Housekeeping enzymes are vital to the metabolism of all plant cells. Two such enzymes adenosine kinase (ADK) and S-adenosylhomocysteine (SAH) hydrolase share a similar function: both sustain S-adenosylmethionine-dependent methylation reactions by removing inhibitory by-products. SAH hydrolase breaks down SAH which is a competitive inhibitor of all methyltransferase activities. ADK phosphorylates the Ado produced by SAH hydrolase and in doing so drives this reversible reaction in the hydrolysis direction. By catalyzing the phosphorylation of Ado into adenosine monophosphate, ADK not only prevents SAH from re-forming but also initiates the recycling of Ado into adenylate nucleotides and cofactors. This thesis documents two distinct research topics related to methyl recycling in plants. The first goal was to identify ADK-deficient mutants to establish the contribution of this enzyme activity to adenosine salvage. The second goal was to determine the subcellular localization of ADK and SAH hydrolase in Arabidopsis thaliana (Columbia). T-DNA insertion lines for highly similar ADK isoforms (ADK1 and ADK2) and silencing lines of overall ADK activity (sADK) were compared to WT Arabidopsis to identify phenotypic abnormalities associated with ADK deficiency. In addition to following their growth, microscopic analysis was performed on the sADK lines. While removal of either ADK1 or ADK2 had no phenotypic effect, lowering ADK levels to 6-20% that of WT lead to several changes including small, wavy rosette leaves, delayed leaf senescence, decreased internode length, reduced branching, clustered inflorescences and lack of petal abscission and silique dehiscence. Further analysis linked the abnormal phenotype to increased levels of hypomethylation throughout the plant (K Engel unpublished data), as was expected; however higher levels of active cytokinin were also observed. Thus ADK appears to be integral in regulating cytokinin levels as well as recycling methylation intermediates. To investigate the relationship between the subcellular localization of SAH production and its metabolism, immunogold labelling was performed on leaf and meristematic tissues of Arabidopsis using antibodies specific for either ADK or SAH hydrolase. As well, ???-glucuronidase and green fluorescent protein translational fusions of each enzyme were examined (S. Lee unpublished data). Results of both the immunogold labelling and fusion lines revealed that all ADK and SAH hydrolase isoforms localize to the cytosol, chloroplast and nucleus. Further analysis of purified chloroplasts has given varying results regarding the targeting of these enzymes to the organelle, and further research will be required before ADK and SAH hydrolase can be conclusively localized to the chloroplast.

Adenosine kinase (AK) is a purine salvage enzyme which catalyses the phosphorylation of the 5'-hydroxyl of adenosine via ATP. AK is a key enzyme which controls the intra and extracellular concentration of adenosine (Ado). Agents which inhibit the activity of AK have been found to attenuate cellular damage, demonstrating therapeutic utility in a variety of disease processes. In order to design inhibitors of AK with increased efficacy, a better understanding of enzyme activity is required. Previously, a number of novel characteristics of mammalian AK had been discovered. It was shown that the activity of AK is influenced by the presence of pentavalent ions (PVD such as inorganic phosphate (Pi), arsenate and vanadate. A detailed study of the influence of Pi on the kinetic parameters of Chinese hamster (CH) and beef liver AK was performed. These studies suggested that the Km (Ado) decreases and the Ki (Ado) increases asymptotically in the presence of increasing concentrations of Pi. Under the same conditions, the Vmax for activity increases hyperbolically. The effect of phosphate is not limited to the mammalian form of AK. Pi, arsenate and vanadate were all found to have similar effects on AK from yeast, spinach and Leishmania donovani AK. PVI as well as the metabolite phosphoenol pyruvate were also found to stimulate the activity of the enzyme ribokinase (RK) from E. coli, which similar to AK, is a member of the PfkB family of carbohydrate kinases. Although AK and RK show little sequence similarity, the residues at the active site and the 3D structues of these two proteins are very similar. Based on sequence alignment of PfkB family members, we have identified a conserved sequence motif, NXXE, which based upon the available structural information appears to be involved in the binding of phosphate. To confirm and understand the role of this motif in Pi binding, the residues at the NXXE site were altered by site-directed mutagenesis and their effect on activity of the recombinant CH AK was examined. Though the residues at the NXXE site do not directly interact with substrate, nor the putative catalytic base, the resulting proteins were found to have greatly altered phosphate requirement, substrate inhibition characteristics and different magnesium requirements. In the AK structure, aspartic acid at position 316 is presumed to act as the catalytic base. This residue was changed to asparagine and glutamic acid by mutagenesis. The resulting proteins were found to be nearly completely devoid of activity, confirming its critical role in AK activity. The amino acid sequence at the extreme N-terminus of AK has been found to exhibit the greatest variability within and among species, though the