Made available in DSpace on 2014-10-09T12:53:38Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:08:26Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Orientador: Marcos Hikari Toyama / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T00:18:07Z (GMT). No. of bitstreams: 1 Fonseca_FabianaVieira_M.pdf: 764391 bytes, checksum: 2ea9b8ea9a806b452272ef5f3e45ba17 (MD5) Previous issue date: 2006 / Resumo: O uso de toxinas isoladas de venenos como ferramentas moleculares na compreensão de diversos eventos fisiológicos e patológicos tem sido comprovadas por vários trabalhos na literatura. A serpente Crotalus durissus cascavella é encontrada nas áreas de caatinga do nordeste do Brasil, desde o Maranhão até norte do estado de Minas Gerais, e apesar de pouco estudada sua picada constitui um importante problema da saúde pública (Martins et al, 1998). A agregação platequetária é bem caracterizada para a convulxina isolada do veneno de Crotalus durissus cascavella e Crotalus durissus terrificus. O objetivo principal do projeto foi avaliar a atividade de agregação plaquetária induzida por outras frações biológicas e farmacologicamente importantes do veneno total de Cotalus durissus cascavella, que neste projeto foram a crotoxina e giroxina. Através de uma combinação de varias metodologias em HPLC como exclusão molecular, troca iônica e de fase reversa conseguimos isolar os principais constituintes da crotoxina (PLA2, crotapotina e as proteínas ¿trombina-like¿) e a L-aminoácido oxidase a partir da giroxina. Durante o fracionamento do veneno total em coluna de HPLC de exclusão molecular foram detectados dois picos de atividade serino protease, um na fração giroxínica e outro na fração crotoxínica, sendo que na fração crotoxínica encontrou-se uma nova protease até então não caracterizada e na fração giroxínica foram monitoradas a atividade L-aminácido oxidase. Através da cromatografia em HPLC de troca iônica em DEAE 5PW obteve-se a fração Laminoácido oxidase cujo grau de homogeneidade molecular foi confirmado por HPLC de fase reversa. Da fração crotoxínica foram obtidos três grupos principais de proteínas (PLA2, crotapotinas e proteases) e da fração proteolítica foram isoladas três isoformas principais denominadas de F201, F202 e F203, sendo a fração F202, a fração majoritária. F202 foi obtida com maior homegeneidade molecular com massa molecular de 28kDa e mostrou uma alta quantidade de ácido aspártico, ácido glutâmico e outros aminoácidos importantes como histidina, cisteína e lisina. Esta proteína mostrou alta especificidade para BApNA e mostrou um comportamento Michaelis-Menten com Vmáx estimado em 5,64 µM/min e um Km de 0,58mM para este substrato. Neste trabalho foi investigada a habilidade desta proteína em degradar fibrinogênio e observou-se que F202 clivou ambas as cadeias a e ß. A atividade enzimática assim como a agregação plaquetária foi inibida fortemente com a incubação com TLCK, um inibidor específico para serinoproteases. O N-terminal da seqüência de aminoácidos de F202 mostrou alta homologia com outras proteínas ¿trombina-like¿, mas foi significantemente diferente da ¿trombina-like¿ isolada da fração giroxina. Crotalus durissus cascavella apresenta uma fração menos estudada denominada giroxina que tem sido descrita como uma proteína ¿trombina-like¿ como relatado por Raw et al. (1986) e Alexander et al. (1988). Neste trabalho foi demonstrado que a giroxina é uma fração heterogênea composta de uma ¿trombina-like¿ e proteína LAO, a qual parece estar envolvida em várias atividades / Abstract: The use of the isolated toxins from poisons as molecular tools in the understanding of diverse physiological and pathological events has been proved by some works in literature. The serpent Crotalus durissus cascavella is found in the areas of Caatinga Northeast of Brazil, since Maranhão until North of the state of Minas Gerais, and in spite of it hasn¿t been studied a lot, its bite constitutes an important problem to the public health (Martins et al, 1998). The platelet aggregation is well characterized to the isolated convulxin of the poison of Crotalus durissus cascavella and Crotalus durissus terrificus. The main objective of the project was to evaluate the activity of platelet aggregation induced by gyroxin and crotoxin that are biological and pharmacological important fractions of the total poison of Cotalus durissus cascavella. Through a combination of various methodologies in HPLC -as molecular exclusion, ionic exchange and the reverse phase- we managed to isolate the main constituent of the crotoxin (PLA2, crotapotin and the thrombin-like proteins) and the L-amino acid oxidase