This thesis describes how the human Alpha class glutathione transferase (GST) A1-1 can be reprogrammed either to function as a multipurpose biosensor for detection of small molecule analytes, or as a handle providing for more efficient protein purification. A novel, user-friendly, and efficient method for site-specific introduction of functional groups into the active site of hGST A1-1 is the platform for these achievements. The designed thioester reagents are glutathione-based and they are able to label one single nucleophile (Y9) and leave the other 50 nucleophiles (in hGST A1-1) intact. The modification reaction was tested with five classes of GSTs (Alpha, Mu, Pi, Theta and Omega) and was found to be specific for the Alpha class isoenzymes. The reaction was further refined to target a single lysine residue, K216 in the hGST A1-1 mutant A216K, providing a stable amide bond between the protein and the labeling group. To further improve the labeling process, biotinylated reagents that could deliver the acyl group to Y9 (wt hGST A1-1) or K216 in the lysine mutant, while attached to streptavidin-coated agarose beads, were designed and synthesized. A focused library of eleven A216K/M208X mutants was made via random mutagenesis to provide an array of proteins with altered micro-environments in the hydrophobic binding site, where M208 is situated. Through the invented route for site-specific labeling, a fluorescent probe (coumarin) was introduced on K216 in all double mutants, with the purpose of developing a protein-based biosensor, akin to the olfactory system. The array of coumarin-labeled proteins responded differently to the addition of different analytes, and the responses were analyzed through pattern recognition of the fluorescence signals. The labeled proteins could also be site-specifically immobilized on a PEG-based biosensor chip via the single C112 on the surface of the protein, enabling development of surface-based biosensing systems. Also, a refined system for efficient detection and purification of GST-fusion proteins is presented. Through a screening process involving A216K and all produced A216K/M208X mutants, two candidates (A216K and A216K/M208F) were singled out as scaffolds for the next generation of fusion proteins. In addition to the features present in commercially available GST fusion constructs, the new mutants can be site-specifically labeled with a fluorophore in bacterial lysates providing quick and sensitive monitoring of expression and purification. Furthermore, the proteins could be labeled with a unique aldehyde moiety providing for a novel protein purification scheme.

The persistence and propagation of bacterial antibiotic resistance presents significant challenges to the treatment of drug resistant bacteria with current antimicrobial chemotherapies, while a dearth in replacements for these drugs persists. The thiopeptide family of antibiotics may represent a potential source for new drugs and thiostrepton, the prototypical member of this antibiotic class, is the primary subject under study in this thesis. Using a facile semi-synthetic approach novel, regioselectively-modified thiostrepton derivatives with improved aqueous solubility were prepared. In vivo assessments found these derivatives to retain significant antibacterial ability which was determined by cell free assays to be due to the inhibition of protein synthesis. Moreover, structure-function studies for these derivatives highlighted structural elements of the thiostrepton molecule that are important for antibacterial activity. Organisms that produce thiostrepton become insensitive to the antibiotic by producing a resistance enzyme that transfers a methyl group from the co-factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the thiostrepton binding site on 23S rRNA, thus preventing binding of the antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to generate point mutations at key active site residues. Ensuing biochemical assays and co-factor binding studies on these variants identified amino acid residues in the active site that are essential to the formation of the AdoMet binding pocket and provided direct evidence for the involvement of an active site arginine in the catalytic mechanism of the enzyme. Certain bacteria that produce neither thiostrepton nor the resistance methyltransferase express the thiostrepton binding proteins TIP-AL and TIP-AS, that irreversibly bind to the antibiotic, thereby conferring resistance by sequestration. Here, it was found that the point mutation of the previously identified reactive amino acid in TIP-AS did not affect covalent binding to the antibiotic, which was immediately suggestive of a specific, high affinity non-covalent interaction. This was confirmed in binding studies using chemically synthesized thiostrepton derivatives. These studies further revealed structural features from thiostrepton important in this non-covalent interaction. Together, these results indicate that thiostrepton binding by TIP-AS begins with a specific non-covalent interaction, which is necessary to properly orient the thiostrepton molecule for covalent binding to the protein. Finally, the synthesis of a novel AdoMet analogue is reported. The methyl group of AdoMet was successfully replaced with a trifluoromethyl ketone moiety, however, the hydrated form (germinal diol) of this compound was found to predominate in solution. Nevertheless, the transfer of this trifluoroketone/ trifluoropropane diol group was demonstrated with the thiopurine methyltransferase.

