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Interação do peptídeo MP-I com micela de SDS em solução aquosa: um estudo por dinâmica molecularValder, Tamára Rodrigues [UNESP] 04 May 2010 (has links) (PDF)
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valder_tr_me_sjrp.pdf: 1809932 bytes, checksum: 8e85d4281d284b8a21374552b1591c82 (MD5) / Moléculas de Dodecil Sulfato de Sódio (SDS) em solução aquosa podem formar aglomerados micelares em condições adequadas de concentrações, pH, temperatura e força iônica, devido às características polar/apolar de suas cabeças/caudas. Essas micelas, aniônicas, mimetizam o ambiente da membrana de células procariontes e são usados como modelo no estudo da interação de peptídeos antimicrobianos com membranas das bactérias. Neste trabalho usamos simulações por dinâmica molecular de equilíbrio para estudar a interação do peptídeo Polybia MP-I com micela de SDS em solução aquosa, considerando tanto a situação em que a micela está pré-formada como a de automontagem da micela na presença do peptídeo. Mostramos que em ambos os casos o peptídeo se insere na micela de forma aproximadamente paralela à membrana, com a cadeia principal enterrada na região de interface entre as caudas e as cabeças polares das moléculas de SDS e os grupos carregados localizados na interface das cabeças polares com a solução. Estes dados confirmam modelos de inserção de peptídeos antimicrobianos em membranas existentes na literatura. Discutimos também a estabilidade da estrutura helicoidal anfipática do peptídeo em função da solvatação de sua cadeia principal, que proporciona a manutenção de uma rede de ligações de hidrogênio do tipo NH --- O entre resíduos do tipo n-(n+4) da cadeia principal, típicos de estruturas em α – hélice. Além disso, os resultados indicam que pontes salinas formadas entre os grupos negativos dos resíduos do ácido aspártico (Asp) 2 e 8 com resíduos de lisinas 5 e 11 são importantes para a manutenção da estrutura do peptídeo. Os resultados indicam também que a estrutura micelar mantém-se aproximadamente esférica com ou sem o peptídeo independente... / The Sodium Dodecyl Sulphate (SDS) molecules can form micellar agregates in aqueous solution under appropriate concentration, pH, temperature and ionic strength conditions due to the polar/apolar features of its head and tail. The anionic micelles formed mimic the environment of the prokaryotic cells membrane of bacteria. In this work we use equilibrium molecular dynamics simulation to study the interaction of the peptide Polybia MP-I with SDS micelles in aqueous solution considering both a preformed micelle and a micelle obtained by self-assembling the SDS molecules in the presence of the peptide. We show that in both cases the peptide inserts in an almost parallel way relative to the micelle surface, with the main chain buried in the interface region between the tails and polar heads of SDS molecules and the charged groups of the peptide located at the interface with the polar heads the solution. These data support models of insertion of antimicrobial peptides in membranes in the literature. We also discuss the stability of the amphipathic helical structure of the peptide as a function of salvation of the main chain, which contributes to the maintenance of the network of hydrogen bonds in the main chain of type NH --- O between residues n and n+4 that are typical of structures in α-helix. Furthermore, the results indicate that salt bridges formed between the negative groups of aspartic acid residues (Asp) 2 and 8 with lysine residues 5 and 11 are importance in maintaining the structure of the peptide. The results also indicate that the micellar structure remains approximately spherical with and without the peptide, independent of the process as it is obtained, whether preformed or self-assembled. The micellar radius increases to accommodate the peptide and in the case of self-assembled micelle... (Complete abstract click electronic access below)
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Interação do peptídeo MP-I com micela de SDS em solução aquosa : um estudo por dinâmica molecular /Valder, Tamára Rodrigues. January 2010 (has links)