rest of the protein remains greatly conserved. To delineate the residues that are involved in the structural stability and activity of AK, systematic deletions of the residues from both the N-and C-terminus were performed, and the structure-activity relationships were examined. It was determined that the first 16 residues of CH AK can be removed without affecting activity. Removal of the next 11 residues resulted in sequential decreases in enzyme stability and activity. These 11 residues are involved in the first B-structure of the protein and are required for the stability of the tertiary structure. All residues at the C terminus were required for activity, and involved in a hydrogen-bonding network necessary for the stability of the ATP binding site. These studies provide novel insight to the structure-activity relationship of mammalian AK as well as the PfkB family of enzymes. Our work has identified a site distal to the catalytic site, that is implicated in the PVI binding and catalytic effect. Further studies should be aimed at understanding how binding of PVI at this site influences the catalytic activity of AK. Development of inhibitors which bind to this site and modulate the activity of AK should prove very useful in this regard. / Thesis / Doctor of Philosophy (PhD)

In the present study, adenosine kinase (AK) was purified from a number of different sources (e.g. bovine liver, Syrian hamster liver, kidney and heart; Chinese hamster ovary cells and human placenta). The enzyme from bovine liver and Syrian hamster liver was purified to apparent homogeneity by a combination of ion-exchange chromatography, affinity chromatography and gel filtration chromatography. The purified enzyme showed a single band of 40 kDa in SDS-polyacrylamide gel electrophoresis, which was similar to that reported from other sources in the literature. A number of biochemical and enzymatic characteristics of AK were investigated. The reliability and reproducibility of the AK assay as established in previous studies was determined. The apparent Km of partially purified AK from Syrian hamster liver for adenosine was determined to be 0.16 uM, which is consistent with earlier reports. A novel result obtained in these studies is that AK activity was found to be completely dependent upon the presence of phosphate or other pentavalent ions. AK from different sources did not show any activity in the Tris-HCl buffer, pH 7.0, but the activity increased dramatically upon the addition of phosphate and it reached a maximum at 2 mM phosphate. There was no inhibition of AK activity when the phosphate concentration was increased up to 100 mM. AK could also be activated by substituting phosphate with either arsenate or vanadate, which have similar chemical structures to phosphate. The temperature inactivation kinetics of AK showed that AK from human fibroblast cells had higher thermal resistance than AK from Chinese hamster ovary cells at 50°C. The presence of phosphate had no effect on the thermal stability of AK. Antibodies to purified AK from Syrian hamster liver were raised in both rabbits and guinea pigs. Antiserum from the rabbit which gave the strongest response was used for further studies. AK was recognized by and reacted specifically with the antiserum at a dilution of up to 1: 16,000. The AK antibody which was covalently bound to Protein A Sepharose beads immunoprecipitated a 40 kDa protein from radio-labelled Chinese hamster ovary or baby hamster kidney cell extracts. In addition, this antibody preparation immunoprecipitated AK activity from Syrian hamster liver extracts. However, immunoblotting showed that all the antisera could not react with AK from Chinese hamster ovary cells and human cells, suggesting a strong species specificity. A partial protein sequence of AK was obtained by microsequencing of a cyanogen bromide fragment of purified AK from Syrian hamster liver. The sequence was Tyr-Val-Asp-Ser-Leu-Phe-Gly-Ala-Glu-Thr-Glu-Ala-Ala-Leu. Degenerate DNA probes for this sequence can now be made and used for either screening of eDNA libraries or carrying out PCR experiments to facilitate the cloning of the AK gene(s). Finally, conditions for selection of revertants from AK⁻ mutants of Chinese hamster ovary cells have been developed. The AK⁺ revertants from AK⁻ mutants can be obtained using adenosine, alanosine, uridine and erythro-9-(2-hydroxyl-3-nonyl) adenine in the growth medium. Three revertants have been isolated. These revertants regained their AK activities and lost their drug-resistance at same time. This method can also be used in the future to clone the AK gene by transfecting AK⁻ mutants with foreign DNA. / Thesis / Master of Science (MSc)

