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PHOTOINITIATED-PROCESSES IN ADENINE OLIGONUCLEOTIDES: EXAMINING THE NATURE OF π-STACKING INTERACTIONS IN MULTI-CHROMOPHORE SYSTEMSSpata, Vincent Anthony January 2016 (has links)
DNA provides the genetic code which is almost universal in all living organisms. When DNA is exposed to ultra-violet light it can cause cell degradation and mutation which are two of the major causes which lead to cancer. The nature of decay in DNA oligomers is a widely studied topic. Fluorescence and Transient Absorption (TA) experiments on polynucleotides which compare the behavior of the decay to the monomer bases have revealed the presence of longer-lived components in the multimeric systems. There has been heated debate over the character of the excited states responsible for the long-lived signals. Theoretical methods are well suited to compliment experiment by providing a description of processes and physical properties on the molecular level. We have studied π-stacked adenines in the gas-phase with Quantum Mechanical (QM) methods, but also in the helical environment using high-level ab initio methods, classical simulations and the combination of the two (QM/MM). Inclusion of the environmental interactions dramatically alters the shape of the potential energy surfaces due to steric interactions from the backbone and interactions with the surrounding bases and environment. This work examines the complete picture of photophysical processes occurring in adenine oligonucleotides within the helical environment after the absorption of a photon: the nature of initial absorption and the subsequent radiative and non-radiative decay pathways. It contributes key discoveries inherent to the mechanisms which govern photo-initiated processes in DNA, and also contributes to our fundamental knowledge of the photophysical behavior of π-stacked chromophores. The work reveals the effects of π-stacking interactions and the environment on photo-initiated processes in oligonucleotides. It reveals that excitonic coupling is responsible for the key differences in features in the absorption spectrum of adenine oligomers compared to the isolated bases, illustrates the role of charge transfer (CT) mixing in both absorption and decay processes, and the importance of bonded excimers in deactivation. The work also illustrates that CT excimers are responsible for the long-lived signals evidenced in Transient Absorption and Fluorescence experiments and that neutral excimers can exist within the DNA helical environment. It also adds to the discussion in the field on the nature of photodecay mechanisms occurring within the DNA helix. / Chemistry
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Regulation of Pyridine Nucleotide Metabolism in Saccharomyces cerevisiaeTing, Haung-yu 05 1900 (has links)
The levels of total nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), and their redox states were determined as the function of growth in S. cerevisiae. Cells growing in a medium containing 0.8% glucose exhibit two phases of exponential growth, utilizing glucose and ethanol, respectively. The NAD pool is 50% reduced during both stages of growth while the NADP pool is 67% reduced in glucose growth and 48% reduced in ethanol growth. The NAD/NADP ratio is constant during growth on glucose and a two-fold increase in the NAD/NADP ratio occurs upon exhaustion of glucose. The increased ratio is maintained during growth on ethanol. This alteration in the regulation of the relative levels of NAD and NADP may be due to a change in the regulation of NAD kinase and/or NADP phosphatase activities. These changes may be related to the redox state of the NADP pool.