from the gyroxin. During the fragmentation of the total poison in column of HPLC of molecular exclusion two peaks of serine protease were found: one in the gyroxin fraction and another one in the crotoxin fraction. In the crotoxin fraction a new protease in not characterized yet and in the gyroxin fraction was found the L-amino acid activity oxidase. Through the chromatography in HPLC of ionic exchange in DEAE 5PW that allowed to the attainment of the fraction L-amino acid oxidase whose degree of molecular homogeneity was confirmed by HPLC of reverse phase. From the crotoxin fraction three main groups of proteins (PLA2, crotapotin and proteases) were isolated, and from the named proteolyitic fraction three isoforms of F201, F202 and F203, noticing that the F202 fraction is the major one. The fraction F202 showed a high quantity of aspartic acid, glutamic acid and others amino acids very important as histidine, cysteine and lysine and so more molecular homogeneity could be obtained and with molecular mass of 28kDa. This protein whose behavior Michaelis-Menten with Vmáx measured in 5,64 µM/min and one Km de 0,58 mM to this substratum showed high specificity to BapNA. In this work was investigated the ability of this protein in degrading the fibrinogen and was observed that the F202 made the cleavage into both chains a and ß. The enzymatic activity as well as the platelet aggregation were strongly inhibited with the incubation with TLCK, a specific inhibitor to serine protease. The N-terminal of the amino acid sequence of F202 showed the high homology with other proteins thrombin-like, but it was significantly different from thrombin-like isolated fro m the gyroxin fraction. Crotalus durissus cascavella presents a fraction less studied named gyroxin that has been described as a protein thrombin-like as related by Raw et al. (1986) and Alexander et al. (1988). In this work was demonstrated that the gyroxin is a composed heterogeneous fraction of one thrombin-like and protein LAO, and this seems to be involved in some activities / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Made available in DSpace on 2014-10-09T12:53:38Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:08:26Z (GMT). No. of bitstreams: 0 / As serino proteases participam de diversos processos fisiológicos (tal como o de coagulação) e patológicos. Essas enzimas estão amplamente distribuídas entre as espécies, são também toxinas dos venenos de serpentes, sendo denominadas SVSPs (snake venom serine proteases). Essas SVSPs são multifuncionais e contêm uma tríade catalítica formada pelos aminoácidos HDS. Algumas SVSPs são comercialmente disponíveis, sendo indicadas para o tratamento de infarto do miocárdio, tromboses e embolia pulmonar. No veneno de Crotalus durissus terrificus estão descritas até o momento, apenas duas SVSPs sendo que a mais estudada é a giroxina que representa cerca de 2,5% do veneno total. No presente estudo foi reportado a clonagem de sete serino proteases amplificadas a partir de uma biblioteca de cDNA de glândula de veneno de um único espécime adulto de Crotalus durissus terrificus. Estes clones foram analisados com relação à organização do cDNA, estrutura e prováveis funções. A construção do modelo tridimensional da giroxina permitiu verificar as similaridades com tripsina, trombina e outras SVSPs. A glicosilação e a presença de muitas pontes dissulfetos dificultam a obtenção das SVSP recombinantes na forma solúvel e com atividade, por expressão em E.coli. Assim, neste trabalho foi abordada a expressão em células de mamífero (que realiza as modificações pós-traducionais) com resultados promissores. Para tanto, o peptídeo sinal de Igk, a seqüência madura e a região 3 UTR da giroxina foram clonados no vetor pED, originando um novo vetor (pED-Giro). Este vetor carrega o peptídeo sinal de Igk, o que possibilitou a secreção da giroxina para o meio de cultura. O vetor pED-Giro foi transfectado em células CHO DXB11 dhfr e COS-7. A giroxina foi detectada no extrato total das células COS-7 por western blot e, em seguida, purificada do meio de cultura com coluna de afinidade (Benzamidina Sepharose) e demonstrado sua integridade pelo ensaio de atividade esterásica. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

