The discovery of cisplatin, cis-[pt(NH3)2Cl2], as an anticancer agent in 1969 by Rosenbert and his colleagues sparked interest in the area of metal complexes as chemotherapeutic agents. Anticancer dimetal complexes such as Re2(O2CCH2CH3)2Br4·2H2O are proposed to prevent replication of cancel cells by coordinating to the purine nucleobases in DNA. To investigate the interaction between dimetal compounds and DNA, dirhenium complexes coordinated to purine dinucleotides were isolated and analyzed. LC/MS, HPLC, 1H NMR, and UV-Visible spectroscopy were used to characterized complexes of Re2(O2CR)2X4·2H2O (R = CH3, CH2CH3; X= Cl, Br) with the purine dinucleotides dApG and dGpG. HPLC, UV-Vis, and 1H NMR are used to investigate the aquation of Re2(O2C2H3)2Cl4μ2H2O which may contribute to its biological activity. Upon reaction of Re22C2H3)2Cl4μ2H2O with dApG or dGpG, the intact dirhenium:dinucleotide complex is observed by LC/MS after two days. In both of these reactions, dirhenium:GMP complexes are also observes. 1H NMR studies show the appearance of new resonances in the aromatic region that cannot be attributed to starting material or hydrolyzed DNA fragments. These resonances are proposed to result from the formation of dirhenium:dinucleotide complexes. Additionally, MS spectra support the conclusion that a complex between the dinuclear rhenium complex and the purine dinucleotides of dApG and dGpG is formed after two days. A dirhenium:nucleotide product is also formed as a result of the dinucleotide hydrolysis.

A identificação de genes relacionados com resistência a compostos antiparasitários tem contribuído para um melhor entendimento do mecanismo de ação de compostos antileishmania. Pouco se sabe sobre o mecanismo de ação do análogo de purina tubercidina (TUB) em Leishmania. Utilizando a estratégia de superexpressão após transfecção gênica, isolamos um locus de Leishmania (Leishmania) major, de 31 kb, capaz de conferir níveis de resistência quatro vezes maior que o parasita selvagem. Várias deleções desse locus foram geradas e a construção de 3 kb (pSNBR/3kbClaI-EcoRI) também conferiu níveis de resistência quando comparado ao parasita elvagem. Através de análises no genoma de L. (L.) major, localizamos esse locus no cromossomo 31 e, no fragmento de 3 kb, um gene que codifica para uma proteína com função desconhecida até o momento (LmjF.31.2010). Esta proteína foi relacionada com resistência a TUB em todas as linhagens transfectadas analisadas (cosTUB2 e pSNBR/3kbClaI-EcoRI), assim denominamos LmjF.31.2010, de proteína relacionada com resistência a TUB (PRRT). A quantificação relativa de transcritos de mRNA na construção pSNBR/3kbClaI-EcoRI apresentou níveis altos de transcritos da PRRT. Foram gerados ainda mutantes de L. (L.) major e L. (L.) amazonensis resistentes a TUB e estes se apresentaram bem adaptados a concentrações altas de TUB, apresentando razão de resistência maior que 200 vezes, quando comparado com os respectivos parasitas selvagens. A PRRT também foi relacionada na resistência a TUB nos mutantes gerados, pois houve amplificação gênica de prrt. Os resultados obtidos neste trabalho fornecem dados para inferir a importância da PRRT no mecanismo relacionado com resistência a TUB. / The identification of genes associated with resistance to antiparasitic compounds has contributed to a better understanding of the mechanism of action of compounds against Leishmania. Little is known about the mechanism of action of purine analog tubercidin (TUB) in Leishmania. Using a strategy of gene overexpression after transfection, we isolated a locus of Leishmania (Leishmania) major, 31 kb, capable of conferring fold resistance four times greater than the wild type parasite. A set of deletions of this locus were generated and a 3 kb construction (pSNBR/3kbClaI-EcoRI) conferred fold resistance twice than the wild type. Analysis of L. (L.) major genome, located this locus on chromosome 31 and on 3 kb fragment we identified a gene encoding a protein with unknown function (LmjF.31.2010). This protein has been related to TUB resistance in all strains analyzed (cosTUB2 and pSNBR/3kbClaI-EcoRI), so we named mjF.31.2010 of protein related with resistance to TUB (PRRT). Relative quantification of mRNA transcripts in the construction pSNBR/3kbClaI-EcoRI showed high levels of PRRT transcripts.Mutants of L. (L.) major and L. (L.) amazonensis resistant to TUB were also generated and these were well adapted to high TUB concentrations, presenting fol resistance greater than 200 times when compared with their respective wild type. The PRRT was also related to TUB resistance mutants generated by PRRT gene amplification. Despite the high fold resistance presented by TUB resistant mutants, the ratio of expression of these mutant PRRT transfected and wild was similar to the wild type.

