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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
241

EPR, ENDOR and DFT Studies on X-Irradiated Single Crystals of L-Lysine Monohydrochloride Monohydrate and L-Arginine Monohydrocloride Monohydrate

Zhou, Yiying 16 July 2009 (has links)
When proteins and DNA interact, arginine and lysine are the two amino acids most often in close contact with the DNA. In order to understand the radiation damage to DNA in vivo, which is always associated with protein, it is important to learn the radiation chemistry of arginine and lysine independently, and when complexed to DNA. This work studied X-irradiated single crystals of L-lysine monohydrochloride dihydrate (L-lysine·HCl·2H2O) and L-arginine monohydrochloride monohydrate (L-arginine·HCl·H2O) with EPR, ENDOR, EIE techniques and DFT calculations. In both crystal types irradiated at 66K, the carboxyl anion radical and the decarboxylation radical were detected. DFT calculations supported these assignments. Specifically, the calculations performed on the cluster models for the carboxyl anion radicals reproduced the proton transfers to the carboxyl group from the neighboring molecules through the hydrogen bonds. Moreover, computations supported the identification of one radical type as the guanidyl radical anion with an electron trapped by the guanidyl group. In addition, the radical formed by dehydrogenation of C5 was identified in the L-arginine·HCl·H2O crystals irradiated at 66K. For both crystal types, the deamination radicals and the dehydrogenation radicals were identified following irradiation at 298K. Different conformations of main-chain deamination radicals were detected at 66K and at 298K. In L-lysine·HCl·2H2O, these conformations are the result of the different rotation angles of the side chain. In L-arginine·HCl·H2O, one conformation at 66K has no O-H dipolar protons while the others have two O-H dipolar protons. In L-lysine·HCl·2H2O, two radicals with very similar sets of hyperfine couplings were identified as the result of dehydrogenation from C3 and C5. Two other radicals in low concentration detected only at 66K, were tentatively assigned as the radical dehydrogenated from C3 and the side-chain deamination radical. In L-argnine·HCl·H2O, the radicals from dehydrogenation at C5 and C2 also were identified. DFT calculations supported these assignments and reproduced conformations of these radicals.Finally, based on the radicals detected in the crystal irradated at 66K and at 298K, the annealing experiments from the irradiation at 66K, and the previous studies on the irradiated amino acids, the mechanisms of the irradiation damage on lysinie and arginine were proposed.
242

Functional Characterization and Surface Mapping of Frataxin (FXN) Interactions with the Fe-S Cluster Assembly Complex

Thorstad, Melissa 16 December 2013 (has links)
In 1996, scientists discovered a connection between the gene for the human protein frataxin (FXN) and the neurodegenerative disease Friedreich’s ataxia (FRDA). Decreased FXN levels result in a variety of aberrant phenotypes including loss of activity for iron-sulfur containing enzymes, mitochondrial iron accumulation, and susceptibility to oxidative stress. These symptoms are the primary focus of current therapeutic efforts. In contrast our group is pursuing an alternate strategy of first defining FXN function at a molecular level then using this information to identify small molecule functional replacements. Recently, our group has discovered that FXN functions as an allosteric activator for the human Fe-S cluster assembly complex. The work presented here helps to further define molecular details of FXN activation and explain how FRDA missense mutants are functionally compromised. First, the FRDA missense mutants L182H and L182F were investigated. Unlike other characterized FRDA missense mutants, the L182F variant was not compromised in its ability to bind and activate the Fe-S assembly complex. The L182H variant exhibited an altered circular dichroism signature; suggesting a change in secondary structure relative to native FXN, and rapidly degraded. Together these studies suggest that L182 variants are less stable than native FXN and are likely prone to degradation in FRDA patients. Second, as a regulatory role of FXN suggests that its function is likely controlled by environmental stimuli, different maturation forms of FXN as well as post-translational modification mimics were tested as mechanisms to control FXN regulation. Here experiments were designed to test if a larger polypeptide form of FXN represents a functional form of the protein. Kinetic and analytical ultracentrifugation studies revealed a complex heterogeneous mixture of species some of which can activate the Fe-S assembly complex. A previously identified acetylation site was also tested using mutants that mimic acetylation. These mutants had little effect on the ability of FXN to bind and activate the assembly complex. Third, mutagenesis experiments were designed in which the FXN surface residues were replaced with alanine and the resulting variants were tested in binding and activity assays. These experiments revealed a localized “hot-spot” on the surface of FXN that suggests small cyclic peptide mimics might be able to replace FXN and function as FRDA therapeutics. Unexpectedly, one of the FXN variants exhibited significantly tighter binding and could have relevance for therapeutic development.
243