A existência de uma família gênica diversa de serino proteinases em Lepidóptera sugere que essas proteinases desempenham um papel importante na adaptação desses insetos à presença de inibidores de proteinases vegetais. Essas enzimas têm se revelado estarem envolvidas no processo digestivo de larvas de insetos. Larvas de Spodoptera frugiperda foram alimentadas com uma dieta suplementada com inibidor de proteinase de soja (IPS) e a expressão gênica de proteinases intestinais foi avaliada através de PCR em tempo real. Análises de transcrição anteriores mostraram a existência de dois grupos de serino proteinases: um grupo de genes constitutivamente expressos em larvas controle que é induzido pela dieta contendo IPS e um segundo grupo que está ausente no controle, mas que é também induzido por uma dieta rica em IPS. No presente trabalho foi observado um terceiro grupo de proteinases que não são nem induzidas nem reprimidas pela presença do IPS na dieta. Essa observação sugere que a adaptação de S. frugiperda ao IPS envolve a síntese de novas proteinases, a indução de enzimas preexistentes e ainda um terceiro grupo insensível à presença dos inibidores. Proteinases dos intestinos de larvas crescidas em dieta com IPS mostraram insensibilidade ao inibidor. As proteinases também foram insensíveis quando a atividade foi verificada com um inibidor de proteinases de amplo espectrum. Os resultados aqui apresentados propõem que a adaptação de S. frugiperda ao IPS segue uma estratégia generalizada, baseada na indução geral de um grande grupo de endoproteinases. / The existence of a diverse serine proteinase gene family in lepidopteran insects has suggested its significant role in the insect adaptation to plant proteinase inhibitors. These enzymes have been shown to be involved in the proteolytic digestion process of insect larvae. Spodoptera frugiperda larvae were fed on a diet supplemented with soybean proteinase inhibitor (SPI) and the gene expression of intestinal proteinases was evaluated by real time PCR. Previous transcription analyses found two groups of intestinal serine proteinases: one group of genes constitutively expressed in the control larvae that is induced by the SPI-containing diet during the experiment, and a second group that is absent in the control but also induced by the SPI rich diet. Herein was observed a third group of proteinases that are neither induced nor repressed by the presence of SPI in the diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis, up regulation of existing enzymes and that there is a third group insensitive to the presence of the inhibitors. Proteinases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteinases were also insensitive when the activity was checked with a broad-spectrum potato proteinase inhibitor. The results here presented propose that adaptation of S. frugiperda to SPI follows a \"shotgun\" approach, based on a general up regulation of a large set of endoproteinases.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-17T18:50:12Z No. of bitstreams: 1 Portela, Aline Cordeiro [Dissertação, 2014].pdf: 9279752 bytes, checksum: 9172b58477a24ce8a80f3405e0759adb (MD5) / Made available in DSpace on 2017-03-17T18:50:12Z (GMT). No. of bitstreams: 1 Portela, Aline Cordeiro [Dissertação, 2014].pdf: 9279752 bytes, checksum: 9172b58477a24ce8a80f3405e0759adb (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A dengue é um problema de saúde pública mundial, acometendo cerca de 50 milhões de pessoas por ano. O HCV é a principal causa de hepatite crônica e estima-se que 200 milhões de pessoas estejam infectadas no mundo. A terapia atual contra o HCV apresenta eficácia limitada e baixa tolerância, enquanto que para a dengue não existe um tratamento antiviral específico. Com o objetivo de explorar essa demanda, o presente trabalho descreve a síntese para a obtenção de quinze compostos peptideomiméticos inéditos, contendo o cerne rígido proveniente do isosorbídeo, como potenciais agentes inibidores da enzima NS3 protease de ambos os vírus. A estratégia de síntese dos peptideomiméticos consistiu inicialmente na obtenção de dez compostos inéditos, das séries 28a-c (derivada de oxazolonas) e 29a-g (derivada de aminoácidos N-protegidos). Os compostos foram inicialmente testados frente à inibição da protease do DENV-2, os quais não apresentaram resultados significativos. Entretanto, os resultados de inibição enzimática dos compostos frente à NS3/4A do HCV-1b foram mais satisfatórios, sendo o composto 28a (1,4:3,6-dianidro-5-[[(2Z)-2-(benzoilamino)-1-oxo-3-(2-tienil)-2-propen-1-il]amino] -2-deoxi-2-O-(fenilmetil)-D-iditiol, o mais ativo, com IC50 = 88μm. Estudos de docking molecular foram realizados para avaliar o modo de interação dos ligantes com a serina protease NS3/4A do HCV. Dentre os compostos sintetizados, 28a foi considerado protótipo para o desenvolvimento de novos compostos com potencial de inibição da enzima em questão. A partir de 28a, a nova série de compostos inéditos 42a-e foi proposta e obtida. Os resultados dos