A identificação de genes relacionados com resistência a compostos antiparasitários tem contribuído para um melhor entendimento do mecanismo de ação de alguns desses compostos. Utilizando a estratégia que permite a indução de super-expressão após transfecção gênica, isolamos dois loci relacionados com resistência ao análogo tóxico de purina, tubercidina (TUB). Em um desses locus identificamos um ortólogo do gene TOR (TOxic nucleoside Resistance) em L. (L.) major (TOR-Lm), capaz de conferir altos níveis de resistência a TUB. A identificação e localização cromossomal do segundo locus foi obtida, mas os testes funcionais em presença de TUB não foram tão significativos quanto os obtidos após a transfecção do TOR-Lm. Na segunda parte desta dissertação avaliamos a eficácia da associação de TUB com um inibidor específico do transporte de nucleosídeos em mamíferos, nitrobenziltioinosina (NBMPR), visando reverter a toxicidade de TUB apenas no hospedeiro. Demonstramos que TUB tem uma potente ação anti-parasitária em culturas de Leishmania spp., e que o inibidor NBMPR é capaz de proteger células mamíferas de camundongos infectados da ação tóxica de TUB. / Gene identification associated with drug resistance has contributed to a better understanding of the mechanism of action of anti parasitic compounds. Using transfection and over-expression selection strategy we isolated two loci related with the resistance of tubercidin (TUB), a toxic analog purine. In the first locus we identified an ortholog of the TOR gene (TOxic nucleoside Resistance) in L. (L.) major (TOR-Lm), capable to render wild cells resistance to TUB after transfection and over-expression. Chromosomal location and identification of the second locus was done, but functional tests in the presence of TUB were not as significant as those obtained after TOR-Lm transfection. In the second part of this work, we evaluate the effectiveness of the association of TUB with an inhibitor specific to the mammals nucleoside transport, as nitrobenzylthioinosine (NBMPR), aimed at reversing the TUB toxicity only on the host. We first demonstrate that TUB has a potent anti-parasitic action in cultures of Leishmania spp. Then, we discuss the capacity of the NBMPR inhibitor to protect infected macrophages from the toxic effects of TUB.