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Aspectos de biologia molecular, estrutural e funcional de adenina fosforribosiltransferase de Homo Sapiens e seu envolvimento na sindrome urolitíase / Functional, structural and molecular biology aspects of the adenine phosphorybosiltransferase from Homo sapiens and its involvement in the urolitiasis syndromeSilva, Márcio 03 June 2005 (has links)
Esta tese apresenta em sua introdução uma revisão bibliografia sobre a importância das enzimas Fosforribosil-transferases (PRTases) para a homeostase tanto em células de mamíferos como em células de protozoários Kinetoplastidas. É ressaltada a estreita relação entre mutações pontuais encontradas no gene da enzima Adenina Fosforribosiltransferase (APRT) e a doença humana Dihidroxiadenina Urolitiase. A doença leishmaniose, causada por Kinetoplastidas do gênero Leishmania, é descrita indicando a possível utilização das enzimas da via de recuperação de purinas como alvos para o desenvolvimento de novos medicamentos contra leishmaniose. Os experimentos descritos nesta tese, visam a produção heteróloga da enzima (APRT) de Homo sapiens, elucidação da estrutura cristalográfica e análise estrutural das mutações encontradas em pacientes com Dihidroxiadenina Urolitiase. A proteína homóloga de Leishmania terentolae também foi objetivo de estudo. Com essa APRT, os experimentos descritos nesta tese visam a elucidação da estrutura tridimensional e a utilização da enzima na busca de novos inibidores de PRTases. Os resultados apresentados nesta tese mostram que os objetivos do estudo foram alcançados. A elucidação da estrutura da APRT de H. sapiens possibilitou discutir as prováveis alterações estruturais provocadas pelas mutações pontuais do gene da APRT. Novos inibidores de PRTases de Leishmania foram obtidos. Adicionalmente, os inibidores das enzimas foram testados em culturas de Leishmania major promastigota e promoveram inibição do crescimento do parasita. / This thesis in its introduction presents a brief bibliografic revision about the importance of the phosphoribosyltransferase enzyme (PRTases) to cellular homeostasis, both in mammalian cell as in the protozoa Kinetoplastida. It is emphasized the close relation between point mutatons found in the gene coding for Adenine phosphoribosyltransferase (APRT) and disease Dihidroxiadenine Urolitiasis. The deseases leishmaniasis, caused by Kinetoplastidae beloing to the Leishmania genera, is described indicating the possible use of the purine salvage pathway (PRTases) as targets for the future development of novel drugs against leishmaniasis. The experience described in this thesis aims at the heterologous production of the enzyme APRT of Homo sapiens, its crystallographic structure elucidation and structural analysis of mutations found in patient with Dihidroxiadenine Urolitiasis. The Leishmania tarentolae homologue was also the aim of this work. With this APRT the experiments describes in this thesis aim at the structural resolution of the enzyme and its use in the screening for novel PRTases inhibitors. The results presented in this thesis show that the objectives of the work have been achieved. The structural elucidation of the human APRT allowed the discussion of the potential modifications caused bu the point mutations in the APRT gene. Novel inhibitors of PRTases of Leishmania major promastigote cultures and promoted the growth inhibition of this parasite.
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Effect of 1-methyladenine on double-helical DNA structures and stabilities.January 2009 (has links)
Yang, Hao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 53-57). / Abstract also in Chinese. / Title Page --- p.i / Thesis Committee --- p.ii / Abstract (English version) --- p.iv / Abstract (Chinese version) --- p.vi / Acknowledgment --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations and Symbols --- p.xv / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- DNA Methylation --- p.1 / Chapter 1.2 --- DNA Methylation Repair --- p.2 / Chapter 1.3 --- Objectives of This Work --- p.2 / Chapter 1.4 --- DNA Structure --- p.3 / Chapter 1.4.1 --- Nomenclature Scheme for DNA --- p.3 / Chapter 1.4.2 --- Base Pair Scheme --- p.4 / Chapter 1.4.3 --- Sugar Conformation --- p.5 / Chapter 