Ischaemic heart disease is a major contributor to premature death and heart failure in the Westernised world. Ischaemic injury within the heart may be beneficially modulated by the nucleoside adenosine. Derived from catabolism of ATP, adenosine was initially known as a potent bradycardic and hypotensive agent. However, more recently the protective function of adenosine has been investigated. The protective effects of adenosine are mediated via activation of adenosine receptors: A1, A2A, A2B, and A3 receptors. Activation of these potentially protective (or retaliatory) adenosine receptors hinges upon accumulation of adenosine during ischaemia-reperfusion. This Thesis examines the role and mechanisms of adenosine mediated cardioprotection in young and aged hearts, exploring endogenous and exogenous adenosine receptor activation, genetic manipulation of A1 receptors and adenosine deaminase and pharmacological manipulation of adenosine metabolism. The effects of age on ischaemic responses and adenosine handling and protection are also assessed. The core approach to assess each of the above issues involved the Langendorff isolated mouse heart preparation. Experiments within Chapter 3 focuses on the contractile effects of adenosine receptors in normoxic hearts. This study indicates A2A receptors have no direct effect on contractility, while adenosine exerts positive inotropy independently of coronary flow and perfusion pressure (i.e. Independent of the Gregg phenomenon). In addition, investigations in genetically modified hearts hint at positive inotropy in response to A1 receptors. Since the latter is only evidenced in modified lines, it is possible A1-mediated inotropy may be abnormal or supraphysiological. In Chapter 4 the impact of genetic 'deletion' of A1ARs and/or adenosine deaminase (ADA) on intrinsic tolerance to ischaemia were studied. Data demonstrate that genetic deletion of A1 receptors significantly limits the ability of the mouse myocardium to withstand injury during ischaemic insult. Thus, providing strong support for a role of A1ARs in determining intrinsic tolerance to ischaemia-reperfusion. ADA KO mice confirm protection afforded by endogenous adenosine and the notion of adenosine metabolism modification as a protective strategy. Interestingly, effects of A1AR KO differ from A1AR overexpression or A1AR agonism in that the latter decrease contractile diastolic dysfunction while A1AR KO selectively increase systolic dysfunction and increase oncosis without altering diastolic injury. This challenges current dogma regarding the action of A1 adenosine receptors on ischaemic injury. In Chapter 5 the effects of adenosine metabolism inhibition (via adenosine deaminase (ADA) and adenosine kinase (AK) inhibitors) were studied. Inhibition of adenosine deaminase with the drug EHNA, and adenosine phosphorylation with iodotubercidin significantly protected mouse hearts from ischaemia-reperfusion, reducing contractile dysfunction and cardiac enzyme efflux. However, inhibitors failed to improve the outcome of the aged myocardium. 8-SPT and 5-HD reduced the protective effects of EHNA and iodotubercidin demonstrating thus; cardioprotection via ADA and AK appears to rely on adenosine receptor activation and involves a mitoK ATP dependent mechanism. Since aging is of considerable importance with regard to outcomes of ischaemic heart disease, experiments in Chapter 6 focused on effects of aging (and gender) on cardiovascular function and injury during ischaemia-reperfusion. In assessing post ischaemic outcomes in hearts from young adult (2-4 months), mature adult (8 months), middle aged (12 months), aged (18 months) and senescent (24-28 months) C57/BL/6J mice, data reveal a substantial age-related decline in ischaemic tolerance (which appears selective for myocardial vs. vascular injury). The decline in ischaemic tolerance is expressed primarily within the initial 12 months in both males and females with relatively little further decline with continued aging. There is evidence of a modest improvement in tolerance in senescence vs. aged hearts possibly reflecting selection of a protected phenotype in senescent populations. In addition, mature and middle-aged female hearts showed improved tolerance to ischaemia-reperfusion compared to males, supporting a role for hormonal changes. Effects of aging and purine metabolism were studied in Chapter 7. Data suggest impaired tolerance to ischaemia-reperfusion in older hearts may stem in part from shifts in myocardial purine catabolism. Data reveal reduced accumulation of salvageable and cardioprotective adenosine and enhanced accumulation of poorly salvaged (and potentially injurious) hypoxanthine and xanthine. These changes may potentially predispose the aged myocardium to ischaemic injury and radical generation via the xanthine oxidase reaction. The final data Chapter of this Thesis describes preliminary data regarding aging, signalling and adenosine mediated protection. It was found that protein kinase C (PKC) and A1 receptors mediate protection in young hearts and also that A1 receptors appear to mediate protection via a PKC LindependentLi signalling cascade. In addition, experiments in aged hearts (attempting to elucidate mechanisms behind impaired adenosinergic protection with age) suggest a PKC-independent A1-mediated protection path may be preserved with aging, since A1 receptors continue to offer some protection while PKC activation does not. It is possible the failure of exogenous adenosine to offer protection in aged hearts may result from a lesion at or downstream of PKC.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