<p>Brief episodes of ischemia and reperfusion render the myocardium more resistant to necrosis from a subsequent, otherwise lethal ischemic insult. This phenomenon is called ischemic preconditioning(IP). Today, much is known about the signalling pathways involved in IP; however, the details of the final steps leading to cardioprotection, remain elusive. Adenosine (a catabolite of ATP) plays a major role in the signalling pathways of IP. Following IP there is an unexplained discrepancy between an increased adenosine production (evidenced by increased 5’-nucleotidase activity) and the successively lower adenosine levels observed in the interstitial space. We propose that this discrepancy in adenosine production vs. availability may be due to an increased metabolic utilisation of adenosine by the IP myocardium. According to our hypothesis, IP induces/activates a metabolic pathway involving deamination of adenosine to inosine. Inosine is further catalysed (in presence of Pi) to hypoxanthine and ribose-1-phosphate. Ribose-1-phosphate can be converted to ribose-5-phosphate in a phosphoribomutase reaction. Ribose-5-phosphate is an intermediate of the hexose monophosphate pathway also operative under anaerobic conditions. Hence the ribose moiety of adenosine can be utilised to generate pyruvate and ultimately ATP (via lactate formation) n.b. without any initial ATP investment. Such cost-effective adenosine utilisation may at least partly explain the cardioprotective effect of IP. Objectives & Methods: In the current studies we investigated the role of adenosine metabolism according to the suggested metabolic pathway by addition of adenosine and inhibition of its metabolism during IP as well as by comparing tissue and interstitial levels of key energy-metabolites following different protocols of IP. Furthermore, we studied the importance of the IP protocol with regard to the number of ischemia and reperfusion cycles for the cardioprotective effect of IP. In addition, the validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism was evaluated. For these purposes the microdialysis technique, tissue biopsies, and planimetric infarct size estimation in an open chest porcine heart-model was used. Results: Addition of adenosine via microdialysis probes enhanced the interstitial release of inosine, hypoxanthine and lactate in the myocardium of IP-subjects during prolonged ischemia. This finding did not occur in non-preconditioned subjects. Similar addition of deoxyadenosine a non-metabolizable adenosine receptor-agonist, did not evoke the same metabolic response. Purine nucleoside phosphorylase (PNP) is responsible for the conversion of inosine to hypoxanthine being a key enzyme in the above mentioned metabolic pathway. Inclusion of 8' aminoguanosine (a competitive inhibitor of PNP) decreased interstitial hypoxanthine release (as a token of PNP inhibition) and increased the release of taurine (marker of cellular injury) in the ischemic IP myocardium. Addition of inosine (a natural substrate of PNP) reverted these changes. Four IP cycles protected the heart more than one IP cycle as evidenced by morphometric and energy-metabolic data.Proportionally more hypoxanthine was found in the myocardium of IP subjects during prolonged ischemia. The ratio of tissue levels of inosine/hypoxanthine (used as an indicator of PNP activity) was significantly smaller in the IP groups. In addition, myocardial interstitial levels of energy-related metabolites (lactate, adenosine, inosine, and hypoxanthine) obtained by the microdialysis technique correlated with tissue biopsy levels of corresponding metabolites. Conclusions: IP activated a metabolic pathway favouring metabolism of exogenous adenosine to inosine, hypoxanthine and eventually lactate. Inhibition of adenosine metabolism following IP (via inhibition of PNP-activity resulted in enhanced cellular injury.</p><p>PNP-activity is proportionally higher in IP-myocardium. Metabolic utilisation of adenosine in IP-myocardium (as outlined above) may represent a costeffective way to produce ATP and at least partly explain the cardioprotective effect of IP. IP protects the myocardium in a graded fashion. Furthermore, we confirmed the validity of the microdialysis technique (in the current setting) for studying dynamic changes of myocardial energy metabolism.</p>