Disrupting the quaternary structure of DHDPS as a new approach to antibiotic design.

Evans, Genevieve Laura January 2010 (has links)
This thesis examined the enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from the pathogen Mycobacterium tuberculosis. DHDPS is a validated antibiotic target for which no potent inhibitor based on substrates, intermediates or product has been found. The importance of the homotetrameric quaternary structure in E. coli DHDPS has been demonstrated by the 100-fold decrease in activity observed in a dimeric variant, DHDPS-L197Y, created by site-directed mutagenesis. This suggested a new approach for inhibitor design: targeting the dimer-dimer interface and disrupting tetramer formation. DHDPS catalyzes the first committed step in the biosynthetic pathway of meso-diaminopimelic acid, a critical component of the mycobacterial cell wall. In this study, wild-type M. tuberculosis DHDPS was thoroughly characterized and compared with the E. coli enzyme. A coupled assay was used to obtain the kinetic parameters for M. tuberculosis DHDPS: KM(S) ASA = 0.43 (±0.02) mM, KMpyruvate = 0.17 (±0.01) mM, and kcat = 138 (±2) s 1. Biophysical techniques showed M. tuberculosis DHDPS to exist as a tetramer in solution. This is consistent with the crystal structure deposited as PDB entry 1XXX. The crystal structure of M. tuberculosis DHDPS showed active-site architecture analogous to E. coli DHDPS and a dimeric variant of M. tuberculosis DHDPS was predicted to have reduced enzyme activity. A dimeric variant of M. tuberculosis DHDPS was engineered through a rationally designed mutation to analyze the effect of disrupting quaternary structure on enzyme function. A single point mutation resulted in a variant, DHDPS-A204R, with disrupted quaternary structure, as determined by analytical ultracentrifugation and gel-filtration chromatography. DHDPS-A204R was found to exist in a concentration-dependent monomer-dimer equilibrium, shifted towards dimer by the presence of pyruvate, the first substrate that binds to the enzyme. The secondary and tertiary structure of DHDPS-A204R was analogous to wild-type M. tuberculosis DHDPS as judged by circular dichroism spectroscopy and X ray crystallography, respectively. Surprisingly, this disrupted interface mutant had similar activity to the wild type enzyme, with a kcat of 119 (±6) s-1; although, the affinity for its substrates were decreased: KM(S) ASA = 1.1 (±0.1) mM, KMpyruvate = 0.33 (±0.03) mM. These results indicated that disruption of tetramer formation does not provide an alternative direction for drug design for DHDPS from M. tuberculosis. Comparison with the recently discovered dimeric DHDPS from Staphylococcus aureus shed further light on the role of quaternary structure in DHDPS. In M. tuberculosis DHDPS-A204R and the naturally dimeric enzyme, the association of monomers into the dimer involves a greater buried surface area and number of residues than found in E. coli DHDPS-L197Y. This provides a framework to discriminate which DHDPS enzymes are likely to be inactive as dimers and will direct future work targeting the dimer-dimer interface of DHDPS as an approach for drug design.
244

Strukturelle und funktionale Analyse der acetylierten kleinen GTPase Ran / Structural and functional analysis of the acetylated small GTPase Ran

Gloth, Daniel 06 March 2015 (has links)
No description available.
245

Structural and Functional Studies on the Escherichia coli Inducible Lysine Decarboxylase: Linking the Acid Stress and Stringent Responses

