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Deuteration is the replacement of a hydrogen atom by deuterium atom in a molecule. The replacement begins at the most acidic hydrogen in the molecule. In ATP, the deshielded hydrogen is C8-H which is the first replaced during deuteration. During ATP deuteration some of the ATP is hydrolysed to ADP concurrently. Using kinetic analysis, it was confirmed that the ATP hydrolysis that occurs is 1st order in ATP concentration, while the hydrogen replacement is 2nd order. The ATP and its C8 deuterated analogue were tested against three enzymes shikimate kinase (SK), acetate kinase (AK) and glutamine synthetase (GS) to determine if a kinetic isotope effect (KIE) exists in these systems. With AK and GS, the KIED increased as the KIEH decreased, while with SK the KIED decreased as the KIEH increased as the concentration of the ATP or deuterated analogue increased. Deuteration of imidazole and purine compounds reduced the specific activity of AK or SK at low concentrations in an enzyme-catalysed reaction. From a library of imidazole-containing compounds that inhibited SK, three compounds were selected and their IC50 values were determined on the SK-catalysed reaction. These compounds show a differential potency and efficiency between their protonated and deuterated analogues when compared in a 1:1 mixture. Synthesized purines incorporating three different substituents at N-9 were tested against AK or SK for their ability to lower the specific activity of the enzymes used / Physics / M. Sc. (Physics)

Ruminant animals consuming low N-diet have been reported to have increased urea reabsorption with the Nguni being categorized as N-recycling ruminant. The enzymes associated with N-cycling are hypothesized to contribute to survival of the Nguni in harsh conditions. Enzymes responsible for such a function needed to be characterized in order to determine their effect in the functioning of the Nguni as opposed to Hereford breed. Crude enzymes from both breeds were separated from most or some contaminants by sephadex G-25, DEAE sephacel, and different affinity column chromatography. CPS and GDH were successfully purified and characterized by LC-MS/MS and further analysed by ProteinPilot™, blasted and matched >95% with those of Bos Taurus. Comparison of characterized enzymes and those which failed to ionise such as ARG, GS and GA was done using kinetics and graphs annotating specific activities. Partial purification and characterization was in part achieved. / Life and Consumer Sciences / M. Sc. (Life Sciences)

The complexity of all biomolecules in existence today can be attributed to the variation of the amino acid repertoire. In nature, 20 canonical amino acids are translated to form these biomolecules, however, many of these amino acids have revealed posttranslational modifications (i.e. acetylation, methylation) after incorporation. Amino acids that exhibit PTM are known for their involvement in cellular processes such as DNA repair and DNA replication; these PTMs are commonly found on histones within the chromatin complex. Utilization of in vivo site-specific incorporation has recently reported functionality of post-translationally modified amino acids.1 xii Here we report the synthesis and in vivo site-specific incorporation of the histone PTM, 2-hydroxyisobutyrl lysine (Khib), with the pyrrolysyl tRNA/ RNA synthetase system. This translational machine can better serve to probe Khib for functional benefits. Additionally, this thesis focuses much of its attention on the development of unnatural amino acids (UAA) with optogenetic characteristics. These UAAs, if site-specifically incorporated, can be used to control enzymes and proteins through rapid light perturbation (365nm UV light). Furthermore, discussed is the synthesis of photo-caged threonine and photo-caged serine as potential substrates for the pyrrolysyl translational machinery.

A targeted approach to the development of antifolate therapies has been sought for many years. Central to the success of such development is an understanding of the molecular mechanisms dictating the sensitivity of cells to antifolates and the fundamental differences of these processes between normal and neoplastic phenotypes. This dissertation addressed transcriptional mechanisms and cell-signaling events responsible for the efficacy of antifolate therapies. Transcriptional processes and cell signaling pathways are often aberrant in neoplastic tissues, providing a potential point of distinction between a normal and neoplastic cellular state. Folylpolyglutamate synthetase (FPGS) catalyzes the formation of poly-γ-glutamate derivatives of folates and antifolates, which permits intracellular retention and accumulation of these compounds. The mouse fpgs gene uses two distant promoters to produce functionally distinct isozymes in a tissue-specific pattern. We questioned how the two promoters were differentially controlled. An analysis of DNA methylation and histone post-translational modifications across the length of the mouse fpgs gene showed that epigenetic mechanisms contributed to the tissue-specific control of the upstream (P1), but not the downstream (P2) fpgs promoter. RNAPII complexes and general transcription factors were present over P1 only when P1 was transcribed, but these components were present over P2 in most tissues, and promoter-proximal pausing was evident in brain. Clear promoter occlusion was found over P2 in liver. These studies concluded that tissue-specific coordination of dual promoters required multiple interacting controls. The mammalian target of rapamycin (mTOR) controls protein translation initiation, and is central to a cell-signaling pathway rich in tumor suppressor and oncogenic proteins. mTOR dysregulation is a common feature of several human cancers and inhibition of this protein has been sought as an ideal cancer drug target. We have determined that antifolates inhibiting the two folate-dependent steps of purine synthesis (GART or AICART) activate AMP-dependent protein kinase (AMPK) and inhibit mTOR. The mechanism of AMPK stimulation appears to be mediated by either nucleotide depletion (GART inhibitors), or ZMP accumulation (AICART inhibitors). These studies discovered a new mechanism for antifolates that surprisingly defines them as molecularly targeted therapeutics.

