processes.</p>