Kanjee, Usheer 30 August 2012 (has links)
The Escherichia coli acid stress response allows the survival of cells over a wide range of pH challenges: down to pH 2.0 with the extreme acid stress response and down to pH 4.0 – 5.0 with the mild acid stress response. The cell employs a number of different acid stress response systems, including a number of structurally related, pyridoxal-5′-phosphate (PLP)-dependent amino acid decarboxylases, including the glutamic acid, arginine, lysine, and ornithine decarboxylases. The decarboxylases are large multi-domain enzymes that exist as homodimers or higher-order oligomers and have various activity optima at different pH values. By the proton-consuming decarboxylation of a target amino acid, these enzymes provide a response to a wide range of pH challenges. The primary focus of this work is the elucidation of the X-ray crystal structure of the inducible lysine decarboxylase LdcI, a homodecameric enzyme that has distinct 5-fold symmetry. A combination of heavy-atom derivatization, anomalous scattering and molecular replacement techniques were used to determine the X-ray structure and the model was refined to a resolution of 2.0 Å. The structure of LdcI revealed that the protein co-crystallized with the stringent response alarmone ppGpp. The stringent response is activated under nutritional and stress conditions and reorganizes cellular transcription and metabolism from exponential-phase growth into stationary phase growth. The primary target of ppGpp is the RNA polymerase, but other classes of enzymes are known to be affected. ppGpp was found to be a potent inhibitor of LdcI both in vitro and in vivo and this role provides the first evidence of a linkage between the stringent response and acid stress response. Among the decarboxylases related to LdcI (the constitutive lysine, the ornithine and arginine decarboxylases), a number of these enzymes were similarly regulated by ppGpp.
246

Nitric oxide, arginine and acute pancreatitis /

Sandström, Per A., January 2004 (has links) (PDF)
Diss. (sammanfattning) Linköping : Univ., 2004. / I publikationen felaktig serie: Linköping studies in health sciences. Härtill 4 uppsatser.
247

Investigating the inhibitor and substrate diversity of the JmjC histone demethylases

Schiller, Rachel Shamo January 2016 (has links)
Epigenetic control of gene expression by histone post-translational modifications (PTMs) is a complex process regulated by proteins that can 'read', 'write' or 'erase' these PTMs. The histone lysine demethylase (KDM) family of epigenetic enzymes remove methyl modifications from lysines on histone tails. The Jumonji C domain (JmjC) family is the largest family of KDMs. Investigating the scope and mechanisms of the JmjC KDMs is of interest for understanding the diverse functions of the JmjC KDMs in vivo, as well as for the application of the basic science to medicinal chemistry design. The work described in this thesis aimed to biochemically investigate the inhibitor and substrate diversity of the JmjC KDMs, it led to the identification of new inhibitors and substrates and revealed a potential combinatorial dependence between adjacent histone PTMs. Structure-activity relationship studies gave rise to an n-octyl ester form of IOX1 with improved cellular potency and selectivity towards the KDM4 subfamily. This compound should find utility as a basis for the development of JmjC inhibitors and as a tool compound for biological studies. The rest of this thesis focused on the biochemical investigations of potential substrates and inhibitors for KDM3A, a JmjC demethylase with varied physiological functions. Kinetic characterisation of reported KDM3A substrates was used as the basis for evaluations of novel substrates and inhibitors. Further studies found TCA cycle intermediates to be moderate co-substrate competitive inhibitors of KDM3A. Biochemical investigations were carried out to study potential protein-protein interactions of KDM3A with intraflagellar transport proteins (IFTs), non-histone proteins involved in the formation of sperm flagellum. Work then addressed the exploration of novel in vitro substrates for KDM3 (KDM3A and JMJD1C) mediated catalysis, including: methylated arginines, lysine analogues, acetylated and formylated lysines. KDM3A, and other JmjC KDMs, were found to catalyse novel arginine demethylation reaction in vitro. Knowledge gained from studies with unnatural lysine analogues was utilised to search for additional novel PTM substrates for KDM3A. These results constitute the first evidence of JmjC KDM catalysed hydroxylation of an Nε-acetyllysine residue. The H3 K4me3 position seems to be required for acetyllysine substrate recognition, implying a combinatorial effect between PTMs. Preliminary results provide evidence that JMJD1C, a KDM3 protein previously reported to be inactive, may catalyse deacetylation in vitro. An additional novel reaction, observed with both KDM3A and JMJD1C, is deformylation of N<sup>ε</sup>-formyllysine residues on histone H3 fragment peptides. Interestingly, H3 K4 methylation was also observed to enhance the apparent deformylation of both KDM3A and JMJD1C catalysed reactions. Overall, findings in this thesis suggest that the catalytic activity of JmjC KDMs extends beyond lysine demethylation. In a cellular context, members of the KDM3 subfamily might provide a regulatory link between methylation and acylation marks. Such a link will further highlight the complex relationships between histone PTMs and the epigenetic enzymes that regulate them. The observed dependency of H3 K9 catalysis on H3 K4 methylation adds another layer of complexity to the epigenetic regulation by histone PTMs.
248