Le virus de l’immunodéficience humaine de type 1 (VIH-1), est un rétrovirus dont le génome est composé de deux molécules d’ARN simple brin. La transcriptase inverse codée par le VIH-1 utilise l’ARNt3Lys de la cellule hôte pour amorcer la réplication de son génome ARN en ADN proviral. L’ARNt3Lys est encapsidé dans les virions lors de l’assemblage; la lysyl-ARNt synthétase (LysRS) cellulaire est impliquée dans ce mécanisme et sert de co-transporteur à l’ARNt3Lys.Chez l’homme, il existe deux formes de LysRS, une forme cytoplasmique (cLysRS) et une forme mitochondriale (pmLysRS) qui donnera la forme mature (mLysRS) après translocation dans la mitochondrie. Les deux LysRS sont issues d’un même gène par épissage alternatif. Il a été démontré que seule la forme mitochondriale est présente dans les particules virales.Nous avons établi un modèle des interactions protéine-protéine impliquées dans la formation du complexe d’encapsidation de l’ARNt3Lys. En recherchant les interactions des précurseurs Gag et GagPol avec les LysRS et leurs domaines, nous avons démontré que seul le domaine Pol du précurseur GagPol a la capacité de s’associer à la LysRS. Ce sont les sous-domaines transframe TF et intégrase IN du domaine Pol qui permettent l’association entre LysRS et GagPol. Cette association se fait via le domaine catalytique de l’enzyme. La sélectivité de l'encapsidation de la forme mitochondriale de LysRS aux dépens de sa forme cytoplasmique pourrait résider dans la stricte compartimentation cellulaire de ces deux formes enzymatiques. Nous avons voulu établir à quel stade l’encapsidation de la LysRS mitochondriale a lieu, soit avant sa translocation mitochondriale sous forme de précurseur pmLysRS, soit après sous forme mLysRS maturée. Nous avons déterminé le site de maturation du précurseur pmLysRS puis caractérisé les deux formes mitochondriales de la LysRS, en déterminant leurs paramètres cinétiques et leur affinité pour l’ARNt3Lys. Alors que la forme pmLysRS ne forme pas de complexe stable avec l’ARNt, la forme maturée mLysRS est la plus apte à interagir avec l’ARNt3Lys. Ce serait donc la mLysRS qui serait impliquée dans le transport de l’ARNt3Lys dans les particules virales lors du bourgeonnement.Comme l'interaction GagPol:LysRS n'est pas spécifique in vitro de la forme mLysRS qui est la seule espèce de LysRS encapsidée, nous avons recherché si d’autres protéines virales pouvaient intervenir dans la formation du complexe d’encapsidation et conférer la spécificité pour la seule mLysRS. Nous avons montré que les protéines auxiliaires Rev et Vpr ont la capacité à s’associer à la LysRS sans distinction d'origine, mais ne peuvent interagir dans le contexte du complexe d'encapsidation GagPol:mLysRS:ARNt3Lys. Les différentes formes de LysRS pourraient ainsi réguler l'activité de Vpr et Rev à d'autres étapes du cycle viral. / The Human immunodeficiency virus type 1 (HIV-1) is a retrovirus with a genome composed of two molecules of single stranded RNA. The reverse transcriptase encoded by HIV-1 uses the cellular tRNA3Lys to prime the replication of its RNA genome into a proviral DNA. The tRNA3Lys is packaged into the viral particles during their assembly; the cellular lysyl-tRNA synthetase (LysRS) is involved in this mechanism as a co-carrier of tRNA3Lys.In human, there are two forms of LysRS, a cytoplasmic form (cLysRS) and a mitochondrial form (pmLysRS) that will be maturated into mLysRS after translocation into the mitochondrion. Both LysRS arise from the same gene by alternative splicing. It was demonstrated that only the mitochondrial species is present in the viral particles.We established a model of the protein-protein interactions which are implied in the formation of the packaging complex of tRNA3Lys. By searching for interactions of the viral precursors Gag and GagPol with the LysRS species and their domains, we demonstrated that only the Pol domain of the GagPol precursor has the capacity to interact with LysRS. The transframe (TF) and integrase (IN) domains of the Pol region of the polyprotein GagPol are required for association of LysRS with GagPol. This association is mediated by the catalytic domain of the enzyme. The selectivity of the packaging of the mitochondrial species of LysRS but not of its cytoplasmic species would rest on the cellular compartmentalization of these two enzyme forms. To establish at