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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. 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 de la chaîne polyphosphate. En effet, ces composés présentent une activité agoniste vis-à-vis du récepteur P2Y11, leurs synthèses sont bien décrites dans la littérature et les produits de départ sont beaucoup moins onéreux qu’en série 2’-désoxy. De plus, ces synthèses permettront de mettre les différentes réactions au point avant de synthétiser les analogues de la 2’-désoxyadénosine. Ces composés présentant une activité agoniste vis-à-vis du récepteur P2Y11 systématiquement supérieure à leurs homologues de la série 2’-OH, leur synthèse fera l’objet de la deuxième partie de ce travail. Deux schémas de synthèse en sept étapes ont été imaginés pour effectuer la synthèse des dérivés [101] à [105], l’un au départ d’adénosine [12] et l’autre au départ de guanosine [39]. Le schéma au départ d’adénosine ne nous a pas permis d’obtenir les analogues désirés en raison des difficultés de reproductibilité des réactions ainsi qu’en raison du faible rendement de la deuxième étape du schéma de synthèse. Le schéma au départ de la guanosine a permis quant à lui d’obtenir les analogues de l’adénosine-5’-triphosphate [101] à [105] avec des rendements globaux compris entre 10 et 20 %. Les tests d’activité agoniste ont montré que le dérivé [105] active le récepteur P2Y11 de la même manière que l’ATPgS avec des concentrations trois fois moindre. La deuxième partie de ce travail consista en la synthèse d’analogues du 2-thiodésoxynucléotide-5’-triphosphate. Un schéma de synthèse analogue à celui utilisé pour la synthèse des dérivés [101] à [105] au départ de la 2’-désoxyguanosine [40] aurait pu permettre l’obtention de tels dérivés. Cependant, les problèmes de dégradation rencontrés que le problème lié à l’introduction d’un motif thioalkyle (troisième étape) nous ont contraint à abandonner cette stratégie de synthèse. Les dérivés [106] à [110] ont finalement été synthétisés au départ du 2-désoxyribose [50]. Nous avons ensuite engagé ces analogues dans la réaction de phosphorylation. C’est lors de cette réaction que nous avons rencontré des problèmes importants de purification et de dégradation. Afin de résoudre ces problèmes, nous avons adopté pour une autre stratégie de synthèse afin d’obtenir les analogues triphosphate. Cette stratégie consiste en l’introduction d’un naphtoate en position 3’ afin de modifier la polarité de la molécule et espérer ainsi pouvoir obtenir une meilleure séparation. Les phosphorylations sur ces dérivés ont été effectuées dans les mêmes conditions que précédemment n’ont pas permis d’aboutir aux monophosphates correspondants. Dans ces conditions, l’ester naphtoïque et le lien glycosidique sont hydrolysés.

Nucleic acids are important bio-macromolecules in living systems. They are involved in important functions like gene expression and regulation. Nucleoside triphosphates serve as precursors for biochemical synthesis of modified nucleic acids and nucleotide coenzymes. The modification of nucleic acids, particularly at nucleobases, can expand the function and chemical properties of nucleic acid. Herein, we report the chemical synthesis of a novel thiol-modified nucleoside S-(3-(acetylthio)propyl)-5-(mercaptomethyl)-uridine and the corresponding nucleotide via a “new synthetic methodology” developed in our laboratory. The synthesized triphosphate was used for RNA transcription. The activity and nuclease resistance of the transcribed RNA is studied. The results showed that the properties of the nucleotide with thiol functionality are as good as the native. The modified RNA can be used for RNA/protein complex structure studies and gold nanoparticles stabilizer. They can also serve as a probe in DNA/RNA microchip surface functionalization for detection of various diseases and pathogens.