ensaios farmacológicos frente à NS3/4A do HCV-1b permitiram-nos inferir que a substituição do tiofeno de 28a pelo furano e 3-metiltiofeno em 42b e 42c, respectivamente, resultou em um aumento de cerca de 30% no perfil de inibição enzimática, com inibição de 70% dessa enzima viral. Dessa forma, podemos identificar esses dois novos compostos como protótipos para o planejamento de futuras moléculas potencialmente inibidoras da enzima serina protease do vírus da Hepatite C / Dengue fever is a worldwide public health concern, affecting approximately 50 million people per year. HCV is the main cause of chronic hepatitis and it is estimated that 200 million people are infected worldwide. Current therapy against HCV has limited efficacy and low tolerance, and there is no specific antiviral treatment for dengue fever. In order to exploit this demand, this work describes the synthesis for obtaining fifteen novel peptidemimetic compounds, isosorbide derivatives as potential inhibitory agents of the NS3 protease enzyme of both viruses. Initially the synthetic strategy of peptidemimetics consisted in obtaining ten novel compounds: 28a-c (derived from oxazolones) and 29a-g (derived from N-protected amino acids) series. The compounds were initially tested on the inhibition of protease DENV-2 , which results were not significant. However, the results of enzymatic inhibition of the compounds against the NS3/4A HCV-1b was more satisfactory, and the compound 28a (1,4 : 3,6- dianhydro -5 - [[( 2Z ) -2- ( benzoylamino) -1 -oxo- 3- (2- thienyl) -2 - propen -1- yl] amino] -2- deoxy -2- O- ( phenylmethyl ) -D- iditiol) , the most active , presenting IC50 = 88μm. Molecular docking studies were performed to assess the compounds mode of interaction with the serine protease NS3 / 4A HCV. Among the compounds synthesized, 28a was considered a prototype for the development of new compounds with a potential inhibition of the enzyme in question. Staring from 28a as a prototype the 42a-e serie was proposed and obtained. The results of pharmacological tests against the NS3/ 4A HCV -1b allowed us to conclude that substitution of the 28a furan thiophene and 3- methylthiophene in 42b and 42c respectively resulted in an increase of 30 % in the profile enzyme inhibition , with inhibition of 70 % of the viral enzyme . Thus , we can identify these new compounds as prototypes for planning future potentially molecule inhibitors of serine protease enzyme of the Hepatitis C virus

Le sujet de cette thèse porte sur la synthèse d'inhibiteurs spécifique de la Sérine-Thréonine phosphatase I (PP1). Un criblage de la chimiothèque de l'institut Curie, réalisé par l'équipe du Dr. Popov a permis d'identifier une 3-aryl-1(2H)isoquinolone, qui perturbe la dynamique des microtubules et qui s’est ensuite avéré être un inhibiteur sélectif de PP1. Dans une première partie, nous avons mis au point une nouvelle méthodologie de synthèse de ces composés hétérocycliques par une réaction tandem d’aminocarbonylation-cyclisation pallado catalysée. L’étude d’une seconde voie de synthèse de ces composés a été étudié par réaction d'arylation direct d'une 1(2H)isoquinolone. Dans le but de trouver d’autres hit, ligand de cette phosphatase, nous avons tenté de développer un test de triple hybride chimique, en collaboration avec la société Hybrigenics. Ce test est basé sur l’interaction de notre inhibiteur hit avec la phosphatase PP1. Pour cela, nous avons synthétisé une sonde à partir de la molécule hit initiale. La deuxième partie a trait à un développement de chimie médicinal pour optimiser le hit initial. Des dérivés de très bonne sélectivité pour l’enzyme cible ont été préparés. / This PhD thesis deals with the synthesis of serine threonine phosphatase I (PPI) inhibitors. This project started with the screening of the Institut Curie’s Library carried out by Dr. Popov team. They identified a 3-aryl-1(2H)isoquinolone (hit molecule) which strongly disturbs the microtubules dynamics. In the first part, we designed an original methodology to prepare those heterocycles, though a tandem palladium catalyzed aminocarbonylation/cyclization reaction. Then, we studied the direct arylation reaction to obtain the desired scaffold. In collaboration with Hybrigenics, we synthesize a probe for a triple hybrid system, based on the specific interaction of the hit molecule with its target PPI. Thanks to this system, one could identify new inhibitors of the targeted phosphatase protein. Eventually, a library of isoquinolones derivatives was synthesized. During the invitro tests, some of those molecules proved to be very specific for the serine threonine phosphatase I.