Orientador: José Roberto Ruggiero / Banca: Mário Sérgio Palma / Banca: Luiz Carlos Gomide Freitas / Resumo: Moléculas de Dodecil Sulfato de Sódio (SDS) em solução aquosa podem formar aglomerados micelares em condições adequadas de concentrações, pH, temperatura e força iônica, devido às características polar/apolar de suas cabeças/caudas. Essas micelas, aniônicas, mimetizam o ambiente da membrana de células procariontes e são usados como modelo no estudo da interação de peptídeos antimicrobianos com membranas das bactérias. Neste trabalho usamos simulações por dinâmica molecular de equilíbrio para estudar a interação do peptídeo Polybia MP-I com micela de SDS em solução aquosa, considerando tanto a situação em que a micela está pré-formada como a de automontagem da micela na presença do peptídeo. Mostramos que em ambos os casos o peptídeo se insere na micela de forma aproximadamente paralela à membrana, com a cadeia principal enterrada na região de interface entre as caudas e as cabeças polares das moléculas de SDS e os grupos carregados localizados na interface das cabeças polares com a solução. Estes dados confirmam modelos de inserção de peptídeos antimicrobianos em membranas existentes na literatura. Discutimos também a estabilidade da estrutura helicoidal anfipática do peptídeo em função da solvatação de sua cadeia principal, que proporciona a manutenção de uma rede de ligações de hidrogênio do tipo NH --- O entre resíduos do tipo n-(n+4) da cadeia principal, típicos de estruturas em α - hélice. Além disso, os resultados indicam que pontes salinas formadas entre os grupos negativos dos resíduos do ácido aspártico (Asp) 2 e 8 com resíduos de lisinas 5 e 11 são importantes para a manutenção da estrutura do peptídeo. Os resultados indicam também que a estrutura micelar mantém-se aproximadamente esférica com ou sem o peptídeo independente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Sodium Dodecyl Sulphate (SDS) molecules can form micellar agregates in aqueous solution under appropriate concentration, pH, temperature and ionic strength conditions due to the polar/apolar features of its head and tail. The anionic micelles formed mimic the environment of the prokaryotic cells membrane of bacteria. In this work we use equilibrium molecular dynamics simulation to study the interaction of the peptide Polybia MP-I with SDS micelles in aqueous solution considering both a preformed micelle and a micelle obtained by self-assembling the SDS molecules in the presence of the peptide. We show that in both cases the peptide inserts in an almost parallel way relative to the micelle surface, with the main chain buried in the interface region between the tails and polar heads of SDS molecules and the charged groups of the peptide located at the interface with the polar heads the solution. These data support models of insertion of antimicrobial peptides in membranes in the literature. We also discuss the stability of the amphipathic helical structure of the peptide as a function of salvation of the main chain, which contributes to the maintenance of the network of hydrogen bonds in the main chain of type NH --- O between residues n and n+4 that are typical of structures in α-helix. Furthermore, the results indicate that salt bridges formed between the negative groups of aspartic acid residues (Asp) 2 and 8 with lysine residues 5 and 11 are importance in maintaining the structure of the peptide. The results also indicate that the micellar structure remains approximately spherical with and without the peptide, independent of the process as it is obtained, whether preformed or self-assembled. The micellar radius increases to accommodate the peptide and in the case of self-assembled micelle... (Complete abstract click electronic access below) / Mestre
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Computational Study of Adiabatic Bubble Growth Dynamics from Submerged Orifices in Aqueous Solutions of SurfactantsDeodhar, Anirudh M. 18 September 2012 (has links)
No description available.
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Enhanced gel electrophoresis (GE) and inductively coupled plasma-mass spectrometry (ICP-MS) based methods for the identification and separation of proteins and peptidesHaider, Syed January 2012 (has links)
The main focus of the PhD study was to develop new gel electrophoresis and ICP-MS based methods to analyze a wide variety of the bio-molecules such as proteins, phosphoproteins and metalloproteins etc. The tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) method is commonly used to resolve low molecular mass proteins, however, it requires a high percentage gel and a very complicated procedure to achieve this separation. This study describes a modification to tricine-SDS-PAGE to make it more effective for the separation of smaller proteins and for coupling to ICP-MS. The modified method employs low percentage PAGE gels and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. This modified method was applied to analyze phosphopeptides. Phosphopeptides are very small in size and difficult to separate using the other techniques such as Laemmli SDS-PAGE, original tricine-SDS-PAGE, immobilized metal affinity chromatography (IMAC), size exclusion chromatography (SEC) etc. In this study a simplified procedure is described based on modifying the original tricine-SDS-PAGE method. A comparative study showed that this modified method successfully resolved a digest mixture of very low to high molecular mass phosphopeptides/peptides. In off-line coupling of this method with ICP-MS, much better recoveries of the peptides from the gel were obtained as compared to traditional methods which indicate the compatibility of this modified method for quantitative studies. An on-line coupling of the modified system with ICP-MS was also demonstrated and it was applied for the separation, detection and quantification of phosphopeptides. Another application of this modified system was the separation of serum proteins. Blood serum contains five major protein groups i.e., albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin. The separation of these five major proteins in a single gel is difficult to achieve using traditional methods. The modified system was shown to be superior for the separation of these serum proteins in a 7% (m/v) native-PAGE gel and a cellulose acetate membrane. A further study was carried out into controlling the factors that cause metal loss and protein fragmentation in SDS-PAGE. Using a reducing sample buffer, and heating to high temperatures (90-100ºC) in alkaline or acidic conditions may cause protein fragmentation and decrease the metal binding affinity. 70ºC was found suitable to prepare the sample at neutral, alkaline or acidic pH as no fragmentation observed. To prevent metal loss, the binding constant (log K) values of metal-amino acids, play the major role. Those metals which have high binding affinities with the amino acids in proteins can also be affected by the variation of the pH so prior information about pH to maintain the binding constant values is essential to minimize metal loss. This was observed in the loss of zinc, and to a lesser extent copper from human serum albumin (HSA) as measured by inductively coupled plasma mass spectrometry (ICP-MS). The method described above was applied for the separation and quantification of the serum proteins obtained from age-related macular degeneration (AMD) patients (where the AMD patients were from Moorfields Eye Hospital, London). Zn and Cu were quantified employing external calibration. Zn concentration showed variation whilst Cu did not show any significant variations in samples from AMD patients. A brief study of the interaction of cisplatin and oxaliplatin with HSA and transferrin was also performed. Cisplatin bound much faster than oxaliplatin with HSA. After 24 hours incubation, cisplatin showed a decrease in signal intensity which indicates that cisplatin binding decreases with time. Cisplatin binding with transferrin as compared to HSA was not significant, which could be the result of unstable Pt-transferrin complex formation. Oxaliplatin did not show high binding to either protein, perhaps due to the presence of the bulky, non polar DACH ligand.
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Poly(Ethylene Oxide) Based Bottle-Brush Polymers and their Interaction with the Anionic Surfactant Sodium Dodecyl Sulphate : Solution and Interfacial PropertiesIruthayaraj, Joseph January 2008 (has links)
The aim of this thesis work is to study the physico-chemical properties of poly(ethylene oxide), PEO, based brush polymers both in solution and at solid/aqueous interfaces. The importance of studying the surface properties of brush polymers can be related to a broad spectrum of interfacial-related applications such as colloidal stability, lubrication, detergency, protein repellency to name a few. In many applications it is desirable to form brush-like structures through simple physisorption. In this context the surface properties of PEO based brush polymers differing in molecular architecture were studied, using ellipsometry and surface force apparatus (SFA), to gain some understanding regarding the effect of molecular architecture on the formation of brush structures. The molecular architecture was varied by varying the charge/PEO ratio along the backbone. This study demonstrates that the formation of a brush structure at solid/aqueous interface is due to interplay between the attraction of the backbone to the surface and the repulsions between the PEO side chains. An optimal balance between the two antagonistic factors is required if one aims to build a well-defined brush structure at the interface. In this study the brush-like structures are formed when 25-50% of the backbone segments carry poly(ethylene oxide) side chains. Scattering techniques such as light and neutron reveal that these brush polymers are stiff-rods up to a charge to PEO ratio of 75:25. These stiff PEO brush polymer easily replace the more flexible linear PEO at the silica/water interface, the reason being that the entropy loss on adsorption is smaller for the brush polymer due to its stiff nature. Polymer-surfactant systems play a ubiquitous role in many technical formulations. It is well known that linear PEO, which adopts random coil conformation in aqueous solution, interact strongly with the anionic surfactant, Sodium Dodecyl Sulphate (SDS). It is of interest to study the interaction between SDS and brush PEO owing to the fact that the PEO side chains have limited flexibility as compared to the linear PEO. The interaction between brush PEO and the anionic surfactant SDS in solution are studied using different techniques such as NMR, tensiometry, SANS and light scattering. The main finding of this study is that the interaction is weaker compared to the linear PEO-SDS interactions which poses an interesting question regarding the role of chain flexibility in polymer-surfactant interactions. / QC 20100813
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Studium interakce záporně nabitých vezikulárních systémů s polykationty / Study of interaction of negatively charged vesicular systems with polycationsRepová, Romana January 2020 (has links)
This diploma thesis deals with the preparation and characterization of negatively charged catanionic vesicular systems and their combination with selected polycations. The catanionic vesicular system was prepared by mixing of two oppositely charged surfactants SDS and CTAB. The negative charge as well as the stability of the vesicular system was provided by the incorporation of phosphatidic acid. Polycations, DEAE and TMC, have been selected for use in a pharmaceutical applications. Characterization of the prepared systems was performed by measuring DLS and ELS. The results indicate that we were able to prepare stable negatively charged vesicles that were eligible to non-covalently interact with selected polycations.
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Characterisation of nicotine binding sites on human blood lymphocytesWongsriraksa, Anong January 2008 (has links)
Nicotine exerts a therapeutic effect in ulcerative colitis (UC) but the mechanism underlying this effect, is not clear. However, this effect may imply that nicotine has some, as yet to be discovered, effect on the immune system. The aim of the work described in this thesis was to characterise the nicotinic acetylcholine receptors (nAChRs) on human peripheral blood lymphocytes in term of receptor subtype. To achieve this, a combination of radioligand binding assays, pharmacological and molecular biological techniques were used. The data obtained from the binding studies suggested that the presence of one binding site for (-)- nicotine on human peripheral blood lymphocytes with a Kd 15 ± 5.759 nM (1.5 ± 5.759 x 10-8 M) and Bmax 2253 ± 409 sites/cell. The competition studies showed that ligands competing with [3H]-(-)-nicotine were (-)-nicotine, epibatidine and α-bungarotoxin, while others ligands for nAChRs displaced radiolabelled nicotine in insignificant quantities. Thus, radioligand-binding experiments suggest that the binding site for nicotine on human peripheral blood lymphocytes is a nAChR containing α7 and possibly α4 or/and b2 containing nAChR subunits. No evidence was obtained to suggest the presence of a non-cholinergic nicotine receptor. Furthermore, considerable subject to subject variation in the specific binding of radiolabelled nicotine was observed. Because of