Thiostrepton (TS: TS; C72H85N19O18S5) is a thiazoline antibiotic that is effective against Gram-positive bacteria and the malarial parasite, Plasmodium falciparum. Tight binding of TS to the bacterial L11-23S ribosomal RNA (rRNA) complex of the large 50S ribosomal unit inhibits protein biosynthesis. The TS producing organism, Streptomyces azureus, biosynthesizes thiostrepton-resistance methyltransferase (TSR), an enzyme that uses S-adenosyl-L-methionine (AdoMet) as a methyl donor, to modify the TS target site. Methylation of A1067 (Escherichia coli ribosome numbering) by TSR circumvents TS binding. The S. azureus tsr gene was overexpressed in E. coli and the protein purified for biochemical characterization. Although the recombinant protein was produced in a soluble form, its tendency to aggregate made handling a challenge during the initial stages of establishing a purification protocol. Different purification conditions were screened to generate an isolation protocol that yields milligram quantities of protein with little aggregation and sufficient purity for crystallographic studies. Enzymological characterization of TSR was carried out using an assay to monitor AdoMet-dependent ([methyl-3H]-AdoMet) methylation of the rRNA substrate by liquid scintillation counting. During the optimization of assay, it was found that, although this method is frequently employed, it is very time and labour intensive. A scintillation proximity assay was investigated to evaluate whether it could be a method for collecting kinetic data, and was found that further optimization is required. Comparative sequence analysis of TSR has shown it to be a member of the novel Class IV SpoUT family of AdoMet-dependent MTases. Members of this class possess a non-canonical AdoMet binding site containing a deep trefoil knot. Selected SpoUT family proteins were used as templates to develop a TSR homology model for monomeric and dimeric forms. Validation of the homology models was performed with structural validation servers and the model was then used as the basis of ongoing mutagenesis experiments. The X-ray crystal structure of TSR bound with AdoMet (2.45 Å) was elucidated by our collaborators, Drs. Mark Dunstan and Graeme Conn (University of Manchester). This structure confirms TSR MTase’s membership in the SpoUT MTase family with a deep trefoil knot in the catalytic domain. The AdoMet bound in the crystal structure is in an extended conformation not previously observed in SpoUT MTases. RNA docking simulations revealed some features that may be relevant to binding and recognition of TSR to the L11 binding domain of the RNA substrate. Two structure-activity studies were conducted to investigate the TS-rRNA interaction and TS solubility. Computational analyses of TS conformations, molecular orbitals and dynamics provided insight into the possible modes of TS binding to rRNA. Single-site modification of TS was attempted, targeting the dehydroalanine and dehydrobutyrine residues of the antibiotic. These moieties were modified using the polar thiol, 2-mercaptoethanesulfonic acid (2-MESNA). Similar modifications had been previously used to improve solubility and bioavailability of antibiotics. The resulting analogue was structurally characterized (NMR and mass spectrometry) and showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus.

Thiostrepton (TS: TS; C72H85N19O18S5) is a thiazoline antibiotic that is effective against Gram-positive bacteria and the malarial parasite, Plasmodium falciparum. Tight binding of TS to the bacterial L11-23S ribosomal RNA (rRNA) complex of the large 50S ribosomal unit inhibits protein biosynthesis. The TS producing organism, Streptomyces azureus, biosynthesizes thiostrepton-resistance methyltransferase (TSR), an enzyme that uses S-adenosyl-L-methionine (AdoMet) as a methyl donor, to modify the TS target site. Methylation of A1067 (Escherichia coli ribosome numbering) by TSR circumvents TS binding. The S. azureus tsr gene was overexpressed in E. coli and the protein purified for biochemical characterization. Although the recombinant protein was produced in a soluble form, its tendency to aggregate made handling a challenge during the initial stages of establishing a purification protocol. Different purification conditions were screened to generate an isolation protocol that yields milligram quantities of protein with little aggregation and sufficient purity for crystallographic studies. Enzymological characterization of TSR was carried out using an assay to monitor AdoMet-dependent ([methyl-3H]-AdoMet) methylation of the rRNA substrate by liquid scintillation counting. During the optimization of assay, it was found that, although this method is frequently employed, it is very time and labour intensive. A scintillation proximity assay was investigated to