Brief episodes of ischemia and reperfusion render the myocardium more resistant to necrosis from a subsequent, otherwise lethal ischemic insult. This phenomenon is called ischemic preconditioning(IP). Today, much is known about the signalling pathways involved in IP; however, the details of the final steps leading to cardioprotection, remain elusive. Adenosine (a catabolite of ATP) plays a major role in the signalling pathways of IP. Following IP there is an unexplained discrepancy between an increased adenosine production (evidenced by increased 5’-nucleotidase activity) and the successively lower adenosine levels observed in the interstitial space. We propose that this discrepancy in adenosine production vs. availability may be due to an increased metabolic utilisation of adenosine by the IP myocardium. According to our hypothesis, IP induces/activates a metabolic pathway involving deamination of adenosine to inosine. Inosine is further catalysed (in presence of Pi) to hypoxanthine and ribose-1-phosphate. Ribose-1-phosphate can be converted to ribose-5-phosphate in a phosphoribomutase reaction. Ribose-5-phosphate is an intermediate of the hexose monophosphate pathway also operative under anaerobic conditions. Hence the ribose moiety of adenosine can be utilised to generate pyruvate and ultimately ATP (via lactate formation) n.b. without any initial ATP investment. Such cost-effective adenosine utilisation may at least partly explain the cardioprotective effect of IP. Objectives &amp; Methods: In the current studies we investigated the role of adenosine metabolism according to the suggested metabolic pathway by addition of adenosine and inhibition of its metabolism during IP as well as by comparing tissue and interstitial levels of key energy-metabolites following different protocols of IP. Furthermore, we studied the importance of the IP protocol with regard to the number of ischemia and reperfusion cycles for the cardioprotective effect of IP. In addition, the validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism was evaluated. For these purposes the microdialysis technique, tissue biopsies, and planimetric infarct size estimation in an open chest porcine heart-model was used. Results: Addition of adenosine via microdialysis probes enhanced the interstitial release of inosine, hypoxanthine and lactate in the myocardium of IP-subjects during prolonged ischemia. This finding did not occur in non-preconditioned subjects. Similar addition of deoxyadenosine a non-metabolizable adenosine receptor-agonist, did not evoke the same metabolic response. Purine nucleoside phosphorylase (PNP) is responsible for the conversion of inosine to hypoxanthine being a key enzyme in the above mentioned metabolic pathway. Inclusion of 8' aminoguanosine (a competitive inhibitor of PNP) decreased interstitial hypoxanthine release (as a token of PNP inhibition) and increased the release of taurine (marker of cellular injury) in the ischemic IP myocardium. Addition of inosine (a natural substrate of PNP) reverted these changes. Four IP cycles protected the heart more than one IP cycle as evidenced by morphometric and energy-metabolic data.Proportionally more hypoxanthine was found in the myocardium of IP subjects during prolonged ischemia. The ratio of tissue levels of inosine/hypoxanthine (used as an indicator of PNP activity) was significantly smaller in the IP groups. In addition, myocardial interstitial levels of energy-related metabolites (lactate, adenosine, inosine, and hypoxanthine) obtained by the microdialysis technique correlated with tissue biopsy levels of corresponding metabolites. Conclusions: IP activated a metabolic pathway favouring metabolism of exogenous adenosine to inosine, hypoxanthine and eventually lactate. Inhibition of adenosine metabolism following IP (via inhibition of PNP-activity resulted in enhanced cellular injury. PNP-activity is proportionally higher in IP-myocardium. Metabolic utilisation of adenosine in IP-myocardium (as outlined above) may represent a costeffective way to produce ATP and at least partly explain the cardioprotective effect of IP. IP protects the myocardium in a graded fashion. Furthermore, we confirmed the validity of the microdialysis technique (in the current setting) for studying dynamic changes of myocardial energy metabolism.