Les récepteurs N-méthyl-D-aspartate (NMDA) sont des récepteurs ionotropiques du glutamate jouant un rôle clé dans la plasticité synaptique et les fonctions cognitives. En conséquence, la perturbation de leur activité est impliquée dans de nombreux troubles neurologiques et psychiatriques tels que l'épilepsie et la schizophrénie. La particularité de ces récepteurs est qu'ils nécessitent pour être activés la liaison simultanée de leur agoniste, le glutamate, et d'un co-agoniste. La glycine fut le premier co-agoniste identifié mais plus récemment, de nombreuses études ont révélé que la D-sérine joue également ce rôle dans de nombreuses régions cérébrales, notamment dans l'hippocampe. Toutefois il restait à définir si les fonctions de ces deux co-agonistes étaient régulées au cours du développement ou si elles étaient spécifiques à certaines synapses. Dans la première partie de mon travail, j'ai montré que la D-sérine est le co-agoniste préférentiel des synapses SC-CA1 matures alors que la glycine est le co-agoniste préférentiel des synapses mPP-DG. De plus, le remplacement des récepteurs NMDA composés de sous-unités GluN2B par des récepteurs contenant GluN2A au cours du développement post-natal survient au même moment qu'un changement dans l'identité du co-agoniste préférentiel des synapses SC-CA1. Dans la seconde partie de mon travail, je me suis intéressée à la contribution de la D-sérine en conditions pathologiques sur un modèle murin d'épilepsie chimio-induite par la pilocarpine. J'ai ainsi montré l'implication de la D-sérine dans l'activité épileptique initiée par la pilocarpine. / N-methyl-D-aspartate (NMDA) receptors are glutamate-gated ionotropic receptors which play a crucial role in synaptic plasticity and cognitive functions. As a consequence, disturbance in their activity is correlated with a broad range of neurological and psychiatric disorders including epilepsy and schizophrenia. The major particularity of NMDA receptors is the requirement of simultaneous binding of their agonist, glutamate, and a co-agonist to be activated. Glycine was the first co-agonist identified but more recently several studies showed that D-serine is also playing this role in many brain areas including the hippocampus. Whether the identity of the co-agonist is synapse specific or developmentally regulated remains unexplored. In the first part of my work I showed that D-serine is the preferred co-agonist at SC-CA1 mature synapses while glycine is the preferred one at mPP-DG synapse. Moreover, we showed that during postnatal development, the replacement of GluN2B by GluN2A-containing NMDA receptors at SC-CA1 synapses parallels a change in the co-agonist identity from glycine to D-serine. In the second part of my work I investigated the contribution of D-serine in pathological conditions. By using a model of acute intoxication of pilocarpine, I demonstrated that D-serine is implicated in epileptiform activity initiated after pilocarpine perfusion.

No description available.