Ribonucleotide reductase (RNR) is a key enzyme for de novo dNTP biosynthesis. We have studied nucleotide-dependent oligomerization of the allosterically regulated mammalian RNR using a mass spectrometry–related technique called Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA). Our results showed that dATP and ATP induce the formation of an α6β2 protein complex. This complex can either be active or inactive depending on whether ATP or dATP is bound. In order to understand whether formation of the large complexes is a general feature in the class Ia RNRs, we compared the mammalian RNR to the E. coli enzyme. The E. coli protein is regarded a prototype for all class Ia RNRs. We found that the E. coli RNR cycles between an active α2β2 form (in the presence of ATP, dTTP or dGTP) and an inactive α4β4 form in the presence of dATP or a combination of ATP with dTTP/dGTP. The E. coli R1 mutant (H59A) which needs higher dATP concentrations to be inhibited than the wild-type enzyme had decreased ability to form these complexes. It remains to be discovered how the regulation functions in the mammalian enzyme where both the active and inactive forms are α6β2 complexes. An alternative way to produce dNTPs is via salvage biosynthesis where deoxyribonucleosides are taken up from outside the cell and phosphorylated by deoxyribonucleoside kinases. We have found that the pathogen Trypanosoma brucei, which causes African sleeping sickness, has a very efficient salvage of adenosine, deoxyadenosine and adenosine analogs such as adenine arabinoside (Ara-A). One of the conclusions made was that this nucleoside analog is phosphorylated by the T. brucei adenosine kinase and kills the parasite by causing nucleotide pool imbalances and by incorporation into nucleic acids. Ara-A-based therapies can hopefully be developed into new medicines against African sleeping sickness. Generally, the dNTPs produced from the de novo and salvage pathways can be imported into mitochondria and participate in mtDNA replication. The minimal mtDNA replisome contains DNA polymerase γA, DNA polymerase γB, helicase (TWINKLE) and the mitochondrial single-stranded DNA-binding protein (mtSSB). Here, it was demonstrated that the primase-related domain (N-terminal region) of the TWINKLE protein lacked primase activity and instead contributes to single-stranded DNA binding and DNA helicase activities. This region is not absolutely required for mitochondrial DNA replisome function but is needed for the formation of long DNA products.

All living cells are dependent on nucleic acids for their survival. The genetic information stored in DNA is translated into functional proteins via a messenger molecule, the ribonucleic acid (RNA). Since DNA and RNA can be considered as polymers of nucleotides (NTPs), balanced pools of NTPs are crucial to nucleic acid synthesis and repair. The de novo reduction of ribonucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs), the precursors for DNA synthesis, is catalyzed by the enzyme ribonucleotide reductase (RNR). In cycling cells the dominant form of mammalian RNR consists of two proteins called R1 and R2. A proteasome-mediated degradation completely deprives postmitotic cells of R2 protein. The nonproliferating cells use instead a p53 inducible small RNR subunit, called p53R2 to synthesize dNTPs for mitochondrial DNA replication and DNA repair. To address the ongoing controversy regarding the localization and subsequently function and regulation of RNR subunits, the subcellular localization of all the mammalian RNR subunits during the cell cycle and after DNA damage was followed as a part of this thesis. Irrespective of the employed methodology, only a cytosolic localization could be observed leading to a conclusion that the dNTPs are synthesized in the cytosol and transported into the nucleus or mitochondria for DNA synthesis and repair. Thus, our data do not support the suggestion that nuclear translocation is a new additional mechanism regulating ribonucleotide reduction in mammalian cells. In an attempt to find a cure for African sleeping sickness, a lethal disease caused by a human pathogen, Trypanosoma brucei, nucleotide metabolism of the parasite was studied. The trypanosomes exhibit strikingly low CTP pools compared with mammalian cells and they also lack salvage of cytidine/cytosine making the parasite CTP synthetase a potential target for treatment of the disease. Following expression, purification and kinetic studies of the recombinant T. brucei CTP synthetase it was found that the enzyme has a higher Km value for UTP than the mammalian CTP synthetase. In combination with a lower UTP pool the high Km may account for the low CTP pool in trypanosomes. The activity of the trypanosome CTP synthetase was irreversibly inhibited by the glutamine analog acivicin, a drug extensively tested as an antitumor agent. Daily injections of acivicin to trypanosome-infected mice were sufficient to suppress the parasite infections. The drug was shown to be trypanocidal when added to cultured bloodstream T. brucei for four days at 1 uM concentration. Therefore, acivicin may qualify as a drug with “desirable” properties, i.e. cure within 7 days, according to the current Target Product Profiles of WHO and DNDi. Trypanosomes lack de novo purine biosynthesis and are therefore dependent on exogenous purines such as adenosine that is taken up from the blood by high-affinity transporters. We found that besides the cleavage-dependent pathway, where adenosine is converted to adenine by inosine-adenosine-guanosine-nucleoside hydrolase, T. brucei can also salvage adenosine by adenosine kinase (AK). The efficient adenosine transport combined with a high-affinity AK yields a strong salvage system in T. brucei, but on the other hand makes the parasites highly sensitive to adenosine analogs such as adenine arabinoside (Ara-A). The cleavage-resistant Ara-A was shown to be readily taken up by the parasites and phosphorylated by the TbAK-dependent pathway, inhibiting trypanosome proliferation and survival by incorporation into nucleic acids and by affecting nucleotide levels in the parasite.