The obesity epidemic is associated with an increase in the prevalence of a number of chronic diseases including type 2 diabetes, cardiovascular disease, hypertension and some cancers and has been described by the World Health Organisation as one of the greatest public health challenges of the 21st century. Obesity is characterised by excessive expansion of adipose tissue mass underpinned by adipocyte hyperplasia. Central to this is the process of adipogenesis, which encompasses the proliferation and terminal differentiation of fibroblastic preadipocytes, contained within adipose tissue, to mature adipocytes. Despite the pivotal role of this process in obesity our understanding of the regulatory mechanisms governing adipocyte development, either in physiological or pathophysiological settings, is limited. Studies aimed at understanding this complex process are integral to development of more effective strategies for the prevention and/or treatment of obesity and obesity related diseases. Our laboratory recently identified a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in the dynamic regulation of lipid accumulation. Upon treatment of a variety of cell types with insulin or oleic acid IMPDH translocates to lipid droplets and inhibition of this translocation is correlated with reduced lipid accumulation. As lipid droplet formation and lipid accretion are defining features of adipogenesis, it was hypothesised that IMPDH may facilitate efficient lipid accumulation during adipose conversion of preadipocytes. In vitro systems have been used extensively to dissect the molecular and cellular events involved in adipogenesis. Therefore the aim of this project was to extend these investigations to examine the requirement for IMPDH activity during adipogenesis, using the well characterised murine 3T3-L1 cell line and primary human preadipocytes (phPAs). IMPDH expression and activity were transiently increased during differentiation of the 3T3-L1 cells although IMPDH did not associate with lipid droplets under these conditions. Pharmacological inhibition of IMPDH, using mycophenolic acid (MPA; 1 µM), reduced intracellular GTP by 60%, and blocked mitotic clonal expansion (MCE) and adipogenesis. Supplementation with guanosine (60 µM), a substrate in the nucleotide salvage pathway, restored both GTP levels and adipogenesis. These observations indicated that IMPDH activity is required for efficient differentiation of 3T3-L1 preadipocytes. Preliminary studies, involving differentiation of phPAs in standard serum-free medium (SFM) suggested that phPAs were resistant to MPA. To afford better comparison between the phPAs and the 3T3-L1 cells, which are differentiated in serum-containing medium (SCM), a modified 3T3-L1 like protocol facilitating efficient differentiation of the phPAs in SCM was established. Under these conditions phPAs displayed considerable variation in sensitivity to MPA which gave a trend towards decreased differentiation (reduced by 26%; p=0.07). Supplementation with guanosine significantly reduced adipogenesis (by 37%; p<0.05) in the phPAs independent of MPA. Furthermore, cells that were MPA resistant were also refractory to guanosine suggesting greater plasticity of guanine metabolism in phPAs from those subjects. A major difference between the cell types was that phPAs differentiated with high efficiency in the absence of MCE. Collectively, these data indicate that MCE is required for efficient differentiation of 3T3-L1 cells but not phPAs, even when differentiated under similar conditions, and suggest that the involvement of MCE underpins the differences in sensitivity to MPA between cell types. The differential effects of guanosine suggest there are additional differences with respect to the effects of manipulation of guanine nucleotides between cell types. In summary, the work presented in this thesis demonstrated inhibition of IMPDH blocked adipogenesis of murine 3T3-L1 cells and reduced differentiation of phPAs in some subjects. These observations provided novel insights into differences between differentiation of 3T3-L1 cells and phPAs, including their relative sensitivities to alterations in guanine nucleotides, and have implications for adipose tissue biology especially those factors involved in guanine metabolism. Ultimately this knowledge may form the basis for development of novel therapeutic strategies aimed at reduction of obesity and associated complications such as insulin resistance and type 2 diabetes.