1.4.4 --- Backbone Conformation --- p.5 / Chapter 2 --- Materials and Methods --- p.7 / Chapter 2.1 --- Sample Design --- p.7 / Chapter 2.2 --- Sample Preparation --- p.7 / Chapter 2.3 --- UV Optical Melting Study --- p.8 / Chapter 2.4 --- NMR Study --- p.9 / Chapter 2.4.1 --- NMR Melting Study --- p.10 / Chapter 2.4.2 --- Resonance Assignment --- p.10 / Chapter 2.4.3 --- Determination of Sugar Conformation --- p.12 / Chapter 2.4.4 --- Determination of Backbone Conformation --- p.13 / Chapter 3 --- Effect of 1-Methyladenine on Double-Helical DNA Structures --- p.14 / Chapter 3.1 --- NMR Resonance Assignments --- p.14 / Chapter 3.1.1 --- TA-oligo Resonance Assignments --- p.14 / Chapter 3.1.2 --- TmlA-oligo Resonance Assignments --- p.16 / Chapter 3.2 --- DNA Double-Helical Structures upon 1-Methylation of Adenine --- p.18 / Chapter 3.2.1 --- Base Pairing Mode --- p.18 / Chapter 3.2.2 --- Sugar Puker --- p.21 / Chapter 3.2.3 --- Backbone Conformation --- p.22 / Chapter 3.3 --- Summary --- p.24 / Chapter 4 --- Effect of 1-Methyladenine on Double-Helical DNA Stabilities --- p.25 / Chapter 4.1 --- Thermodynamic Studies --- p.26 / Chapter 4.1.1 --- Influence of m6A on UV Melting Studies --- p.26 / Chapter 4.1.2 --- Thermodynamics by NMR Melting Studies --- p.28 / Chapter 4.2 --- "NMR Structural Studies on Gm1A-, Am1A- and Cm1A-oligo" --- p.33 / Chapter 4.2.1 --- Gml A-oligo --- p.33 / Chapter 4.2.1.1 --- Gm1A-oligo Resonance Assignments --- p.33 / Chapter 4.2.1.2 --- Base Pair Structures of Gm1A-oligo --- p.35 / Chapter 4.2.2 --- AmiA-oligo --- p.37 / Chapter 4.2.2.1 --- Am1A-oligo Resonance Assignments --- p.37 / Chapter 4.2.2.2 --- Base Pair Structures of Am1A-oligo --- p.39 / Chapter 4.2.3 --- Cm1A-oligo --- p.43 / Chapter 4.2.3.1 --- Cm1A-oligo Resonance Assignments --- p.43 / Chapter 4.2.3.2 --- Base Pair Structures of Cm1A-oligo --- p.45 / Chapter 4.3 --- Summary --- p.46 / Chapter 5 --- Conclusion and Future work --- p.47 / Appendix I Proton chemical shift values of TA-oligo --- p.48 / Appendix II Proton chemical shift values of TmlA-oligo --- p.49 / Appendix III Proton chemical shift values of GmlA-oligo --- p.50 / Appendix IV Proton chemical shift values of Am1A-oligo --- p.51 / Appendix V Proton chemical shift values of CmlA-oligo --- p.52 / References --- p.53
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Purification and characterization of NADH oxidase and peroxidase from Lactobacillus caseiBarstad, Louise Ann 01 January 1978 (has links)
A protein exhibiting NADH oxidase and NADH peroxidase activity has been isolated from Lactobacillus casei in high purity. Evidence obtained through gel filtration, electrophoresis, and electrofocusing indicates that the two activities are characteristic of a single tetrameric protein with an approximate molecular weight of 240,000.
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Aspectos de biologia molecular, estrutural e funcional de adenina fosforribosiltransferase de Homo Sapiens e seu envolvimento na sindrome urolitíase / Functional, structural and molecular biology aspects of the adenine phosphorybosiltransferase from Homo sapiens and its involvement in the urolitiasis syndromeMárcio Silva 03 June 2005 (has links)
Esta tese apresenta em sua introdução uma revisão bibliografia sobre a importância das enzimas Fosforribosil-transferases (PRTases) para a homeostase tanto em células de mamíferos como em células de protozoários Kinetoplastidas. É ressaltada a estreita relação entre mutações pontuais encontradas no gene da enzima Adenina Fosforribosiltransferase (APRT) e a doença humana Dihidroxiadenina Urolitiase. A doença leishmaniose, causada por Kinetoplastidas do gênero Leishmania, é descrita indicando a possível utilização das enzimas da via de recuperação de purinas como alvos para o desenvolvimento de novos medicamentos contra leishmaniose. Os experimentos descritos nesta tese, visam a produção heteróloga da enzima (APRT) de Homo sapiens, elucidação da estrutura cristalográfica e análise estrutural das mutações encontradas em pacientes com Dihidroxiadenina Urolitiase. A proteína homóloga de Leishmania terentolae também foi objetivo de estudo. Com essa APRT, os experimentos descritos nesta tese visam a elucidação da estrutura tridimensional e a utilização da enzima