La phosphorylation de protéines correspond à l’addition covalente d’un groupement phosphate (PO4 3-) par une protéine kinase sur un substrat. Cette réaction est réversible : la déphosphorylation est catalysée par des protéines phosphatases. Chez S. aureus, la sérine/thréonine kinase Stk1 phosphoryle des acides aminés sérines et thréonines et a été montrée impliquée dans la régulation de la virulence du pathogène : nous avons approfondi les connaissances sur ce mécanisme en identifiant trois nouveaux substrats et en étudiant les effets de la phosphorylation sur leur activité : - l’enzyme LuxS, responsable de la synthèse de l’AI-2 impliqué dans la communication intra bactérienne, voit son activité enzymatique drastiquement diminuée en étant phosphorylée par Stk1 sur un site thréonine unique (T14) ; - le régulateur CcpA, dont la fixation sur l’ADN module l’expression de nombreux gènes de virulence, est phosphorylée par Stk1 sur deux sites (T18 et T33) et cette phosphorylation diminue l’affinité de la protéine régulatrice CcpA pour l’ADN de ses gènes cibles ; - l’élément réponse du système à deux composants SaeR est phosphorylé sur deux sites thréonines (T87 et T192) de la région impliquée dans l’affinité de SaeR pour l’ADN. Les rôles de la kinase Stk1 sont donc multiples et liés à la régulation de la virulence de S. aureus. Les autres voies mettant en jeu la phosphorylation de protéines bactériennes, comme les systèmes à deux composants ou le système CcpA/HPr, sont couplées à cette phosphorylation par la sérine/thréonine kinase : ces résultats soulignent à la fois la diversité et la complexité de la régulation des mécanismes responsables de la virulence de S. aureus / Protein phosphorylation consists in the catalyzed addition of a phosphate group on a substrate. This reversible reaction is ensured by both kinase and phosphatase proteins. S. aureus is a human prokaryote pathogen and a part of its virulence is known to be regulated by the serine/threonine kinase Stk1, which phosphorylates serine or threonine residues of its substrates. We investigated the mechanisms of this virulence regulation and newly identified three substrates of Stk1: the quorumsensing LuxS protein, the catabolite carbon protein CcpA and the two components system response element SaeR. LuxS is phosphorylated on a unique threonine residue in position 14 and phosphorylation dramatically influences its enzymatic activity on AI-2 production. CcpA phosphorylation on two threonine residues in the DNA-binding region of the protein (T18 and T33) decreases the affinity of the protein for its targeted DNA sequences. Besides, Stk1 also phosphorylates the response element SaeR on two threonine residues (T87 and T192) in the DNAbinding region. Therefore Stk1 kinase plays numerous roles in S. aureus virulence regulation and the complexity of this regulation pattern increases when considering that three of the phosphorylation pathways in prokaryotes are crossed over: the two components system phosphorylation, the HPr/HPrK system and the serine/threonine kinase proteins phosphorylation. These results highlight the need to focus on Stk1 as a key element in the complexity of virulence regulation in S. aureus

It is increasingly important to identify the nature of the interfacial water in biology in order to explain how biological functions and systems work. It is not simply a matter of which biomolecules are present in a cell, but also of how these biomolecules interact with one another. This body of work uses neutron scattering techniques to explain the nature of the vibrational dynamics of water interacting with biomolecules and systems that mimic the biological molecular crowding environment of a cell. Recent work in science has seen the synthesis of periodic mesoporous organosilicas with organic groups attached. In the first paper in this thesis, the use of one of these materials is highlighted to look at confined water, equivalent to the water found in a crowded cellular environment. Here it is shown that the properties of the water within the pores and water molecules around the surface were shown to be different and then identified as interfacial and bulk water respectively. In order to develop the investigation of interfacial water with biological matter, it seemed appropriate to start with the most basic molecules, amino acids. The second paper presents a complete survey of the 20 biologically important amino acids using one of the world's highest resolution neutron scattering spectrometer (TOSCA at ISIS, Rutherford Appleton Laboratory). Computer simulation of the experimental work through molecular dynamics, allows many vibrational modes to be assigned for the first time and correlated with the broader vibrational peaks previously observed for proteins. Comparison of the dry states with the hydrated states of amino acids, gives some insight into the sites within the amino acid side chains where water molecules are likely to bind. For serine this is the hydroxyl group in the side chain. The third paper focuses on IINS data of serine in more detail and discusses several low energy vibrational modes that have been assigned and for the first time, shows how the presence of water molecules changes the dynamic behaviour of librational and torsional modes differently. The combination of these studies allows a clearer picture of how water in biology interacts with biomolecules and of the importance of water to our existence.