Submitted by Luciana Sebin (lusebin@ufscar.br) on 2016-10-11T12:04:16Z No. of bitstreams: 1 DissACMF.pdf: 2760486 bytes, checksum: 27824647d350872bddb22f28a9206da8 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T13:38:00Z (GMT) No. of bitstreams: 1 DissACMF.pdf: 2760486 bytes, checksum: 27824647d350872bddb22f28a9206da8 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T13:38:07Z (GMT) No. of bitstreams: 1 DissACMF.pdf: 2760486 bytes, checksum: 27824647d350872bddb22f28a9206da8 (MD5) / Made available in DSpace on 2016-10-20T13:38:15Z (GMT). No. of bitstreams: 1 DissACMF.pdf: 2760486 bytes, checksum: 27824647d350872bddb22f28a9206da8 (MD5) Previous issue date: 2016-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The mansoni schistosomiasis is the most important of human helminthiasis. Despite advances in its control this disease continues to spread to new geographical areas. It currently affects more than 250 million people. However, limited options are available for and Praziquantel is the drug of choice. Various authors have been searching new drugs and vaccines to control schistosomiasis. This study aimed to evaluate the effects of a prior immunization with recombinant enzymes of Schistosoma mansoni: Adenosine Kinase (AK) and Hypoxanthine-guanine Phosphoribosyltransferase (HGPRT), which are important for parasite purine metabolism, as well as a MIX of these enzymes, and subsequent challenge with cercariae of the parasite in the control of murine infection. Female Balb/c mice were divided into 5 groups. The groups were enzyme-immunized in three doses and 15 days after the last immunization, animals were infected with S. mansoni. After infection in the 47º day egg count were carried in mice faeces and in the 48º day mice were sacrificed for evaluation of leukocyte numbers (blood and peritoneal cavity), worm burden, antibodies production, cytokines quantification and histopathological analysis of the liver of these animals. Our results strongly suggest that, immunization with a MIX originated in these animals reduction in the number of eggs in faeces by 46% when compared with the animals of the infected group. Animals of the groups immunized with AK, HGPRT and/or MIX seem to induce a reduction in the number of eosinophils in the peritoneal cavity when compared to the animals of the infected group. Concerning worm burden, the animals of the MIX group presented greater reduction (31.27%) when compared to the animals of the infected group. The animals of the immunized groups, AK, HGPRT and/or MIX were capable of producing IgG1 antibodies and IgE anti the enzymes and anti the parasite proteins. The animals of the immunized group MIX showed a slight increase in IL-4 production and observed reduction of IL-10, and in the HGPRT group induced a slight increase on IFN-γ production when in compared with the infected group. In addition, the animals of the AK group showed a decrease in the number of hepatic granulomas in tissue (44,55%) and the eggs present in liver (42,31%). Therefore, it suggests that immunization with these enzymes can contributes to schistosomiasis control, as well as it might helps to modulate experimental infection inducing reduction of physiopathology of this parasitosis. / A esquistossomose mansônica é a mais importante das helmintíases humanas. Apesar dos avanços no seu controle continua se espalhando para novas áreas geográficas. Atualmente afeta mais de 250 milhões de pessoas. Entretanto, opções limitadas estão disponíveis para o tratamento da doença e o único fármaco de escolha é o Praziquantel. Assim, vários estudos têm sido propostos para encontrar novos fármacos e vacinas para combater a esquistossomose. Dessa forma, o presente estudo teve como proposta avaliar os efeitos da imunização prévia com as enzimas recombinantes de Schistosoma mansoni Adenosina Quinase (AK) e Hipoxantina-Guanina Fosforibosiltransferase (HGPRT), que participam do metabolismo de purinas do parasito, bem como com o MIX das duas enzimas, e posterior desafio com cercárias do parasito, para o controle da infecção murina. Camundongos fêmea