evaluate whether it could be a method for collecting kinetic data, and was found that further optimization is required. Comparative sequence analysis of TSR has shown it to be a member of the novel Class IV SpoUT family of AdoMet-dependent MTases. Members of this class possess a non-canonical AdoMet binding site containing a deep trefoil knot. Selected SpoUT family proteins were used as templates to develop a TSR homology model for monomeric and dimeric forms. Validation of the homology models was performed with structural validation servers and the model was then used as the basis of ongoing mutagenesis experiments. The X-ray crystal structure of TSR bound with AdoMet (2.45 Å) was elucidated by our collaborators, Drs. Mark Dunstan and Graeme Conn (University of Manchester). This structure confirms TSR MTase’s membership in the SpoUT MTase family with a deep trefoil knot in the catalytic domain. The AdoMet bound in the crystal structure is in an extended conformation not previously observed in SpoUT MTases. RNA docking simulations revealed some features that may be relevant to binding and recognition of TSR to the L11 binding domain of the RNA substrate. Two structure-activity studies were conducted to investigate the TS-rRNA interaction and TS solubility. Computational analyses of TS conformations, molecular orbitals and dynamics provided insight into the possible modes of TS binding to rRNA. Single-site modification of TS was attempted, targeting the dehydroalanine and dehydrobutyrine residues of the antibiotic. These moieties were modified using the polar thiol, 2-mercaptoethanesulfonic acid (2-MESNA). Similar modifications had been previously used to improve solubility and bioavailability of antibiotics. The resulting analogue was structurally characterized (NMR and mass spectrometry) and showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus.

Studies presented in this dissertation were conducted to gain more insight into the role of phospholipid asymmetry and amyloid-β (Aβ)-induced oxidative stress in brain of subjects with amnestic mild cognitive impairment (aMCI) and Alzheimer disease (AD). AD is a largely sporadic, age-associated neurodegenerative disorder clinically characterized by the vast, progressive loss of memory and cognition commonly in populations over the age of ~65 years, with the exception of those with familial AD, which develop AD symptoms as early as ~30 years-old. Neuropathologically, both AD and FAD can be characterized by synapse and neuronal cell loss in conjunction with accumulation of neurofibrillary tangles and senile plaques. Elevated levels of oxidative stress and damage to brain proteins, lipids, and nucleic acids are observed, as well. Likewise, aMCI, arguably the earliest form of AD, displays many of these same clinical and pathological characteristics, with a few exceptions (e.g., no dementia) and to a lesser extent. Studies in this dissertation focused on the contributions of oxidative stress to the exposure of phosphatidylserine (PtdSer) to the outer-leaflet of the lipid membrane, how and when PtdSer asymmetric collapse contributes to the progression of aMCI, AD, and FAD, and the role played by methionine-35 (Met-35) of Aβ in oxidative stress and damage, as measured in a transgenic mouse model of Aβ pathology. Normally, the PtdSer is sequestered to the cytosolic, inner-leaflet of the bilayer by the adenosine triphosphate (ATP)-dependent, membrane-bound translocase, flippase, which unidirectionally transports PtdSer inward against its concentration gradient. Oxidative stress-induced modification of flippase and/or PtdSer, however, leads to prolonged extracellular exposure of PtdSer on the outer membrane leaflet, a known signal for both early apoptosis and selective recognition and mononuclear phagocytosis of dying cells. Within the inferior parietal lobule (IPL) of subjects with aMCI and AD, a significant collapse in PtdSer asymmetry was found in association with increased levels of both pro- and anti-apoptotic proteins, Bax, caspase-3, and Bcl-2. Moreover, a significant collapse in PtdSer asymmetry was also found in whole brain of human double-mutant knock-in mouse models of Aβ pathology, together with significantly reduced Mg2+ATPase activity, representing flippase activity, and increased levels of pro-apoptotic caspase-3. Significant PtdSer externalization corresponded to the age at which significant soluble Aβ(1-42) deposition occurs in this particular mouse model (9 months), and not of plaque deposition (12 months), suggesting that elevated levels of Aβ(1-42), together with increasing oxidative stress and apoptosis, may contribute to altered PtdSer membrane localization. Also in this dissertation, transgenic mice carrying Swedish and Indiana mutations on the human amyloid precursor protein (APPSw,In) and