na busca de novos inibidores de PRTases. Os resultados apresentados nesta tese mostram que os objetivos do estudo foram alcançados. A elucidação da estrutura da APRT de H. sapiens possibilitou discutir as prováveis alterações estruturais provocadas pelas mutações pontuais do gene da APRT. Novos inibidores de PRTases de Leishmania foram obtidos. Adicionalmente, os inibidores das enzimas foram testados em culturas de Leishmania major promastigota e promoveram inibição do crescimento do parasita. / This thesis in its introduction presents a brief bibliografic revision about the importance of the phosphoribosyltransferase enzyme (PRTases) to cellular homeostasis, both in mammalian cell as in the protozoa Kinetoplastida. It is emphasized the close relation between point mutatons found in the gene coding for Adenine phosphoribosyltransferase (APRT) and disease Dihidroxiadenine Urolitiasis. The deseases leishmaniasis, caused by Kinetoplastidae beloing to the Leishmania genera, is described indicating the possible use of the purine salvage pathway (PRTases) as targets for the future development of novel drugs against leishmaniasis. The experience described in this thesis aims at the heterologous production of the enzyme APRT of Homo sapiens, its crystallographic structure elucidation and structural analysis of mutations found in patient with Dihidroxiadenine Urolitiasis. The Leishmania tarentolae homologue was also the aim of this work. With this APRT the experiments describes in this thesis aim at the structural resolution of the enzyme and its use in the screening for novel PRTases inhibitors. The results presented in this thesis show that the objectives of the work have been achieved. The structural elucidation of the human APRT allowed the discussion of the potential modifications caused bu the point mutations in the APRT gene. Novel inhibitors of PRTases of Leishmania major promastigote cultures and promoted the growth inhibition of this parasite.
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Biochemical Characterization Of An Acid-Adaptive Type III DNA Methyltransferase From Helicobacter Pylori 26695 And Its Biological SignificanceBanerjee, Arun 07 1900 (has links) (PDF)
Enzyme DNA methylation is an important biochemical process that imprints DNA with additional information. DNA methylation is catalyzed by S-adenosyl-L-methionine (AdoMet)-dependent methyltraferases (MTases). Prokaryotic DNA MTases are usually components of restriction-modification(R-M) systems that enable cells to resist propagation of foreign genomes that would otherwise kill them. Based on the position methyl group transfer on the bases in DNA, MTases are classified into two groups-exocyclic or amino MTases and endocyclic or ring MTases. The amino MTases methylate exocyclic amino nitrogen to form either N6-methyladenine or n4-methycytosine. N6-methyaladenine is mostly found in the genomes of bacteria, archaea protists and fungi.
Helicobacter pylori is a gram-negative, flagellated, fastidious bacterium that colonizes the highly acidic environment of the gastric mucosa. Frequently and persistence of H.paylori infection in humans make it attractive model for studying the host- pathogen interaction mechanisms.
Analysis of the genome sequence of H.pylori strains 26695, J99.HPAGI, and G27 revealed an abundance of restriction-modification (R-M) systems. Most of the R-M system genes are either conserved among the strains or specific to each strain. Strain specific genes are responsible for different phenotypes in several host adapted pathogens such as H.pylori. Many of the R-M gene homologues exhibit different usages of condon bias and lower G+C content from the average genes suggesting horizontal transfer of the R-M system genes in H. Pylori. Genome analysis of strain 26695 showed the presence of three putative type III R-M systems and hp0592-hp0593 constitutes one such type III R-M system. Based on the conserved motif arrangements, HP0593 MTases belongs to the subgroups of MTases. The amino acid sequence of HP0593 MTases has 38% sequence identity to Ecop11 MTases and EcoP151 MTase, both of which belongs to type IIIR-M systems therefore, it was important to study in detail previously unexplored role of this putative type III DNA MTase (HP0593) in H. Pylori.