which step the mitochondrial LysRS is packaged, either as the pmLysRS precursor before its mitochondrial translocation, or after as the mature mLysRS, we determined the site of maturation of the pmLysRS precursor, then we characterized both mitochondrial forms of LysRS, by determining their kinetic parameters and their affinity for tRNA3Lys. Whereas the pmLysRS species did not form a stable complex with tRNA, the mature pmLysRS species did. Thus, mLysRS is the only LysRS species which could be implied in the transport of tRNA3Lys into the viral particles during the budding step. In vitro, the interaction GagPol:LysRS is not specific for the mLysRS species, but only the mitochondrial LysRS is packaged into the viral particles. We determined if another viral protein could impact the specificity of mLysRS packaging. We showed that the auxiliary proteins Rev and Vpr have the capacity to interact with LysRS but this intercation is not recovered in the context of the GagPol:mLysRS:tRNA3Lys packaging complex. These data suggest that the different forms of LysRS might regulate the activity of Vpr and Rev at other steps of the viral cycle.

A porfiria aguda intermitente é uma doença autossômica dominante, decorrente de um distúrbio na via biossintética do heme, causado pela redução dos níveis da enzima uroporfirinogênio-I-sintetase. As manifestações clínicas envolvem o sistema nervoso periférico e o central. O diagnóstico baseia-se na excreção urinária elevada dos precursores das porfirinas ácido d-aminolevulínico e porfobilinogênio. O objetivo deste estudo foi analisar o quadro clínico apresentado por pacientes do Hospital das Clínicas de São Paulo com porfiria aguda intermitente, atendidos no período de janeiro de 1979 a dezembro de 1999. Foram avaliados 37 pacientes, com idades entre 6 e 48 anos, na proporção de 2,7 mulheres:1 homem. A faixa etária na qual ocorreu o maior número de crises foi a terceira década. Os pacientes apresentaram 63 crises, sendo que 13 deles também tiveram manifestações de caráter crônico. As manifestações clínicas mais freqüentes foram: dor abdominal, alteração da cor da urina, mudança no ritmo intestinal, déficit motor ou sensitivo-motor, vômitos, alteração do nível de consciência ou confusão mental, crises convulsivas, quadros disautonômicos cardio-vasculares e distúrbios psiquiátricos. As crises foram classificadas em leves, moderadas e graves, segundo critérios previamente estabelecidos. Todas as manifestações crônicas foram caracterizadas como leves. A neuropatia periférica motora ou sensitivo-motora nunca foi a manifestação inicial da crise de porfiria aguda intermitente. Houve correlação entre o tipo e o número de fatores precipitantes e a manifestação da neuropatia periférica motora ou sensitivo-motora, verificando-se que as crises nas quais ela está ausente foram em geral desencadeadas por apenas um fator, mais comumente de origem endócrina ou metabólica endógena, como período menstrual e jejum, enquanto que nas crises com neuropatia periférica houve a participação de vários fatores concomitantemente, sendo estes principalmente de origem exógena, como medicamentos. Os tratamentos mais utilizados nos surtos foram a administração de glicose, aumento da ingestão de carboidratos e o uso de fenotiazínicos. / Acute intermittent porphyria is an autosomal dominant disease, caused by a disturbance in the heme biosynthetic pathway, secondary to the reduction on the levels of uroporphyrinogen-I-synthetase enzyme. Clinical manifestations involve central and peripheral nervous system. The diagnosis is based on the elevated urinary excretion of porphyrins precursors d-aminolevulinic acid and porphobilinogen. The aim of this study was to analyze the clinical manifestations of acute intermittent porphyria in patients of the Hospital das Clínicas of São Paulo, seen between January 1979 and December 1999. 