Malaria is a life-threatening parasitic disease endemic throughout the world. Control methods for malaria are becoming less reliable; thus, efforts to develop a safe and effective vaccine are critical. Immunity to malaria requires both cell- and humoral-mediated immunity, CMI and HMI, respectively. CD4+ T cells play a central role in protection against blood stage Plasmodium infection. Given that clinical features of malaria are caused by blood stages, a vaccine against this stage will be very effective in reducing morbidity and mortality. During the blood stage, purine nucleotides, which are essential for parasites’ survival and proliferation, are in high demand. The inability of the parasite to engage in de novo synthesis of purine nucleotides makes the enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) an essential nutrient salvage enzyme. HGXPRT is located in electron-dense regions in merozoites and in vesicles in the red cell cytoplasm. In contrast to other blood stage antigens, those located on the merozoite surface are targets of HMI. To advance HGXPRT as a malaria vaccine candidate, fermentation and purification of the protein from Plasmodium falciparum (PfHGXPRT) was performed using facilities at Q-Gen, the Queensland Institute of Medical Research (QIMR). Escherichia coli carrying PfHGXPRT gene were a gift in-kind from the University of Queensland (UQ). Recombinant PfHGXPRT expressed in E.coli was purified using anion exchange liquid chromatography and gel filtration techniques. Three methods were used to confirm the Q-Gen PfHGXPRT identity: (1) Western blotting showing identical bands of UQ PfHGXPRT and Q-Gen PfHGXPRT at 26 kDa; (2) N terminal sequencing was compatible with the PfHGXPRT sequence; and (3) mass spectrometry showed homogeneity by giving a subunit molecular mass of 26,231 Da. The purification method used is reproducible and affordable, yielding reasonably pure protein for animal experimentation. Following purification of PfHGXPRT, its efficacy as a subunit vaccine candidate in a rodent model of infection was examined. Multiple rodent models of malaria infection were assessed and it was determined that Plasmodium chabaudi AS (P. chabaudi AS) exhibited the highest cross-reactivity against PfHGXPRT in mice. Hence, P. chabaudi AS was chosen as the appropriate rodent model for study in this thesis. Natural immunity against PfHGXPRT during a blood stage P. chabaudi AS infection was assessed by testing sera and splenocyte responses to PfHGXPRT. IFN- and IL-4, as well as antibodies specific for PfHGXPRT, could be detected after infection, suggesting that PfHGXPRT is a target of natural immunity during the blood stage infection. Therefore, further studies of protective immunity generated by immunisation with PfHGXPRT were conducted, specifically to determine their protective efficacy and to determine immune mechanisms elicited by immunisation. Mice immunised with PfHGXPRT and challenged with P. chabaudi AS developed a slightly reduced parasitaemia. T-cell proliferation, but not antibody responses, was detected after immunisation. Protective mechanism(s) were assessed by adoptively transferring immune CD4+ T cells, B cells or sera to naïve SCID mice followed by parasite challenge. Only recipients of immune CD4+ T cells showed extended survival. Nevertheless, immunisation with PfHGXPRT followed by sub-patent infection induced better protection than immunisation with PfHGXPRT alone, which appeared to be related to CD4+ T cells. Reduction of parasitaemia, as well as augmentation of T cell proliferation and IFN-γ production, was evident in PfHGXPRT and sub-patent infected immunised mice. Recipients of CD4+ T cells from PfHGXPRT and sub-patent infection immune mice also showed some degree of protective immunity. PfHGXPRT was shown to induce natural and acquired immunity to P. chabaudi AS. HGXPRT is highly conserved in parasites and humans; therefore, it is essential to define minimal protective epitopes that could be included in a vaccine. Hence, 22 overlapping peptides (termed P1 P22) corresponding to the entire P. chabaudi AS HGPRT sequence were used to define minimal protective epitopes. Following immunisation of mice with seven pools of peptides (P1 P3, P4 P6, P7 P9, P10 P12, P13 P15, P16 P18 and P19 P22), three immunogenic peptides (P11, P13, and P17), which stimulated significant proliferative and IFN-γ responses were chosen for immunisation studies. Peptide P9 (position 76-95 from N-terminal), which induced the highest IFN- levels during P. chabaudi AS infection was also included in the pool of peptides. Mice immunised with P9, P11, P13 and P17 had significantly decreased parasitaemia. Antibody mediated immunity had a partial effect on suppressing parasite growth. CMI, on the contrary, played a central role in adoptively transferred protection by significantly reducing parasitaemia and prolonging survival of recipient SCID mice. Strong T cell proliferation and IFN- secretion were also detected after stimulation of splenocytes from immune mice with P. chabaudi AS antigen. CMI response was significantly increased after immunisation with the peptides followed by sub-patent infection. The findings that four short epitopes of HG(X)PRT confer strong CMI protection suggest that homologues of such epitopes could be included in a multi-component malaria vaccine.