Investigation of methyltransferease activity and sequence specifically of putative DNA adenine MTase (HP0593)
HP0593 (N6-adenine) - DNA MTase is a member of a type III R-M system in H. pylori strain 26695. HP0593 MTase has been cloned, over expressed and purified heterologously in Escherichia coli. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was carried out with purified HP0593 and profile showed a single peak with expected molecular mass of 70.6kDa. The protein was determined as-5.8. HP0593 MTase exits predominantly as monomer and a small fraction as dimer in solution as determined by size exclusion chromatography and glutaraldehyde cross-linking studies.
The recognition sequence of the purified MTase was determined as 5’GCAG-3’ and the target base of methylation is adenine. Dot-blot assay using antibodies that reacted specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine specific MTase. Exocyclic MTase have a conserved catalytic motif (D/N/S/SPPY/F/W). Most interestingly, the amino acid sequence analysis of HP0593 MTase revealed the presence of a PCQ-like motif, which is the catalytic motif for C5-cytosine MTase in addition to DPPY motif. In order to check the role of both these MTase by glycine. HP0593 –Y107G and C54G mutant proteins were purified to near homogeneity. It was found that the Y107G mutant protein was catalytically inactive as compared to wild-type HP0593 MTase. On the other hand the C54G mutant protein was found to be as active as the wild-type HP0593 MTase indicating that HP0593 MTase is an adenine MTase and not a C5- cytosine MTase.
Kinetic and catalytic properties of HP0593 DNA adenine methyltransferase
DNA binding studies were carried out by electrophoretic mobility shift assay using DNA having cognate site and either in absence or presence of AdoHcy or sinefungin. In all the three cases two different DNA-protein complexes were observed-a fast running complex I and a slow running complex 2. It can be surmised that the fast running complex could be HP0593 monomer-DNA and the slow running complex could be a HP0593 dimer-DNA complex. With non specific DNA (lacking 5’-GCAG-3’ sequences) no complexes were formed even in the presence of cofactors. Based on the above observations it is suggested that a specific interactions of HP0593 MTase with DNA occurs on cognate recognition site.
The activity of HP0593 MTase is optional at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. When initial velocities were plotted against varying concentrations of duplex DNA having a single 5’GCAG-3’ site a rectangular hyperbola was obtained confirming that HP0593 MTase obeys michaelis menten kinetics. From non-linear regression analysis of the plot of initial velocity versus DNA concentration Km (DNA) and kcat were calculated. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of methylation activity on enzyme concentration indicated that more than one molecule of enzyme is required for its activity. Metal ion cofactors such as CO 2, Mn2+ and Mg2+ stimulated the HP09593 MTase activity. As Mn2+ showed maximum stimulation of methyaltion activity compared to other metal ions, surface plasmon resonance spectroscopy was used to determine the kinetics of DNA binding by HP0593 MTase in the absence and presence of Mn2+. In the presence of Mn2+, HP0593 MTase showed~1000-fold increase in affinity to duplex DNA.
DNA MTase bind substrates in random or sequential order. Preincubation study demonstrated that the preformed enzyme-DNA complex is competent than the preformed enzyme-AdoMet complex. This suggests that MTase binds to DNA first followed by AdoMet. Isotope partitioning analysis indicated that HP0593 MTase shows a distributive mechanism of methylation DNA having more than one recognition site.
Effects of inactivation of HP0593 DNA MTase in Helicobacter pylori 26695 strain and its functional role.
DNA dot-blot assay using hp0593 gene specific primer showed that this gene is present in 25.15% of the clinical strains checked suggesting that hp0593 is strain-specific gene. Strain-specific genes in many host-adapted pathogene impart strain specific phenotype.
Wild-type 26695 strain grew slightly faster at the initial phase of growth in PH 4.5 compared to pH 7.4. A~5-fold enhanced level of hp0593 mRNA expression was growth under acidic condition HP0593 MTase could play an important role in H. pylori physiology through methylation.