Adenosine 5’ triphosphate (ATP) is a molecule central to life for its role as the cellular energy currency, and a purine nucleotide which serves as a building block of RNA. Thus, on the backdrop of an indispensible intracellular role of ATP, its identification as an extracellular signaling molecule in early 1970s came as a surprise. A novel doctrine, termed as ‘purinergic signaling’, was thus put forth. By definition, purinergic signaling consists of the signaling events triggered by binding of extracellular ATP- a purine nucleotide, and its breakdown products (viz., ADP, AMP, and adenosine) to their cognate receptors, which in turn are termed as ‘purinergic receptors’. Based on their ligand affinity, purinergic receptors are classified into two groups- P1 and P2 receptors. P2 receptors are further subclassified as P2X and P2Y receptors. Till date, four P1 receptors (viz. A1, A2a, A2b, and A3), seven P2X receptors (P2X1-7), and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been cloned and characterized. Conceptually, the first step of purinergic signaling is the release of ATP from an intact cell on encountering a stimulant or a modulator. The main mechanisms of such cellular ATP release include vesicular exocytosis and the release through conductive channels. ATP thus released, binds to its cognate receptors (i.e. P2X receptors, and certain P2Y receptors) and triggers the ‘purinergic signaling’ pathway that modulates the cellular response. In addition to purinergic receptors, cells also express ATP degrading enzymes on their surface, which break ATP down into ADP, AMP, and adenosine. ADP and adenosine, in turn, bind to their cognate receptors (certain P2Y receptors, and P1 receptors respectively) and further contribute to shaping the cellular response to a given cue. Thus, purinergic signaling is a highly dynamic process with pleiotropic downstream effects. First demonstrated in the context of neurotransmission, the phenomenon of purinergic signaling is now widely recognized and has been shown to play a role in regulating functional responses of cells of diverse origins, immune cells being one of them. Purinergic signaling in lymphocytes- an important subset of immune cells- is a common thread for the present research exercise, wherein we have addressed two sets of questions, one of academic curiosity and the other of clinical interest. In the former and the major part, we have examined whether purinergic signaling plays a role in functional aspects of ‘gamma delta (γδ) T cells’, which represent a unique subset of lymphocytes. Whereas, the latter part elaborates on the already identified involvement of purinergic signaling in T cell stimulatory action of ‘hypertonic saline (HS)’, which is used to treat trauma patients. The thesis, thus, is divided into five parts- the ‘Introduction’, ‘Aims and Scope of the study’, ‘Chapter 1’, ‘Chapter 2’, and ‘Summary of the work’. Understanding the questions posed in the present context, strategy designed to answer them, and eventually the experimental results answering these questions invoke basic knowledge of purinergic signaling, which has been attempted to be conferred through the ‘Introduction’ section. The discovery of purinergic signaling, its central theme, and individual molecular players involved in this signaling pathway are highlighted here. From the viewpoint of the present research endeavor, salient findings from the current literatureabout the involvement of purinergic signaling in the functional activities of various subsets of immune cells- are reviewed towards the end of this section. The ‘Introduction’ is followed by definition of the objectives for the present exercise, which are enlisted under ‘Aims and scope of the study’. Here, a brief overview of the background data that led us towards these objectives precedes the actual list of questions which we have approached. Purinergic signaling has been shown to play a role in the activation of ‘conventional αβ T’ cells. So we asked whether a similar purinergic signaling pathway also operates in unconventional γδ T cells. Thus, ‘Chapter 1’ is dedicated to answering the first set of questions about the role of purinergic signaling in γδ T cell activation. The chapter starts off by introducing γδ T cells. The topics such as discovery of γδ T cells, ontology, development, diversity, and distribution of these cells, and most importantly- their antigenic specificity and response are reviewed herein. The details of the experimental procedures employed to answer the defined objectives follow this introduction. We have carried out our experiments on γδ T cells in human circulation. For in vitro stimulation, we have used anti-CD3 + anti-CD28-coated beads (beads) or isopentenyl pyrophosphate (IPP), a γδ T cell specific