APPSw,In mice carrying a Met35Leu mutation on Aβ were derived to investigate the role of Met-35 in Aβ(1-42)-induced oxidative stress in vivo. Oxidative stress analyses revealed that Aβ-induced oxidative stress requires the presence of Met-35, as all indices of oxidative damage (i.e., protein carbonylation, nitration, and protein-bound 4-hydroxy-2-trans-nonenal [HNE]) in brain of Met35Leu mice were completely prevented. Moreover, immunohistochemical analyses indicated that the Met35Leu mutation influences plaque formation, as a clear reduction in Aβ-immunoreactive plaques in Met35Leu mice was found in conjunction with a significant increase in microglial activation. In contrast, behavioral analyses suggested that spatial learning and memory was independent of Met-35 of Aβ, as Met35Leu mice demonstrated inferior water-maze performance compared to non-transgenic mice. Differential expression and redox proteomic analyses to pinpoint proteins significantly altered by the APPSw,In and Met35Leu mutations was performed, as well. Expression proteomics showed significant increases and decreases in APPSw,In and Met35Leu mouse brain, respectively, in proteins involved in cell signaling, detoxification, structure, metabolism, molecular chaperoning, protein degradation, mitochondrial function, etc. Redox proteomics found many of these same proteins to be oxidatively modified (i.e., protein carbonylation and nitration) in both APPSw,In and Met35Leu mouse brain, providing additional insights into the critical nature of Met-35 of Aβ for in vivo oxidative stress in a mammalian species brain, and strongly suggesting similar importance of Met-35 of Aβ(1-42) in brain of subjects with aMCI and AD. Taken together, studies presented in this dissertation demonstrate the role of oxidative stress-induced alteration of PtdSer asymmetry and Met-35 in Aβ-induced oxidative stress in aMCI, AD, and FAD brain.

The persistence and propagation of bacterial antibiotic resistance presents significant challenges to the treatment of drug resistant bacteria with current antimicrobial chemotherapies, while a dearth in replacements for these drugs persists. The thiopeptide family of antibiotics may represent a potential source for new drugs and thiostrepton, the prototypical member of this antibiotic class, is the primary subject under study in this thesis. Using a facile semi-synthetic approach novel, regioselectively-modified thiostrepton derivatives with improved aqueous solubility were prepared. In vivo assessments found these derivatives to retain significant antibacterial ability which was determined by cell free assays to be due to the inhibition of protein synthesis. Moreover, structure-function studies for these derivatives highlighted structural elements of the thiostrepton molecule that are important for antibacterial activity. Organisms that produce thiostrepton become insensitive to the antibiotic by producing a resistance enzyme that transfers a methyl group from the co-factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the thiostrepton binding site on 23S rRNA, thus preventing binding of the antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to generate point mutations at key active site residues. Ensuing biochemical assays and co-factor binding studies on these variants identified amino acid residues in the active site that are essential to the formation of the AdoMet binding pocket and provided direct evidence for the involvement of an active site arginine in the catalytic mechanism of the enzyme. Certain bacteria that produce neither thiostrepton nor the resistance methyltransferase express the thiostrepton binding proteins TIP-AL and TIP-AS, that irreversibly bind to the antibiotic, thereby conferring resistance by sequestration. Here, it was found that the point mutation of the previously identified reactive amino acid in TIP-AS did not affect covalent binding to the antibiotic, which was immediately suggestive of a specific, high affinity non-covalent interaction. This was confirmed in binding studies using chemically synthesized thiostrepton derivatives. These studies further revealed structural features from thiostrepton important in this non-covalent interaction. Together, these results indicate that thiostrepton binding by TIP-AS begins with a specific non-covalent interaction, which is necessary to properly orient the thiostrepton molecule for covalent binding to the protein. Finally, the synthesis of a novel AdoMet analogue is reported. The methyl group of AdoMet was successfully replaced with a trifluoromethyl ketone moiety, however, the hydrated form (germinal diol) of this compound was found to predominate in solution. Nevertheless, the transfer of this trifluoroketone/ trifluoropropane diol group was demonstrated with the thiopurine methyltransferase.