Decoding lysine-11 signals in ubiquitination

Grice, Guinevere January 2018 (has links)
The diverse outcomes of ubiquitination primarily relate to the flexibility of ubiquitin in forming homo- or heterotypic chains on each of its seven lysine residues which in turn stimulate distinct downstream signaling pathways. These ubiquitin signals must be selectively initiated on the substrate protein and subsequently decoded to facilitate the desired cellular function. These initiation and decoding steps often involve additional post-translational modifications and ubiquitin receptor proteins, but the enzymes and ubiquitin chains involved for many ubiquitinated substrates are not clear. Here, I have explored the initiation and decoding of ubiquitin signals, focusing on lysine-11 (K11) linked polyubiquitin chains and their role in protein degradation. I established in vitro assays to understand how K11-chains are decoded and whether these chains act as a signal for proteasome-mediated degradation. Pure homotypic K11-chains did not bind the proteasome or its associated ubiquitin binding proteins, but did bind to the mitophagy ubiquitin receptors, MyosinVI and TAX1BP1. Heterotypic K11/K48 linkages not only bound the proteasome but also stimulated degradation of the cell cycle substrate, cyclin B1. To further explore the functions of K11-chains I focused on the hypoxia inducible transcription factor (HIF) pathway, as K11-ubiquitination had been implicated in proteasome-independent degradation of the transcription factor. I established an in vitro assay to initiate HIF ubiquitination, via prolyl hydroxylation, and determine the type of ubiquitin chains involved. Recombinant HIF isoforms were rapidly hydroxylated when incubated with cell extracts. Moreover, the levels of iron and small molecule metabolites within the lysates regulated HIF hydroxylation. However, this hydroxylation was insufficient to reproducibly promote HIF ubiquitination or determine the ubiquitin chains involved. While the nature of the polyubiquitin chains formed in the HIF pathway remain elusive, my studies identify distinct roles for homotypic and heterotypic K11-polyubiquitination in proteasome-mediated degradation.
249

Influência da suplementação de lisina no terço final da gestação sobre o desempenho de primíparas suínas e sua leitegada / Influence of lysine suplementation in late gestation on sow and piglet performance