stimulant. We observed that, circulating human γδ T cells rapidly release ATP on stimulation with beads or IPP. Pannexin-1 and connexin hemichannels, as well as vesicular exocytosis contribute to the ATP release. Real time RT-PCR data revealed that γδ T cells predominantly express purinergic receptors A2a, P2X1, P2X4, P2X7, and P2Y11. Of these, the inhibition of P2X4 receptors downregulated cytokine expression by γδ T cells post- in vitro stimulation, and also inhibited cytotoxic activity of γδ T cells towards Daudi cells. Selective translocation of P2X4 receptors to the immunological synapse was seen to be the underlying mechanism for these effects. Collectively, these data suggested that autocrine/paracrine purinergic signaling through P2X4 receptors indeed plays an important role in the functional aspects of circulating human γδ T cells. The experimental results are compiled in ‘Chapter 1’; which concludes with the ‘Discussion’ on the mentioned findings, and possible in vivo applications. ‘Chapter 2’ deals with the role of purinergic signaling in HS resuscitation. In addition to restoring the hemodynamic parameters, fluid replacement with small volumes of concentrated NaCl solution (HS) has been reported to reverse the suppression of T cells commonly found in the trauma subjects. Through an in vitro study using Jurkat cells as a model for primary human T cells, it has been shown earlier that, on HS exposure T cells release ATP- which binds to P2X7 receptors and promotes calcium influx. HS treatment also elicits phosphorylation of p38; and put together, Ca2+ influx and phosphorylated p38 synergize with TCR-induced stimulation resulting in the enhancement of transcriptional upregulation of IL-2. However, the mechanism of release of ATP on HS treatment and the possible involvement of P2X1 and P2X4 receptors expressed by T cells had not been addressed in this study. These very questions thus formed the objectives of the second part of present work. Experiments aimed to answer these questions showed that on HS treatment, Jurkat cells release ATP through pannexin-1 hemichannels. The released ATP binds to purinergic receptors P2X1, P2X4, and P2X7. This in turn triggers the downstream signaling cascade leading to phosphorylation of p38 and upregulation of IL-2 transcription, hence augmenting the T cell function. An overview of HS resuscitation, experimental protocols and results, and the discussion on the pathophysiological relevance of these findings comprise ‘Chapter 2’. Hence, we have found the answers to the questions we began with. The results are listed in a point-wise manner under the ‘Summary of the work’. Taken together, our data shows that: (i) Purinergic signaling does play a role in the functional aspects of circulating human γδ T cells. The release of ATP by γδ T cells post-stimulation, and autocrine/paracrine signaling through P2X4 receptors are the main components in this context. (ii) ATP release through pannexin-1 hemichannels, and autocrine/paracrine signaling through P2X1, P2X4, and P2X7 receptors underlie the mechanism of action of HS.

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. It was initially found that comparing to wild type cells, gsk3- cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.

Malaria is a fatal tropical disease affecting billions of people in impoverished countries world-wide. An alarming fact is that a child in Africa dies of malaria every 30 seconds that amounts to 2500 children per day (www.who.int/features/factfiles). Malaria is caused by the intraerythrocytic forms of Plasmodium species, notably P. falciparum, P. vivax, P. ovale and P. malariae (Hyde 2007). The spread of drug-resistant strains, failure of vector control programs, rapid growth rate of the parasite, and lack of a vaccine have further exacerbated the effects of malaria on economic development and human health. It is therefore imperative that novel drug targets are developed or current antimalarial drugs optimized (Foley and Tilley 1998). One such target is folate biosynthesis, given that folates and their derivatives are required for the survival of organisms (Muller et al. 2009). DHFR and DHPS are currently the only folate targets exploited however, their antifolate drugs are almost useless against parasite resistant strains. As such, guanosine-5’triphosphate cyclohydrolase I (GTPCHl) among other antifolate candidates are considered for intervention (Lee et al. 2001). Knock-out studies (of P. falciparum gtpchI) resulted in the suppression of DHPS activity (Nzila et al. 2005). Additionally, gtpchI amplified 11-fold in P. falciparum strains resistant to antifolates due to mutations in dhps and dhfr and this may be a mechanism for the compensation of reduced flux of folate intermediates (Kidgell