The androgen receptor (AR) fulfils important roles for both sexes. By mediating the biological function of androgens, the AR has remained the target for endocrine therapies treating prostate cancer. The AR also determines the effectiveness of medroxyprogesterone acetate (MPA) in treating AR positive breast cancer. Every man will be affected by prostate cancer if he lives long enough. Prostate cancer continues to be a leading cause of death for males despite research into this cancer covering more than 60 years since Huggins' seminal 1941 study showing that androgens play a key role in this cancer. Unfortunately, significant advances have not been forthcoming and the effect of treatment has remained largely the same over past decades, whereby initial treatment provides temporary remission but eventually advanced cases become refractory to further intervention and the disease recurs in a more aggressive form. A plethora of factors are exquisitely sensitive to minute changes in the AR's structural profile, which can be altered by a single mutation, resulting in aberrant activity. A principal feature of these variant ARs associated with prostate cancer, is enhanced capacity to bind a number of molecules other than its cognate ligand, dihydrotestosterone (DHT). The promiscuous activity of this receptor leads to continued AR signalling and stimulus for the cancer despite low androgen levels induced by treatment regimes. A key question is whether mutations occurring within the AR occur as a result of cancer or contribute to the propagation of the cancer. Recent research has demonstrated that treatments incorporating anti-androgens such as flutamide, which are designed to impede prostate cancer progression by inhibiting AR activity, may actually provide selective pressure favouring somatic mutation of the receptor to take place. The specific changes to the AR which are responsible for gains of function have not been resolved as their crystal structures, which are used to provide conformational analysis of proteins, are tremendously problematic to produce with little success found in literature. Generating representative crystals of the AR protein involves producing soluble recombinant protein. Unfortunately the AR is prone to aggregation and is highly unstable, especially in the presence of antagonistic molecules or absence of a stabilising ligand, preventing the protein from being maintained in the soluble state required for crystallization. In order to produce sufficient quantities of soluble material for crystallization, the androgen receptor's ligand binding domain (LBD) was produced as a recombinant protein in Escherichia coli bacteria strain BL21 (DE3) in the presence of DHT, flutamide, as well as in the absence of ligand. Since soluble unbound AR-LBD has not been produced until now, the bacterial culture containing no ligand was further processed to the stage of cleaving the purification tag from the recombinant protein and represents considerable progress into producing soluble material for crystallizing the troublesome yet considerably important AR in the absence of ligand. Although distinct from prostate cancer in males, AR activity in breast tissue is also a factor determining the action of drugs, such as MPA, included in therapies aimed at breast cancer. The use of MPA has declined primarily due to its adverse effects including unsuccessful generation of a biological response, as well as the advent of other drugs administered for hormonal therapies treating breast cancer. Alternative drugs are needed when breast cancer therapies fail as tumours develop resistance to primary drugs. Although there are a number of drugs on the market, success would be maximised if the determined therapy is matched with the patient, based for example, on their genetic makeup. There is a conundrum whereby some patients with an AR do not respond to MPA, a drug normally recognised by the receptor. In clinical trials it was discovered that an AR with threonine instead of methionine at residue 780 (M780T) fails to activate in response to MPA, but the exact mechanism has remained elusive and needs to be answered at the molecular level. The X-ray crystallographic studies that generate 3D images of macromolecules and wet chemistry, which have traditionally been used to provide insight into science in these dimensions, are incorporated with computer based molecular simulation. This is both complementary and distinct to traditional scientific methodologies, enabling further elucidation of protein-protein interactions, and the influence applied to such inter-relations by natural and drug ligands. This approach has been used, and is continually developed, to understand the binding mechanisms of current drugs as well as designing new drugs. In order to produce a receptor representing the M780T variant, the crystal structure representing the AR-LBD was mutated in silico, into which MPA was then docked. It was found that MPA binds into the M780T AR-LBD with considerably more spatial displacement compared to the position of DHT in the crystal structure, and is predicted to be the primary reason for the inability of MPA to activate this variant AR. The clarification of MPA binding and failure to elicit a response from the variant AR is significant for a cohort of breast cancer patients, as not only does the presence of an AR in the tumour determine the effectiveness of MPA, but correct composition of the AR, specifically, the absence of a M780T mutation. In the absence of this AR mutation, MPA could effectively be used either as an alternative to primary drugs, or in secondary therapies when primary therapies fail. Aberrant activity of variant ARs in response to MPA should also be taken into consideration when analysing drug studies about the effectiveness of MPA. The findings on the loss of response to MPA by the M780T variant AR have been included in the journal article &quotDecreased Androgen Receptor Levels and Receptor Function in Breast Cancer Contribute to the Failure of Response to Medroxyprogesterone Acetate" appearing in the September 2005 issue of Cancer Research journal.