37 patients were studied, from 6 to 48 years old, with a rate of 2,7 women: 1 man. The age in which most of the crisis occurred was the third decade. The patients presented 63 crisis, and 13 of them presented also with chronic manifestations. The commonest clinical presentations were: abdominal pain, change in urine color, change in bowel habits, motor or sensory-motor deficit, vomiting, alteration of consciousness or mental confusion, convulsions, dysautonomic cardiovascular signs and psychiatric disorders. The crisis were classified as mild, moderate and severe, following criteria previously established. All chronic manifestations were characterized as mild. The peripheral motor or sensory-motor neuropathy was never the initial manifestation. Correlation was found between the kind and the number of precipitating factors, and the absence of peripheral neuropathy was in general related to just one factor, more commonly of endogenous endocrine or metabolic origin, like menstrual period and starvation, while in the crisis with peripheral neuropathy multiple factors were involved at the same time, these being of exogenous origin, like drugs. The most commonly used treatments were glucose administration, elevation of carbohydrate intake, and phenothiazines use.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-05T14:23:17Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) MARIANE TRINDADE DE PAULA.pdf: 5612709 bytes, checksum: a2f34c2760ece6fed258912c3f6d2442 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-05T14:23:44Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) MARIANE TRINDADE DE PAULA.pdf: 5612709 bytes, checksum: a2f34c2760ece6fed258912c3f6d2442 (MD5) / Made available in DSpace on 2017-06-05T14:23:44Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) MARIANE TRINDADE DE PAULA.pdf: 5612709 bytes, checksum: a2f34c2760ece6fed258912c3f6d2442 (MD5) Previous issue date: 2017 / Os efeitos deletérios de dietas como, por exemplo, as dietas ricas em gorduras (DRG) estão, cada vez mais, sendo estudados uma vez que eleva os níveis de triglicerídeos (TGC), causando um acúmulo de gordura, diminuindo a tolerância ao estresse e, consequentemente, propiciando o desenvolvimento de doenças e complicações que levam a diminuição da expectativa de vida e/ou morte precoce dos indivíduos. A hesperidina (Hesp), um flavonóide cítrico, já demonstrou ter propriedades antioxidantes representando um potencial agente protetor contra danos ligados à obesidade. Assim, o objetivo deste estudo foi investigar as alterações comportamentais, bioquímicas e genéticas induzidas por uma DRG em exemplares adultos e descendentes da mosca da fruta Drosophila melanogaster, bem como os efeitos terapêuticos da Hesp frente a estas alterações. Através deste estudo avaliou-se os danos oxidativos, a atividade e expressão de enzimas antioxidantes, como a superóxido dismutase (SOD) e catalase (CAT), e expressão de mRNA de enzimas envolvidas no metabolismo dos ácidos graxos como acetil-CoA sintetase (ACeCS 1) e acil-CoA sintetase (ACSL 1) e modulação da via de sinalização de estresse. Como resultados, nosso estudo demonstrou pela primeira vez que as moscas progenitoras adultas alimentadas por sete dias com DRG têm um desempenho locomotor reduzido, um aumento da ACeCS 1 e ACSL 1, aumento na produção de espécies reativas e substâncias reativas ao ácido tiobarbitúrico, aumento da expressão da proteína de choque térmico 83 (HSP83) e proteína quinase ativada por mitógenos 2 (MPK2), encurtando a vida útil destas moscas. Além disso, DRG quando adicionada durante todo o período de desenvolvimento embrionário, causa nas moscas descendentes um aumento nos níveis de TGC e glicose, juntamente com um aumento no nível de expressão de mRNA de DILP6, uma diminuição na enzima ACeCS1 presente no metabolismo de ácidos graxos, SOD e CAT em níveis de expressão de mRNA e diminuição da atividade de CAT. Os resultados observados a partir do co-tratamento com a Hesp e a DRG, este composto mostrou ser eficaz na redução de alguns parametros de alterações metabólicas, melhorou os níveis de TGC, glicose, parametros de estresse oxidativo, manteve a viabilidade celular e mitocondrial, melhorou o desempenho locomotor e atividade da enzima acetilcolinesterase (AChE), aumentando a expectativa de vida das moscas progenitoras. Em relação às moscas descendentes, a Hesp melhorou os níveis de TGC, diminuiu os níveis de glicose protegendo contra um possível desenvolvimento de sintomas pré-diabéticos, melhorando a taxa de eclosão dos ovos e expectativa de vida. Portanto, através deste estudo revelamos um eficiente papel protetor do flavonóide Hesp no tratamento de moscas progenitoras ou descendentes que receberam DRG demonstrando assim que este composto é capaz de atenuar alterações metabólicas, estresse oxidativo e sinalização de proteínas causadas pela obesidade.