Inosine monophosphate dehydrogenase (IMPDH) catalyses the key step in de novo guanine nucleotide biosynthesis at the branch point of GTP and ATP production. Mammals have two ubiquitous, catalytically indistinguishable isoforms, IMPDH type I and type II, and these are considered functionally interchangeable. Each contains a Bateman domain known to serve as energy-sensing / allosteric regulatory modules in a range of unrelated proteins. Mutations in the Bateman domain of type I, which do not affect catalytic activity, cause the retina-degenerative disease, retinitis pigmentosa (RP). The central hypothesis of this thesis is that IMPDH is regulated. In particular, that regulation occurs in an isoform specific manner and that mutations causal to RP affect enzyme regulation. Here we have visualised, including in real-time, the redistribution or clustering of IMPDH into linear macrostructures in a time-dependent manner which appeared to be intimately associated with changes in intracellular nucleotide levels. Data presented suggest the significance of IMPDH clustering is unlikely to be associated with substrate channelling, via interaction with other proteins in the de novo biosynthesis pathway, or enhanced protein stability. Although both isoforms responded similarly to fluctuations in intracellular nucleotide levels, type I had a higher propensity to spontaneously cluster into macrostructures compared to type II. This propensity to cluster was found to be conferred by the N-terminal 244 amino acids, which includes the Bateman domain, using a series of type I / type II chimera proteins. A comparative and novel approach revealed isoform-specific purine nucleotide binding characteristics. Type I bound ATP and type II bound AMP, via a mechanism involving the Bateman domain, resulting in conformational changes in IMPDH. This nucleotide binding was not associated with allosteric activation of IMPDH catalytic activity. The RP-causing mutation, R224P, abolished ATP binding and this correlated with an altered propensity to cluster. Collectively these data (i) show IMPDH distribution is regulated by the intracellular environment (ii) demonstrate that the IMPDH isoforms are modulated in a differential manner by AMP and ATP by a mechanism involving the Bateman domain, (iii) indicate communication between the Bateman domain and the active site and (iv) demonstrate that a RP-causing mutation compromises such regulation. From a broader perspective, this work raises the possibility that the nucleotide sensing properties of the Bateman domain in IMPDH serve to regulate IMPDH and co-ordinate nucleotide homeostasis, thereby giving rise to cellular plasticity in an isoform-specific manner to meet the requirements of the cellular environment.

A identificação de genes relacionados com resistência a compostos antiparasitários tem contribuído para um melhor entendimento do mecanismo de ação de alguns desses compostos. Utilizando a estratégia que permite a indução de super-expressão após transfecção gênica, isolamos dois loci relacionados com resistência ao análogo tóxico de purina, tubercidina (TUB). Em um desses locus identificamos um ortólogo do gene TOR (TOxic nucleoside Resistance) em L. (L.) major (TOR-Lm), capaz de conferir altos níveis de resistência a TUB. A identificação e localização cromossomal do segundo locus foi obtida, mas os testes funcionais em presença de TUB não foram tão significativos quanto os obtidos após a transfecção do TOR-Lm. Na segunda parte desta dissertação avaliamos a eficácia da associação de TUB com um inibidor específico do transporte de nucleosídeos em mamíferos, nitrobenziltioinosina (NBMPR), visando reverter a toxicidade de TUB apenas no hospedeiro. Demonstramos que TUB tem uma potente ação anti-parasitária em culturas de Leishmania spp., e que o inibidor NBMPR é capaz de proteger células mamíferas de camundongos infectados da ação tóxica de TUB. / Gene identification associated with drug resistance has contributed to a better understanding of the mechanism of action of anti parasitic compounds. Using transfection and over-expression selection strategy we isolated two loci related with the resistance of tubercidin (TUB), a toxic analog purine. In the first locus we identified an ortholog of the TOR gene (TOxic nucleoside Resistance) in L. (L.) major (TOR-Lm), capable to render wild cells resistance to TUB after transfection and over-expression. Chromosomal location and identification of the second locus was done, but functional tests in the presence of TUB were not as significant as those obtained after TOR-Lm transfection. In the second part of this work, we evaluate the effectiveness of the association of TUB with an inhibitor specific to the mammals nucleoside transport, as nitrobenzylthioinosine (NBMPR), aimed at reversing the TUB toxicity only on the host. We first demonstrate that TUB has a potent anti-parasitic action in cultures of Leishmania spp. Then, we discuss the capacity of the NBMPR inhibitor to protect infected macrophages from the toxic effects of TUB.