To elucidate the possible role(s) played by the MTase in H.pylori physiology, an hp0593 knock-out in 26695 strain was generated by chloramphenecol cassette mediated insertional gene inactivation. Growth kinetic study was carried out with both wild-type and hp0593 knock-out strain at pH7.4, the growth of the hp0593 strain. At pH 4.5 no major differences were observed in the growth compared to the wild-type hp0593 knock-out strain. To further investigate the effect of the knock-out, cell-morphology study was carried out after growing the strains at pH 7.4 till mid-exponential phase. Transmission electron
microscopy studies reveled changes in cell shape, presence of sheathed structure and production of outer membrane vesicles (OMVs) in the hp0593 knock out strain. OMVs contain effectors molecules during infection helps in pathogenicity caused by H.pylori.This is the first report where inactivation of DNA MTase causes shedding of vesicles. OMVs are also known to modulate the production of IL-8 by gastic epitheial cells. To check weather H.pylori strains could produce IL-8, both wild-type and hp0593 knock-out strains were co-cultured with AGS cell infected with the hp0593 knock out strain. This was further confirmed by semi-quantitative RT-PCR analysis.
To analyze the different phenotypes observed in the hp0593 knock-out strain, transcriptome profile were compared by microarray and RT-PCR analysis. In thehp0593 knock-out strain peptidologlycan and murein synthesis genes like pbp2, murC and neu4 showed upregulation which could be responsible for the changes in cell shape presence of sheathed structure and OMVs production. The RT-PCR data showed ~9-fold down-regulation of dank chaperone which might play a key role in slow growth phenotype in the hp0593 knock-out strain.
Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes such as dank, neuA, murC, fliH, filP and cag5, the results presented in this study provide impetus for exploring the role of HP0593 DNA MTase in the cellular processes of H.pylori. However, R-M systems are not absolutely essential, but different methylation patterns may contribute to strain-specific epigenetic gene regulation and may contribute to variability among the strains.
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Application of magnetic resonance for non-invasive phenotyping of mice with altered metabolismFaller, Kiterie Maud Edwige January 2011 (has links)
Changes in myocardial energetics have been implicated in the pathophysiology of heart failure (HF). However, the precise contribution of creatine (Cr) / phosphocreatine (PCr) / creatine kinase (CK) energy buffer and transfer remains unclear. The aim of this thesis was to study the effects on murine cardiac function of both impairment and enhancement of creatine metabolism. In order to longitudinally follow the cause and effect relationship of myocardial creatine concentration, a non-invasive method of quantification was required. Cardiac Cr levels measured in vivo by 1H-MRS were therefore compared with gold-standard invasive HPLC and found to correlate over a wide-range (r2=0.91). 1H-MRS was reproducible for measuring Cr levels in the heart, brain, and skeletal muscle. The cardiac phenotype of a novel model of creatine depletion, the AGAT-/- mouse, was characterized using in vivo MRI, 1H-MRS and LV catheterisation, under conditions of gradually reducing Cr concentrations; zero Cr; and attempted phenotype rescue with dietary Cr. For the first time in the heart, the rate of Cr turnover was quantified (~3 % per day) and demonstrated that cardiac function was preserved even when creatine levels reduced by ~70-90%. Total absence of myocardial Cr induced impairment of inotropic and lusitropic cardiac function and reduced inotropic reserve. Cardiac dysfunction was only partially rescued by replenishment of the Cr pool, suggesting this to be a consequence of long-term adaptations to chronic low Cr. Finally, we tested the hypothesis that combined elevation of myocardial creatine and ribose would be beneficial in a mouse model of chronic HF by increasing cardiac energy availability. Despite an increase in myocardial ribose concentration, this did not prevent loss of total adenine nucleotides (TAN), and there was no improvement in post-infarct LV remodeling or function. Future studies are needed to explore alternative approaches for maintaining TAN in combination with total creatine.