MELO, Wanessa David Canedo. Transições de fase em monocristais de D-Metionina. 2012. 86 f. Tese (Doutorado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2012. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2014-11-13T20:33:27Z No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2014-11-13T20:38:19Z (GMT) No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) / Made available in DSpace on 2014-11-13T20:38:19Z (GMT). No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) Previous issue date: 2012 / In this work we have been investigated the vibrational properties of the D-methionine single crystals using the Raman and Infrared spectroscopies. The Raman spectroscopy measurements were performed under varying hydrostatic pressure from 0 GPa up to 7.0 GPa, indicated two transitions, one around 2.0 GPa and another in 2.5GPa, which were detected by changes of the vibrationals modes of the carboxilic (CO2-), amino (NH3+), CS, CSC, CH2 and CH3, respectively. These vibrationals modes also have been sensitized under low temperatures, whose changes have been helpful to identify the phase transition with the measurements of the infrared scattering from 298K up to 84K. In the measurements of the Raman scattering realized between 298K and 84K, the transition was identified by emerging of the translational mode in 91 cm-1 around 84K. Further, in this work we have been compared changes presented by crystal lattice between D-methionine and L-methionine under extremes conditions of the pressure. / Nesse trabalho investigamos as propriedades vibracionais em monocristais D-Metionina, utilizando as espectroscopias Raman e Infravermelho. As medidas de espectroscopia Raman foram realizadas sob a variação de pressão hidrostática no intervalo de 0GPA até 7GPa, mostraram duas transições de fase, uma por volta 2.0GPa e outra em 2.5GPa, as quais foram detectadas no espectro Raman através das mudanças exibidas pelos picos associados principalmente às vibrações atribuídas à carboxila (CO2-), amina (NH3+), , ligações CS e CSC e grupos CH2 e CH3. Esses modos vibracionais também foram sensibilizados em baixas temperaturas, sendo que suas mudanças serviram como referência para identificar a transição de fase nas medidas de espalhamento infravermelho no intervalo de temperatura de 298K a 84K. Nas medidas de espalhamento Raman de 298K a 13K, a transição foi identificada pelo surgimento do modo translacional em 91 cm-1 por volta de 84K. Além disso, nesse trabalho comparamos o comportamento das mudanças apresentadas na rede cristalina dos monocristais de D-metionina de L-metionina sob condições extremas de pressão.

MELO, Wanessa David Canedo. Transições de fase em monocristais de D-Metionina. 2012. 86 f. Tese (Doutorado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2012. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2015-10-22T18:15:02Z No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2015-10-22T21:31:24Z (GMT) No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) / Made available in DSpace on 2015-10-22T21:31:24Z (GMT). No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) Previous issue date: 2012 / In this work we have been investigated the vibrational properties of the D-methionine single crystals using the Raman and Infrared spectroscopies. The Raman spectroscopy measurements were performed under varying hydrostatic pressure from 0 GPa up to 7.0 GPa, indicated two transitions, one around 2.0 GPa and another in 2.5GPa, which were detected by changes of the vibrationals modes of the carboxilic (CO2-), amino (NH3+), CS, CSC, CH2 and CH3, respectively. These vibrationals modes also have been sensitized under low temperatures, whose changes have been helpful to identify the phase transition with the measurements of the infrared scattering from 298K up to 84K. In the measurements of the Raman scattering realized between 298K and 84K, the transition was identified by emerging of the translational mode in 91 cm-1 around 84K. Further, in this work we have been compared changes presented by crystal lattice between D-methionine and L-methionine under extremes conditions of the pressure. / Nesse trabalho investigamos as propriedades vibracionais em monocristais D-Metionina, utilizando as espectroscopias Raman e Infravermelho. As medidas de espectroscopia Raman foram realizadas sob a variação de pressão hidrostática no intervalo de 0GPA até 7GPa, mostraram duas transições de fase, uma por volta 2.0GPa e outra em 2.5GPa, as quais foram detectadas no espectro Raman através das mudanças exibidas pelos picos associados principalmente às vibrações atribuídas à carboxila (CO2-), amina (NH3+), , ligações CS e CSC e grupos CH2 e CH3. Esses modos vibracionais também foram sensibilizados em baixas temperaturas, sendo que suas mudanças serviram como referência para identificar a transição de fase nas medidas de espalhamento infravermelho no intervalo de temperatura de 298K a 84K. Nas medidas de espalhamento Raman de 298K a 13K, a transição foi identificada pelo surgimento do modo translacional em 91 cm-1 por volta de 84K. Além disso, nesse trabalho comparamos o comportamento das mudanças apresentadas na rede cristalina dos monocristais de D-metionina de L-metionina sob condições extremas de pressão.

Made available in DSpace on 2014-10-09T12:38:39Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:00:11Z (GMT). No. of bitstreams: 1 06041.pdf: 4212913 bytes, checksum: fd1c8026a141fe44d8a936d7cfcd904d (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP