The phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine. This study presents the use of kinetic isotope effects (KIE) as mechanistic probes to compare the reactivity of CvPheH and that of the eukaryotic aromatic amino acid hydroxylases. This study also describes the use of different spectroscopic and kinetic techniques to identify the hydroxylating intermediate for this enzyme and the assignment of the NMR backbone resonances of CvPheH. Kinetic isotope effects on aromatic and benzylic hydroxylation were used to establish that bacterial and eukaryotic phenylalanine hydroxylases have similar reactivity. The observed KIE on aromatic hydroxylation of 1.4 was shown to be a combination of an inverse isotope effect on the hydroxylation of the amino acid and a normal isotope effect on a subsequent step in the reaction. An isotope effect on benzylic hydroxylation of 10 was found for CvPheH. This result establishes the similar reactivity for CvPheH and the eukaryotic aromatic amino acid hydroxylases and suggests the involvement of a common hydroxylating intermediate. Kinetic isotope effects were used to study the hydroxylation of the aliphatic substrate cyclohexylalanine. The Dkcat value with [1,2,2,3,3,4,4,5,5,6,6-2H11]- cyclohexylalanine is unity with wild-type CvPheH, suggesting that chemistry is not ratelimiting with this substrate. The intramolecular isotope effect calculated using [1,2,3,4,5,6-2H6]-cyclohexylalanine yields a value of 14. This result is evidence for the involvement of a reactive iron species capable of abstracting a hydrogen atom from the aliphatic carbon in cyclohexylalanine. Analysis of the CvPheH reaction using freeze-quench Mössbauer spectroscopy allowed the detection of an Fe(IV) species in the first turnover of the enzyme. Chemical quench and stopped-flow spectrophotometric methods were used to establish the kinetic competency of the Fe(IV) intermediate as the hydroxylating species. The NMR amide backbone resonances in the HSQC spectrum of CvPheH were assigned to the corresponding amino acid residues using a suite of TROSY-based threedimensional triple resonance experiments. We were able to assign 224 residues out of the 278 assignable residues in CvPheH, this constitutes 81 percent of the assignable protein sequence.

Polycondensation reaction was carried out for synthesizing rigid-rod polymer hPBI. Various molar ratios (50:1, 25:1, and 15:1) of 2-hydroterephthalic acid and 5-hydroisophthalic acid were also introduced in the synthesis for articulated rigid-rod polymer a-hPBI. The polymers were further derivatized with 1,3-propanesulton for pendants of lithium ionomer to become water soluble polyelectrolytes hPBI-PS(Li+) and a-hPBI-PS(Li+), respectively. Lithium salt doped cast film of the rigid-rod polyelectrolyte hPBI-PS(Li+) showed a room-temperature DC conductivity parallel to film surface as high as 4.02¡Ñ10-3 S/cm. Molecular weight of the rigid-rod polyelectrolyte was low indicating a small molecular aspect ratio. In cast film, the molecules were randomly distributed and highly isotropic facilitated Li cations mobility for a high film conductivity. The conductivity was also insensitive to the anion of lithium salt. No apparent layered structure was revealed by scanning electron microscope suggesting that the cast films had near three-dimensionally isotropic structure and conductivity.

The work presented in this thesis deals with the analysis of protein crystal structures with an emphasis on the stereochemical aspects of the folded conformation of proteins. The various analyses described have been performed on a data-set of 250 high resolution and non-homologous protein structures derived from the Protein Data Bank. The overall objective of the work has been to analyse conformational features of the non-secondary structural regions in proteins and identify structural motifs present therein. The results can be useful in the three-dimensional modelling of proteins, altering the stability of proteins, design of peptide mimics and in understanding the structural rules that guide protein folding. The contents of this thesis can be broadly classified into three parts, (a) Conformational preferences of amino acid residues to occur in the partially allowed regions of the Ramachandran map, (b) conformational features of structural motifs formed by side-chain/main-chain hydrogen bonds by polar residues and (c) analysis and characteristic features of isolated β-strands. Chapter 1 of the thesis gives an introduction, briefly discussing the conformation of polypeptide chains, structural features of globular proteins and applications of protein structural analysis etc. Chapter 2 describes the occurrence of left-handed α-helical conformation in protein structures. A data-set of 250 high resolution (< 2.0A) non-homologous protein crystal structures derived from the Protein Data Bank (PDB) has been analysed for occurrences of left-handed α-helical (αL) conformations. A total of 2,573 αL residues were identified from the data-set. About 59% of the observed examples of at conformations were found to be glycyl residues and about 41% non-glycyl. Continuous long stretches of αL residues are seldom found in protein structures. They are most commonly found as singlets represented by 78% of the observed αL examples. The doublets, triplets and quadruplets account for a very minor fraction of the observed examples. There is only a single example of a stretch of four contiguous αL residues, from the protein thermolysin, which forms a single turn of a left-handed α-helix. A majority of the αL residues are nevertheless part of well-defined substructures in proteins. They play singular roles as part of β-turns and helix termination sites in maintaining the characteristic main-chain hydrogen bonds needed for the stability of these structures. They are also found to be effective in the termination of β-strands. The stereo-chemistry and sequence environment around such structures are discussed. The analysis of the side-chain torsion angles of αL residues indicate that the g+ rotamer is highly unfavourable due to stereo-chemical violations posed by the atoms of the side-chain with those of the backbone. The αL residues are highly conserved by residue type as well as conformation among related proteins indicating their vital importance in protein structures Chapter 3 provides an explanation for the unusual preference of glycyl residues to occur in the bridge regions of the Ramachandran map. The Ramachandran steric map and energy diagrams for the glycyl residue are fully symmetric. Though a plot of the (Φ,Ψ) angles of glycyl residues derived from a data-set of 250 non-homologous and high-resolution protein structures is also largely symmetric, there is a clear aberration in the symmetry. While there is a cluster of points corresponding to the right-handed a-helical region, the "equivalent" cluster is shifted to centre around the (Φ,Ψ)values of (90°, 0°) instead of being centred at the left-handed a-helical region of (60°, 40°). An analysis of glycyl conformations in small peptide structures and in "coil" proteins, which are largely devoid of helical and sheet regions, shows that glycyl residues prefer to adopt conformations around (±90°, 0°) instead of right and left handed a-helical regions. Using theoretical calculations, such conformations are shown to have highest solvent accessibility in a system of two-linked peptide units with glycyl residue at the central Cα atom. This is found to be consistent with the observations from 250 non-homologous protein structures where glycyl residues with conformations close to (±90°, 0°) are seen to have high solvent accessibility. Analysis of a sub-set of non-homologous structures with very high resolution (1.5A or better) shows that water molecules are indeed present at distances suitable for hydrogen bond interaction with glycyl residues possessing conformations close to (±90°, 0°). It is concluded that water molecules play a key role in determining and stabilising these conformations of glycyl residues and explains the aberration in the symmetry of glycyl conformations in proteins. Chapter 4 discusses an analysis of backbone mimicry performed by polar side-chains in protein structures. Backbonemimicry bythe formation of closed loop C7, C10, C13 (mimics of γ-, β- and α-turns) conformations through side-chain main-chain hydrogen bonds by polar groups is found to be a frequent observation in protein structures. A data-set of 250 non-homologous and high-resolution protein structures was used to analyse these conformations for their characteristic features. Seven out of the nine polar residues (Ser, Thr, Asn, Asp, Gin, Glu and His) have hydrogen bonding groups in their side-chains which can participate in such mimicry and as many as 15% of all these polar residues engage in such conformations. The distributions of dihedral angles of these mimics indicate that only certain combinations of the involved dihedral angles aids the formation of these mimics. The observed examples have been categorised into various classes based on these combinations resulting in well-defined motifs. Asn and Asp residues show a very high capability to perform such backbone secondary structural mimicry. The most highly mimicked backbone structure is of the Cio conformation by the Asx residues. The mimics formed by His, Ser, Thr and Glx residues are also discussed. The role of such conformations in initiating the formation of regular secondary structures during the course of protein folding seems significant. Chapter 5 presents a description of deterministic features of side-chain main-chain hydrogen bonds as observed in protein structures. A total of 19,835 polar residues from the data set of 250 non-homologous and highly resolved protein crystal structures were used to identify side-chain main-chain (SC-MC) hydrogen bonds. The ratio of the total number of polar residues to the number of SC-MC hydrogen bonds is close to 2:1, indicating the ubiquitous nature of such hydrogen bonds. Close to 56% of the SC-MC hydrogen bonds are local involving side-chain acceptor/donor (‘i’) and a main-chain donor/acceptor within the window i-5 to i+5. These short-range hydrogen bonds form well defined conformational motifs characterised by specific combinations of backbone and side-chain torsion angles. Some of the salient features of such hydrogen bonds are as follows, (a) The Ser/Thr residues show the greatest preference in forming intra-helical hydrogen bonds between the atoms Oyi and Oi-4 Such hydrogen bonds form motifs of the form αRαRαRαR(g") and are most commonly observed at the middle of α-helices. (b) These residues also show great preference to form hydrogen bonds between OYi and Oi-3, which are closely related to the previous type and though intra-helical, these hydrogen bonds are more often found at the C-termini of helices than at the middle. The motif represented by αRαRαRaR(g+) is most preferred in these cases, (c) The Ser, Thr and Glu (between the side-chain and main-chain of the same residue), (d) The side-chain acceptor atoms of Asn/Asp and Ser/Thr residues show high preference to form hydrogen bonds with acceptors two residues ahead in the chain, which are characterised by the motifs β(tt’)αR and β(t)αR, respectively. These hydrogen bonded segments referred to as Asx turns, are known to provide stability to type I and type I’ β-turns. (e) Ser/Thr residues often form a combination of SC-MC hydrogen bonds, with the side-chain donor hydrogen bonded to the carbonyl oxygen of its own peptide backbone and the side-chain acceptor hydrogen bonded to an amide hydrogen three residues ahead in the sequence. Such motifs are quite often seen at the beginning of a-helices, which are characterised by the β (g+)αRαR motif. A remarkable majority of all these hydrogen bonds are buried from the protein surface, away from the surrounding solvent. This strongly indicates the possibility of side-chains playing the role of the backbone, in the protein interiors, to satisfy the potential hydrogen bonding sites and maintaining the network of hydrogen bonds which is crucial to the structure of the protein. Chapter 6 provides a detailed characterisation of isolated β-strands. Reason for the formation of β-strands in proteins is often associated with the formation of β -sheets. However β-strands, not part of β-sheets, commonly occur in proteins. This raises questions about the structural role and stability of such isolated β-strands. Using a data set consisting of 250 proteins, 518 isolated β-strands have been identified from 187 proteins. The two important features that distinguish isolated β-strands from p-strands occurring in β-sheets are (i) the high preponderance of prolyl residues to occur in isolated β-strands and (ii) their high solvent exposure. It is shown that the high propensity for proline residues to occur in isolated β-strands is not due to the occurrence of polyproline type segments in the data-set. The propensities of other amino acids to occur in isolated β-strands follows the same trend as those for β-sheet forming β-strands. Isolated β-strands are characterised often by their main-chain amide and carbonyl groups involved in hydrogen bonding with polar side-chains or water. They are often flanked by irregular loop structures indicating that they are part of long of loops. Analysis of the conservation of such strands among families of homologous protein structures indicates that a sizeable fraction of them are highly conserved. It is suggested that though the formation of isolated β-strands are driven by the intrinsic preferences of amino acid residues, they have many characteristics like loop segments but with repetitive (Φ,Ψ) values falling within the β-region of the Ramachandran map. In addition of the material described in the six chapters above, the thesis also contains the details of work carried out on an aspect slightly different from the main theme of the thesis. This pertains to the comparative analysis of the members of a family of cytokine receptors to derive information to model new members of the family. The three dimensional modelling of the leptin receptor has been used as a case study and the details are included as an appendix. Appendix describes the 3-dimensional model of the satiety factor receptor (the leptin receptor) modelled using principles of homology modelling. Recessive mutations in the mouse obese (ob) and diabetes (db) genes result in obesity and diabetes in a syndrome resembling human obesity. Data from parabiosis (cross circulation) experiments suggested that the ob gene coded, and was responsible for the generation of a circulating factor called leptin which regulated energy balance and the db gene encoded the receptor for this factor. While the structure of the leptin has been determined that of its cognate receptor is as yet unknown. The leptin receptor shows low but clear sequence similarity to the members of the interleukin type 6 family of receptors. The structures of the members of this family are characterised by two p-sandwich like domains connected by a short 4-residue helical linker. The 3-dimensional models for the N- and C-terminal domains of the leptin receptor was generated using the corresponding structures of the signal transducing component of gpl30, the erythropoetin receptor and the prolactin receptor. Further using the evidence that the leptin binds to its receptor with a stoichiometry of 1:1, the relative orientation of the two domains was modelled based on the structure of the human growth hormone receptor, which also binds its ligand with similar stoichiometry. The complex of leptin with its receptor was also modelled based on the structure of human growth hormone/receptor complex. The final energy minimised model of the complex elucidates the mode of interaction between the leptin and its receptor.

<p>Proteins are among the most complex entities known to science. Composed of just 20 fundamental building blocks arranged in simple linear strings, they nonetheless fold into a dizzying array of architectures that carry out the machinations of life at the molecular level.</p><p>Despite this central role in biology, we cannot reliably predict the structure of a protein from its sequence, and therefore rely on time-consuming and expensive experimental techniques to determine their structures. Although these methods can reveal equilibrium structures with great accuracy, they unfortunately mask much of the inherent molecular flexibility that enables proteins to dynamically perform biochemical tasks. As a result, much of the field of structural biology is mired in a static perspective; indeed, most attempts to naively model increased structural flexibility still end in failure.</p><p>This document details my work to validate alternative protein conformations beyond the primary or equilibrium conformation. The underlying hypothesis is that more realistic modeling of flexibility will enhance our understanding of how natural proteins function, and thereby improve our ability to design new proteins that perform desired novel functions.</p><p>During the course of my work, I used structure validation techniques to validate conformational alternatives in a variety of settings. First, I extended previous work introducing the backrub, a local, sidechain-coupled backbone motion, by demonstrating that backrubs also accompany sequence changes and therefore are useful for modeling conformational changes associated with mutations in protein design. Second, I extensively studied a new local backbone motion, helix shear, by documenting its occurrence in both crystal and NMR structures and showing its suitability for expanding conformational search space in protein design. Third, I integrated many types of local alternate conformations in an ultra-high-resolution crystal structure and discovered the combinatorial complexity that arises when adjacent flexible segments combine into networks. Fourth, I used structural bioinformatics techniques to construct smoothed, multi-dimensional torsional distributions that can be used to validate trial conformations or to propose new ones. Fifth, I participated in judging a structure prediction competition by using validation of geometrical and all-atom contact criteria to help define correctness across thousands of submitted conformations. Sixth, using similar tools plus collation of multiple comparable structures from the public database, I determined that low-energy states identified by the popular structure modeling suite Rosetta sometimes are valid conformations likely to be populated in the cell, but more often are invalid conformations attributable to artifacts in the physical/statistical hybrid energy function.</p><p>Unified by the theme of validating conformational alternatives by reference to high-quality experimental structures, my cumulative work advances our fundamental understanding of protein structural variability, and will benefit future endeavors to design useful proteins for biomedicine or industrial chemistry.</p> / Dissertation

Conducting polymer is a rapidly developing area of research due to its potential in combining the physical properties of polymers with electrical properties previously found only in inorganic systems. These conducting polymers owe their unique properties to a conjugated polymer backbone and become conducting upon oxidation or reduction. Melanin, a biopolymer, possess a conjugated backbone required of a conducting polymer, and has shown properties of an amorphous semiconductor. However, there has not been much study done in this area despite its potential, and this is partially due to the lack of processing methods as melanin is generally synthesised as an intractable powder. Thus, a better synthetic method was required, and a possible solution is the use of electrochemical synthesis. In our previous study we have shown that melanin can be synthesised electrochemically as a free-standing film, which was the first step towards the use of melanin as a bulk material. This project aims to continue from this preliminary work, investigating the various synthetic parameters and possible modifications as well as investigating possible applications for the electrochemically synthesised melanin film.

No description available.

Proteins are one of the most important molecules in biology. The wide range of functions of different proteins is also important for medical physics. Proteins are assembled by amino acids. These amino acids are connected by peptide bonds to form a protein. The function of a protein is decided by the composition and configuration of peptides, amino acids and their peptide bonds. Successful experiments with Xray Free-electron laser has lead to progress in structural biology, however there is still a need to crystallized samples in these experiments. In this project we have investigated three amino acids. These three amino acids are included in several protein that are hard to crystallize, glycine, valine and alanine. We have investigated their separate interatomic bonds by performing density functional calculations and evaluating the susceptibility of the bonds breaking in a typical time range of Xray Free-electron laser pulses. The results shows the fast dissipation of hydrogen atoms, bond shifting within the molecules during certain ionization degrees and the dissociation of the protein backbone after 20 fs.

The world statistics state that 80% of the population encounter pains in the back. These problems were accelerated by modern times, that thanks, to the technical comfort, brought to many people health troubles resulting from the lack of exercise. I chose the subject {\clqq} Training of the back`` because I work as physiotherapist and the people feeling pains in the back belong to my profession every day. I occupy myself with the given problems. Considering the present state of the given problems, I concentrated on the most important connection concerning the training of the back. First, I described the backbone as a part of the locomotory system. Another guideline that was interconnection of muscular groups of the back, chest, abdomen and of the pelvis, that build the muscular corset. I described locomotory stereotypes and disorders there of in particular relations. Integral part of the present state that is the origin of the pain, its cause, diagnosis and therapy. In the theoretical part I dealt with general principles as to the training of the back and with the importance of this training for the therapy of vertebrogenous patients. The aim of the work and of the research was to discover whether a change in locomotory stereotypes before and after therapy took place at patients with pains in lumbosacral part of the backbone and to propose for three different professions the therapy inclusive of necessary regiment measures. The hypothesis was proclaimed that the therapy including also the training of the back will bring about the change locomotory habits of the patients. Secondary analysis of data from health documentation in combination with directly watching the patents were used as technology of the research. The last form of the research is the questionnaire interviewing of the patients suffering from pains in the lumbosacral part of the backbone. It concerns an integral research with prevailing technologies of quantitative research. The basic check group, that are clients of the physiotherapeutical clinic, who suffer from back pains in the lumbosacral part of the backbone. 100 patients altogether underwent the therapy. They were given the same number of questionnaires. All of them were returned. Theoretical questionnaire investigation was under way from October 2007 to March 2008. I used diagrams in processing the collected data and show the results in percentages. I chose three different professions in the course of therapy, and that secretary, forester and student doing sport and I proposed for them the therapy of back pain, that included also the training of their backs and regimen measures as pain prevention. The proclaimed hypothesis was confirmed in connection with the aim of the work. The research work, I carried out, present analysis of particular locomotory stereotypes. The results showed that locomotory habits had been improved after the therapy in direction to psychology. If the patients observe the stereotypes, they learnt and continue the physiotherapy, I chose, and the part of which is also the training of the back, helped not only the patients themselves, but that it brought benefit for the whole physiotherapeutical clinic. The work will be exploited for organization of seminars on the given place of work.

Orientador: Neusa Maria Costa Alexandre / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T13:14:18Z (GMT). No. of bitstreams: 1 Vigatto_Ricardo_M.pdf: 4076945 bytes, checksum: edca34fee371b306dd3b9984b10e2154 (MD5) Previous issue date: 2006 / Resumo: A literatura tem identificado a necessidade de utilização de instrumentos de medida padronizados e confiáveis que avaliam a dor lombar. O questionário Oswestry tomou-se um dos principais instrumentos utilizados para avaliar os distúrbios da coluna vertebral. O objetivo desse estudo foi traduzir e adaptar a versão do questionário Oswestry para o português e avaliar sua validade e confiabilidade. A adaptação cultural foi realizada de acordo com a metodologia recomendada internacionalmente, usando as seguintes fases: tradução, retro-tradução, revisão por um comitê de especialistas e pré-teste. Primeiramente, o questionário foi traduzido para o português por dois tradutores bilíngües, de uma forma independente. Posteriormente, dois outros tradutores profissionais que possuíam como língua materna o inglês, fizeram a retro-tradução independentemente um do outro. Um comitê composto por seis especialistas revisou as versões obtidas e desenvolveram a versão final do instrumento. Essa versão foi pré-testada em 40 pacientes com lombalgia. A confiabilidade foi avaliada através da estabilidade (teste-resteste) e da consistência interna. A validade foi obtida comparando-se a pontuação do Oswestry com outros instrumentos de medida: o questionário Roland-Morris, o questionário SF-36 e uma escala numérica de dor. As propriedades psicométricas da versão traduzida foram avaliadas aplicando o questionário em 120 sujeitos com dor lombar. Os resultados mostraram uma boa validade e consistência interna (Alfa de Cronbach=0,87). No teste-resteste os resultados apontaram uma alta correlação intraclasse (r=0,99). O questionário Oswestry demonstrou possuir uma moderada correlação com a dor, usando-se uma escala numérica de dor (r=0,66). Uma correlação relativamente alta foi encontrada entre as pontuações do Oswestry e do Roland-Morris (r=0,81). Houve uma correlação significante (p<0,001) entre a pontuação do Oswestry e as oito dimensões do questionário SF-36. Os coeficientes de correlação mais altos foram com a capacidade funcional (r=0,83), com a dor (r=0,58) e com os aspectos físicos (r=0,53). Este estudo demonstrou que o processo usado para a adaptação do questionário Oswestry foi realizado com sucesso e que a versão adaptada possui excelentes propriedades psicométricas / Abstract: Reports in the literature have identified a need for internationally standardized and reliable measurements to analyse back pain. The Oswestry questionnaire has become one of the principal outcome measures used in the management of spinal disorders. The objective of this study was to translate and adapt a version of the Oswestry questionnaire into Brazilian Portuguese and evaluate its reliability. The cross-cultural adaptation was performed according to the internationally recommended methodology, using the following guidelines: translation, back-translation; revision by an expert committee and pretesting. First the questionnaire was independently translated into Portuguese by two bilingual translators. Second, two other professional translators whose mother tongue was English performed a back-translation independently from one another. A committee consisting of six specialists was brought together and developed a final version. This version was pretested on 40 subjects suffering from low back pain. Reliability was estimated through stability (test-retest) and homogeneity assessment. The validity was tested comparing scores of the Oswestry with the following measures: The Roland-Morris Disability Questionnaire, the SF-36 questionnaire and a numerical pain scale. The psychometric properties of the translated version were evaluated by administering the questionnaire to 120 subjects with back pain. Results indicated good content validity and internal consistency (Cronbach alpha = 0.87). Intraclass correlation coefficient for test-retest reliability was r = 0.99. The Oswestry questionnaire showed moderate correlation with pain measure using a numerical pain scale (r = 0 66). Relatively high correlation was also found between the Oswestry and the Roland - Morris scores (r = 0.81). There was significant correlation (p< 0.001) between the Oswestry scores and the eight scales of the SF - 36 questionnaire. The highest correlation coefficients were for physical functioning (r = 0,83); bodily pain (r = 0,58) and role physical (r = 0,53). This study confirmed that the process used for adaptation of the Oswestry questionnaire was well succeeded and that this version had excellent psychometrical properties / Mestrado / Enfermagem e Trabalho / Mestre em Enfermagem

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