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The Association of HLA Class II Genetic and Expression Level Variation with Response to the Hepatitis B Vaccine in South African Laboratory WorkersGoldfein, Hadassa 01 December 2017 (has links)
Master of Science / The hepatitis B virus (HBV) vaccine has contributed greatly to decreasing the HBV epidemic. However, it remains unclear why 5-10% of individuals do not mount an adequate antibody response. Previous studies have shown that genetic variation influences HBV vaccine response. Since such studies are lacking in South African individuals, we examined the associations between HBV vaccine response and genetic variation in HLA-DPB1, additional candidate genes and HLA-DPB1 expression levels in a South African cohort. HLA-DPA1 and -DPB1 allele typing was performed using Luminex technology, twenty-four candidate SNPs were typed by MassArray Analysis and HLA-DPB1 mRNA expression levels were measured by qPCR. HLA-DPB1*01:01, *04:01:01G and *09:01 and SNPs and haplotypes in IL1B, IL4, IL12B, IFNG and the HLA region were significantly associated with HBV vaccine response. A trend of lower HLA-DPB1 expression associating with better anti-HBs response was observed, although this was not significant. Response to the HBV vaccine is multi-genic but HLA-DP plays an important role. / CR2017
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Adenina fosforibosiltransferase de Schistosoma mansoni: proposta de detalhamento do mecanismo catalítico por dinâmica molecular / Adenine phosphoribosyltransferase from Schistosoma mansoni: insights into the catalytic mechanism via molecular dynamicsCaldas, Victor Emanoel Armini 12 August 2011 (has links)
A Adenina Fosforibosiltransferase (APRT E.C. 2.4.2.7) pertence à família de enzimas Fosforibosil Transferases (PRTase) do Tipo I , que catalisa a conversão reversível de Adenina e 5-fosfo-α-D-ribose-1-difosfato (PRPP) em difosfato e adenosina monofosfato, um importante precursor energético da célula. A APRT integra a via de salvação de purinas, única forma de suprir o balanço de purinas em Schistosoma mansoni. Este trabalho apresenta o isolamento, clonagem, expressão heteróloga e purificação da APRT de S. mansoni a fim de caracterizá-la quanto seus parâmetros físico-químicos. Não se obtendo cristais de proteína, foram elaborados modelos tridimensionais por homologia para estudos de dinâmica molecular e avaliação conformacional via tCONCOORD. A estrutura de APRT humana foi usada como controle nas simulações. Os dados computacionais e de biologia molecular foram comparados entre si para validação mútua e, verificou-se que a análise cuidadosa de dados computacionais é capaz de fornecer informações críticas sobre a APRT, auxiliando e guiando os estudos experimentais. Ainda, as simulações de dinâmica molecular foram capazes de evidenciar a abertura de loops do sítio ativo, explicitar a importância da análise de rotâmeros em modelos, permitindo, então, rearranjar da forma correta resíduos erroneamente modelados. Por fim, um estudo envolvendo mecanismo catalítico sugere a participação de uma molécula de água abstraindo o próton ligado ao N9 da adenina e, para efeito de comparação, um mecanismo alternativo independente desta participação também foi descrito. Ambas as observações expandem a corrente visão sobre o mecanismo catalítico de APRTs. / The Adenine Phoshoribosyltransferase (APRT E.C.2.4.2.7) belongs to the Phosphoribosyltransferase enzyme family (PRTase) of type I which catalyzes the reversible conversion of adenine and 5-phospho-α-D-ribose-1-diphosphate (PRPP) in diphosphate and adenosine monophosphate, an important source of this compound for the cell. APRT is involved in the purine salvage pathway, the only way that Schistosoma mansoni has to supply its purine demands. This work shows the isolation, cloning, heterologous expression and the purification of APRT from S. mansoni in order to establish its physical chemical and kinetics parameters. Since crystallographic structure was not obtained, we built homology models to perform molecular dynamics experiments and conformational evaluation via tCONCOORD. The human APRT structure was used as control in the simulation experiments. The computational and molecular biology data were compared for consistency and the detailed analysis of the former allowed us to improve experimental manipulation of APRT. The molecular dynamics experiments showed the opening of the loops of the catalytic binding site and the key function of rotamers. Finally, we were able to suggest that a water molecule may catch the proton of adenine N9. Both observations improve the current view of APRT catalytic mechanism.
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