A identificação de genes relacionados com resistência a compostos antiparasitários tem contribuído para um melhor entendimento do mecanismo de ação de compostos antileishmania. Pouco se sabe sobre o mecanismo de ação do análogo de purina tubercidina (TUB) em Leishmania. Utilizando a estratégia de superexpressão após transfecção gênica, isolamos um locus de Leishmania (Leishmania) major, de 31 kb, capaz de conferir níveis de resistência quatro vezes maior que o parasita selvagem. Várias deleções desse locus foram geradas e a construção de 3 kb (pSNBR/3kbClaI-EcoRI) também conferiu níveis de resistência quando comparado ao parasita elvagem. Através de análises no genoma de L. (L.) major, localizamos esse locus no cromossomo 31 e, no fragmento de 3 kb, um gene que codifica para uma proteína com função desconhecida até o momento (LmjF.31.2010). Esta proteína foi relacionada com resistência a TUB em todas as linhagens transfectadas analisadas (cosTUB2 e pSNBR/3kbClaI-EcoRI), assim denominamos LmjF.31.2010, de proteína relacionada com resistência a TUB (PRRT). A quantificação relativa de transcritos de mRNA na construção pSNBR/3kbClaI-EcoRI apresentou níveis altos de transcritos da PRRT. Foram gerados ainda mutantes de L. (L.) major e L. (L.) amazonensis resistentes a TUB e estes se apresentaram bem adaptados a concentrações altas de TUB, apresentando razão de resistência maior que 200 vezes, quando comparado com os respectivos parasitas selvagens. A PRRT também foi relacionada na resistência a TUB nos mutantes gerados, pois houve amplificação gênica de prrt. Os resultados obtidos neste trabalho fornecem dados para inferir a importância da PRRT no mecanismo relacionado com resistência a TUB. / The identification of genes associated with resistance to antiparasitic compounds has contributed to a better understanding of the mechanism of action of compounds against Leishmania. Little is known about the mechanism of action of purine analog tubercidin (TUB) in Leishmania. Using a strategy of gene overexpression after transfection, we isolated a locus of Leishmania (Leishmania) major, 31 kb, capable of conferring fold resistance four times greater than the wild type parasite. A set of deletions of this locus were generated and a 3 kb construction (pSNBR/3kbClaI-EcoRI) conferred fold resistance twice than the wild type. Analysis of L. (L.) major genome, located this locus on chromosome 31 and on 3 kb fragment we identified a gene encoding a protein with unknown function (LmjF.31.2010). This protein has been related to TUB resistance in all strains analyzed (cosTUB2 and pSNBR/3kbClaI-EcoRI), so we named mjF.31.2010 of protein related with resistance to TUB (PRRT). Relative quantification of mRNA transcripts in the construction pSNBR/3kbClaI-EcoRI showed high levels of PRRT transcripts.Mutants of L. (L.) major and L. (L.) amazonensis resistant to TUB were also generated and these were well adapted to high TUB concentrations, presenting fol resistance greater than 200 times when compared with their respective wild type. The PRRT was also related to TUB resistance mutants generated by PRRT gene amplification. Despite the high fold resistance presented by TUB resistant mutants, the ratio of expression of these mutant PRRT transfected and wild was similar to the wild type.