Magnabosco, Diogo January 2011 (has links)
O estudo avaliou a inclusão de Lisina, o primeiro aminoácido limitante na dieta dos suínos, no terço final da gestação de primíparas suínas, e os efeitos em relação ao desempenho das fêmeas e de suas leitegadas ao nascer e o impacto no período lactacional subsequente. Foram selecionadas 298 leitoas, aos 85 dias de gestação, distribuídas uniformemente em dois grupos de acordo com idade de cobertura, peso, escore corporal visual (ECV) e espessura de toucinho (ET), sendo os grupos: Controle - 3,3 kg/ração/fêmea/dia, com 0,84% de lisina, correspondendo a um consumo diário de 28 g/lisina/fêmea e Lisina – 3,3 kg/ração/fêmea/dia, com 0,84% de lisina, mais o acréscimo de 7 g de lisina (correspondendo a um consumo diário de 35 g/lisina/fêmea). O fornecimento de lisina foi feito de forma “on top” dos 85 dias de gestação até o momento de transferência para a maternidade (110 dias de gestação). Foram efetuadas as medidas de peso e ET no momento de transferência para a maternidade. No parto, os leitões nascidos vivos (NV) e natimortos (NAT) foram quantificados e pesados individualmente em até 12 horas após o nascimento, e os fetos mumificados (MUM) foram contabilizados e incluídos no número de leitões nascidos totais (NT). Na lactação foi avaliado o desempenho de 60 fêmeas e seus lactentes. Para isto, logo após o parto foram selecionadas matrizes com base no peso aos 85 d de gestação e tamanho de leitegada ao nascer (NV + NAT). O consumo voluntário de ração do período lactacional foi mensurado por acompanhamento diário da quantidade individual ingerida. Não houve efeito (p>0,10) da suplementação com lisina durante a gestação no ganho de peso e de ET das fêmeas, no tamanho da leitegada, no percentual de mumificados e no peso ao nascimento. Fêmeas do grupo Lisina apresentaram menor percentual de natimortos (p=0,077). O coeficiente de variação do peso ao nascimento dentro da leitegada (p=0,094) e o percentual de leitões com peso inferior a 1100 gramas (p=0,082) foi menor nas fêmeas do grupo Lisina do que naquelas do grupo Controle. Do subgrupo de 60 fêmeas selecionadas para serem acompanhadas durante o período de lactação, não foram observadas diferenças entre os grupos (p>0,10) na perda de peso e perda de ET durante a lactação, consumo voluntário de ração, intervalo desmame-estro e número de leitões desmamados. O aumento de 28 g/dia para 35 g/dia de lisina total consumidas no terço final da gestação de leitoas não exerceu influência sobre o desempenho das fêmeas ao parto e na lactação subsequente. Entretanto, foram encontradas tendências de diminuição no percentual de leitões natimortos, leitões com peso inferior a 1100 g e menor coeficiente de variação no peso das leitegadas de fêmeas que receberam maior quantidade de lisina. / In the present study we evaluated the addition of lysine, the first limiting amino acid in the swine diet, in late gestation of pregnant gilts, and the effects on the performance of the sows and their piglets at birth and subsequent impact on the lactational period. We selected 298 nulliparous pregnant sows on the 85th day of gestation, that were distributed into two groups according to breeding age, weight, body condition score and backfat thickness (BT): Control - 3.3 kg / feed / sow / day, with 0.84% lysine, corresponding to a daily consumption of 28 g / lysine / sow and Lysine - an increase of 7 g lysine (corresponding to a daily consumption of 35 g / lysine / sow). The lysine was supplied from the 85th day of gestation until the transfer to maternity (110 days of gestation). Weight and BT was measured at the time of transfer and farrowing, the piglets born alive (BA) and stillbirths (SB) were counted and weighed individually within 12 hours after birth, and mummified fetuses (MUM) were counted and included in the total number of piglets born (PB). Lactation performance was evaluated in 60 females and their piglets. For this, sows were selected on the basis of weight at 85 d of gestation with litter size (BA + SB) similar for both groups immediately after farrowing. The voluntary food intake of the lactational period was measured by monitoring of the individual amount ingested on a daily basis. We found no effects (p> 0.10) on weight gain and backfat of the sows, litter size, percentage of mummified and piglet's birth weight. The lysine female group had lower percentage of stillbirths (p = 0.077). The coefficient of variation in within litter birth weight (p = 0.094) and percentage of piglets weighing less than 1100 grams (p = 0.082) had lower in the Lysine group than the Control group. The analysis performed in the subgroup of 60 sows observed during the lactation period, indicated that there is no difference between the groups (p> 0.10) in weight and backfat loss during lactation, voluntary food intake, weaning-oestrus and number of piglets weaned. The increase of 28 g / day to 35 g / day of total lysine consumed in the late gestation of gilts had no influence on the performance of sows at birth and on subsequent lactation. However, there was a trend of decrease in the percentage of stillborn piglets, piglets weighing less than 1100 g and lowest coefficient of variation in weight of the litters of sows that received a greater amount of lysine.
250