et al. 2006; Nair et al. 2008). Over-expression of P. falciparum proteins in E. coli remains a challenge mainly due to the A+T rich Plasmodium genome resulting in a codon bias. This results in the expression of recombinant proteins as insoluble proteins sequestered in inclusion bodies (Carrio and Villaverde 2002; Mehlin et al. 2006; Birkholtz et al. 2008a). Comparative expression studies were conducted of native GTPCHI (nGTPCHI), codon optimized GTPCHI (oGTPCHI) and codon harmonized (hGTPCHI) in various E. coli cell lines, using alternative media compositions and co-expression with Pfhsp70. The nGTPCHI protein did not express because the gene consisted of codons rarely used by E. coli (codon bias). The expression levels of purified hGTPCHI were a greater in comparison to oGTPCHI using the different expression conditions. This is because codon-harmonization involves substituting codons to replicate the codon frequency preference of the target gene in P. falciparum, as such the translation machinery matches that of Plasmodium (Angov et al. 2008). Furthermore, greater expression levels of GTPCHI were achieved in the absence of Pfhsp70 due to expression of a possible Nterminal deletion product or E. coli protein. Purification conditions could be improved to obtain homogenous GTPCHI and further analysis (mass spectrometry and enzyme activity assays) would be required to determine the nature of soluble GTPCHI obtained. To improve the expression of soluble proteins the wheat germ expression system was used as an alternate host. However, GTPCHI expression was not effective, possibly due to degradation of mRNA template or the absence of translation enhancer elements. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

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Made available in DSpace on 2016-03-15T14:17:03Z (GMT). No. of bitstreams: 2 7.pdf: 1019934 bytes, checksum: df1b248bb9c258918248c73b73272de2 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / Uridina 5'-trifosfato trissódica (UTPt) e citidina 5'-monofosfato dissódica (CMPd) são nucleotídeos pirimidínicos do ácido nucleico. Eficácia e segurança de fármacos baseados na UTPt e CMPd, usados no tratamento para neuropatias periféricas já foram estudadas, no entanto informações sobre farmacocinética desses fármacos ainda não são conhecidas. O objetivo deste estudo foi propor metodologias para quantificar UTPt e CMPd em matrizes biológicas, baseando-se numa revisão sistemática da literatura. Levando em consideração que a biodisponibilidade das pirimidinas, durante as neuropatias periféricas é diferente da observada em voluntários sadios, os dados disponíveis acerca das concentrações plasmáticas do UTPt e CMPd não devem ser usados para estimar a dose de fármacos baseados nessas pirimidinas. Para diferenciar pirimidinas endógenas e exógenas em matrizes biológicas, estas últimas devem ser marcadas, antes da administração, com material radioativo tais como trício [3H] ou carbono 14 [14C]. Além disso, a cromatografia líquida de alta performance é a técnica mais aplicada para identificação e quantificação de pirimidinas radioativas. Nós concluímos que a radiomarcação de UTPt e CMPd, seguida de separação cromatográfica e detecção por UV e cintilografia líquida, seria uma metodologia factível para estudos de detecção e quantificação de derivados de UTPt e CMPd em matriz biológica / Pyrimidines uridine 5'-triphosphate trisodium (UTPt) and cytidine 5'-monophosphate disodium (CMPd) are standard nucleosides which make up nucleic acids. Efficacy and safety from UTPt and CMPd based drugs on peripheral neuropathies has already been studied. However, information regarding pharmacokinetics of UTPt and CMPd based drugs during pathological condition remains unknown. The aim of this study was to propose methodologies to quantify UTPt and CMPd in biological matrices, based on a systematic literature review. Concerning that the bioavailability of pyrimidines during peripheral neuropathies is different of observed in healthy volunteers, the available data regarding plasmatic levels of UTPt and CMPd should not be used to estimate the dose of UTPt and CMPd based drugs. Furthermore, to differentiate endogenous and exogenous pyrimidines in biological matrices the exogenous pyrimidines must be labeled with [3H] or [14C] before administration. Next, high-performance liquid chromatography (HPLC) has been the most applied technique for identification and quantitation of radiolabeled pyrimidines. We concluded that UTPt and CMPd radiolabelling, followed by chromatographic separation and detection by UV and liquid scintigraphy, is a feasible methodology for detection and quantitation of UTPt and CMPd derivatives in biological matrices.

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.