Balb/c foram divididos em 5 grupos. Os grupos imunizados receberam três doses das enzimas e após 15 dias da última imunização, os animais foram infectados com S. mansoni. Após a infecção, no 47° dia foi realizada a contagem de ovos nas fezes e no 48° dia foi realizada a eutanásia dos animais para avaliação de resposta leucocitária (sangue e lavado da cavidade peritoneal), carga parasitária, produção de anticorpos, quantificação de citocinas e análise histopatológica do fígado desses animais. Os resultados demonstraram que, a imunização com o MIX promoveu nesses animais redução do número de ovos nas fezes de 46% quando comparado com os animais do grupo somente infectado. Os animais dos grupos imunizados com AK, HGPRT e/ou MIX apresentaram diminuição na quantidade de eosinófilos na cavidade peritoneal quando comparados com os animais do grupo somente infectado. Em relação à carga parasitária, os animais do grupo imunizado com o MIX apresentaram maior redução (31,27%) quando comparados aos animais do grupo somente infectado. Os animais dos grupos imunizados com AK, HGPRT e/ou MIX foram capazes de produzir anticorpos IgG1 e IgE anti as enzimas e anti as proteínas do parasito. Os animais do grupo imunizado com o MIX apresentaram aumento discreto de IL-4 e foi observada redução de IL-10, e no grupo imunizado com HGPRT houve aumento discreto de IFN-γ, quando comparados com os animais do grupo somente infectado. Além disso, os animais do grupo imunizado com AK apresentaram redução do número de granulomas hepáticos (44,55%) e de ovos no fígado (42,31%), quando comparados com o grupo somente infectado. Assim, sugere-se que a imunização com essas enzimas pode contribuir para o controle da esquistossomose, bem como auxiliar na modulação da infecção experimental, induzindo redução da fisiopatologia desta parasitose.

Orientador: Ljubica Tasic / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T12:26:39Z (GMT). No. of bitstreams: 1 Rodrigues_FabioHenriquedosSantos_M.pdf: 24628982 bytes, checksum: f6b1490bc6bf2bf5497e5cad40a40daa (MD5) Previous issue date: 2011 / Resumo: A Adenosina Quinase (ADK) é uma enzima importante (EC 2.7.1.20), cuja ação pode estar relacionada a diversas doenças, tais como inflamações, derrame, infarto, entre outras. Desse modo, a inibição de sua atividade é de grande importância, e desperta interesse científico. Na tentativa de inibir a ação da ADK, houve busca por compostos orgânicos cuja capacidade inibitória seja superior comparando-se com inibidores da ADK existentes. Desse modo, derivados de 4-anilinoquinazolinas mostraram-se alvos interessantes. Foi sintetizada uma série de 22 derivados de 8-metóxi-4-anilinoquinazolinas, substituídas nas posições 3'e 4'do anel anilínico. Os compostos sintetizados foram caracterizados e testados frente à ADK, de forma a verificar seu potencial inibitório, principalmente através da técnica de fluorescência de emissão. Da série de compostos, seis apresentaram-se promissores na inibição da ADK. Ensaios in silico também foram realizados, buscando-se uma melhor compreensão do mecanismo de inibição do sistema compostos/ADK / Abstract: The Adenosine Kinase (ADK) is an important enzyme (EC 2.7.1.20) that might be related to several diseases, such as inflammation, stroke and infarct, and many others. Therefore, its activity inhibition is of great importance, arising significant scientific interest. Aiming ADKs inhibition, a search for suitable organic species was realized, in such way that 4-anilinoquinazoline derivatives showed themselves interesting targets. A serie of 22 8-methoxy-4-anilinequinazoline derivatives, substituted on the aniline ring at 3'and 4'positions, was synthesized. The compounds were characterized and tested in in vitro ADKs inhibition, in order to verify their inhibitory potentials, mainly applying emission fluorescence technique. Six compounds of this serie presented promising properties in ADKs inhibition. In silico assays were also conducted, in order to better explain the inhibitory mechanism of the system compounds/ADK / Mestrado / Quimica Organica / Mestre em Química