this only tentative conclusions could be drawn from radioligand binding data. Polymerase chain reaction (RT-PCR) was then used to demonstrate mRNA for the subunits of nAChRs suggested by radioligand binding studies. Data obtained show that the human peripheral blood lymphocytes tested, expressed mRNAs for α4, α5, α7, β2 neuronal nAChRs subunits and β1 muscle nAChR subunit. Expression of the α5 mRNA subunit of nAChR was observed in the lymphocytes in each sample of lymphocytes tested. In contrast, the expression pattern of mRNAs for α4, α7, β1, and β2 mRNAs subunits of nAChRs, varied between individuals. Finally, Western blot analysis was used to confirm that mRNA expression resulted in the expression of protein for nAChR subunits in human peripheral lymphocytes using monoclonal antibodies against α4, α5, α7, and β2 nAChR subunits, which had been detected by RT-PCR. The results obtained from the Western blot analysis show that protein for α4, α5, and α7 nAChR subunits was expressed in most, but not all of the human peripheral blood lymphocyte samples tested and some of the bands obtained were faint. In contrast, protein for the β2 nAChR subunit was observed in a few samples tested and the bands were faint. From the results obtained in this study, it is possible to conclude that human peripheral blood lymphocytes may contain nAChRs with subunit compositions of α4β2, α4β2α5, and/or α7. However, further studies are necessary to show whether or not the single binding site for nicotine demonstrated by radioligand binding experiments is due to one or all of these nAChRs. Thus, the findings of the present study suggest the presence of nAChR on human peripheral blood lymphocytes. Nicotine and its effect may occur through these non- neuronal nAChRs mechanisms. Such a mechanism of action could account for the beneficial of nicotine in ulcerative colitis. Furthermore, a compound that acts on these receptors, but not on nAChRs found on other cells may have therapeutic utility in the treatment of inflammation.
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A study of type-3 copper proteins from arthropodsBaird, Sharon January 2007 (has links)
Arthropod hemocyanin and phenoloxidase are members of a group of proteins called the Type-3 copper oxygen-binding proteins, both possessing a highly conserved oxygen-binding site containing two copper atoms each coordinated by three histidine residues (Decker and Tuczek, 2000). Despite similarities in their active site, these proteins have very different physiological functions. Phenoloxidase possesses both tyrosinase and o-diphenoloxidase activity, and is predominantly involved in reactions which protect insects from infection (Kopàcek et al., 1995). Hemocyanin is a large multi-subunit protein with a primary function as a respiratory protein, reversibly binding and transporting molecular O2 (Decker and Rimke, 1998; Decker and Tuczek, 2000). Recently, it has been demonstrated in vitro that arthropod hemocyanin possesses an inducible phenoloxidase activity when incubated with denaturants, detergents, phospholipids or proteolytic enzymes. This activity appears to be restricted to only a few subunit types, and it has been hypothesised that it may be accompanied by conformational change which opens the active site increasing access for larger phenolic substrates (Decker and Jaenicke, 2004; Decker et al., 2001; Decker and Tuczek, 2000). This possibly suggests a dual role of hemocyanin in arthropods. The presented thesis deals with two distinct aims. The first was to isolate and sequence a phenoloxidase gene from the insect Spodoptera littoralis (Egyptian Cottonleaf Worm). Despite efforts, progress was hindered by a number of experimental problems which are outlined within the relevant chapters. The second aim was to characterise the mode of SDS induced phenoloxidase activity in arthropod hemocyanin from the ancient chelicerates Limulus polyphemus (horseshoe crab) and Eurypelma californicum (tarantula) and the more modern chelicerate Pandinus imperator (scorpion), using a number of biophysical techniques. The results indicated that the SDS induced phenoloxidase activity is associated with localised tertiary and secondary conformational changes in hemocyanin, most likely in the vicinity of the dicopper centre, thus enhancing access for larger phenolic substrates. Experiments indicate that copper remains associated with the protein during these structural changes; however the nature of the association is unclear. SDS concentrations approximating the CMC appeared critical in causing the necessary structural changes required for a significant increase in the detectable phenoloxidase activity to be exhibited.
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Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic ProductsMcCanna, David January 2009 (has links)
The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes.
The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.
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Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic ProductsMcCanna, David January 2009 (has links)
The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes.
The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.
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