Relação lisina digestível: energia metabolizável em dietas para suínos dos 50 aos 100 kg de peso corporal

Gandra, Érika Rosendo de Sena [UNESP] 01 June 2012 (has links) (PDF)
Made available in DSpace on 2014-06-11T19:33:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-06-01Bitstream added on 2014-06-13T20:27:49Z : No. of bitstreams: 1 gandra_ers_dr_botfmvz.pdf: 231357 bytes, checksum: 92d3c168799da775daa0fbd8f6879a6d (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Objetivou-se estimar o melhor nível de lisina digestível e sua relação com a energia metabolizável em dietas de suínos dos 50 aos 70 kg de peso vivo. Para tanto, foram realizados dois ensaios de desempenho, um com 72 machos castrados e outro com 72 fêmeas, com pesos médios iniciais de 49,75±0,41 kg e de 46,05±0,38 kg, respectivamente. O delineamento experimental usado nestes experimentos foi em blocos ao acaso com seis tratamentos, seis repetições e dois animais por unidade experimental (baia). No final dos experimentos mediu-se área de olho de lombo e espessura de toucinho na 10ª costela, por meio de ultrassonografia, em todos os animais. Também colheu-se sangue para leucograma de quatro animais dentro de cada tratamento, de forma aleatória, totalizando 24 machos castrados e 24 fêmeas, considerando delineamento inteiramente ao acaso, com quatro repetições por sexo e o animal era a unidade experimental. Os tratamentos foram 7,00; 8,00; 9,00; 10,00; 11,00 e 12,00 g de lisina digestível/kg de ração que continham 14,25 MJ de energia metabolizável/kg de ração e, em média, 160,92 g de proteína bruta/kg de ração. Realizou-se também um ensaio de digestibilidade e metabolismo, com 12 machos castrados e 12 fêmeas, em que foram avaliadas quatro dietas (7,00; 9,00; 10,00 e 12,00 g de lisina digestível/kg de ração), com três repetições e um animal por unidade experimental (gaiola). No ensaio de desempenho das fêmeas observou-se aumento linear na conversão alimentar, no consumo de proteína bruta (g/dia) e na eficiência de proteína bruta no período experimental de 0 a 16 dias. No período experimental de 0 a 32 dias observou-se aumento linear no consumo de proteína bruta (g/dia) e na eficiência de proteína bruta, bem como efeito quadrático no ganho de peso relativo (%) e na... / This study aimed to estimate the optimum level of lysine and its relation to metabolizable energy in diets for pigs from 70 to 100 kg live weight. For this, were two performance tests, one with 72 barrows and other with 72 females, with average initial weight 76.86 ± 0.90 kg and 76.92 ± 0.96 kg, respectively, the experimental design was a randomized block with six treatments and six replicates of two animals per experimental unit (pen). At the end the experiments was measured loin eye area and backfat thickness at the 10th rib by means of ultrasound in all animals. Also picked up blood WBC to four animals in each treatment, totaling 24 males castrated and 24 female maturation, being considered the completely randomized design, with four replications for sex and the animal was the experimental unit. After fasting for 12 hours, were chosen at random, 24 castrated males and 24 females (four per treatment), which were sent to slaughter in the abattoir for commercial Ratings and carcass boning. The treatments were 7.00, 8.00, 9.00, 10.00, 11.00 and 12.00 g of lysine per kg diet containing 14.25 MJ of ME per kg feed and on average 155.20 g crude protein per kg feed. In assay digestibility and metabolism were used 12 castrated males and 12 females, with evaluation of four diets (7.00, 8.00, 11.00 and 12.00 g of lysine / kg) with three replicates and one animal per experimental unit (cage). In the performance test females observed linear... (Complete abstract click electronic access below)

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